METHODS OF PROGNOSIS AND TREATMENT OF PATIENTS SUFFERING
FROM CANCER
FIELD OF THE INVENTION:
The present invention is in the field of medicine, in particular oncology.
BACKGROUND OF THE INVENTION:
Chronic obstructive pulmonary disease (COPD) is a commonly occurring condition primarily caused by long term exposure to cigarette smoke. This pathologic state is characterized by airways remodeling and destruction of the alveolar wall which leads to an irreversible airflow obstruction (1). This disease has been identified as an independent risk factor for the development of lung cancer, with one-third of COPD patients succumbing to this disease (2). However, the mechanisms leading to this observation are not completely elucidated.
One common mechanism to these two pathologies is the inflammation linked to the chronic exposure to cigarette smoke and aging. This chronic inflammation is sustained by senescent cells which participates to the cancer development. Indeed, senescent cells promotes the growth of cancer cells in vitro and the development of tumor in vivo with a more important effect than non-senescent cells (3). Moreover, the removal of senescent cells in mice model aged to 12 and 18 months delayed the formation of cancer and reduced the occurring of metastasis (4). Furthermore, several studies showed that the secretome of senescent cells referred as SASP induced the epithelial mesenchymal transition (EMT) and the tumor progression (5). However, the role of SASP in lung tumor progression in COPD patients still remain to be confirmed.
Recent research has shed light on the significance of the tumor microenvironment (TME) in the progression of lung cancer, revealing that factors such as inflammation and oxidative stress play crucial roles in promoting cancer growth. The TME is heterogeneous and varies according to different cancer types of localizations. It is formed by an extracellular matrix and different cell populations among which we can highlight cancer-associated fibroblasts (CAFs), endothelial cells as well as immune cells such as dendritic cells, macrophages and lymphocytes. Besides not in the immediate proximity of cancer cells pulmonary fibroblasts could also play a role in the promotion of carcinogenesis through exocrine or paracrine secretions. Indeed, fibroblasts activate the invasion of lung cancer cell line (6). Furthermore, the fibroblasts from breast favored the proliferation of cancer cells in mice model and in organoids (7,8). Should these effects be less efficient than those observed for CAF, it suggests that fibroblasts could share common mechanism and favor the growth of residual cancer cells. These date reinforces the need to understand the role of fibroblasts in lung tumor progression.
In COPD patients, the chronic exposure to cigarette smoke lead to the accumulation of senescent cells especially fibroblasts. These cells secrete several inflammatory mediators such as CXCL12, HGF, IL-1, IL-8, or MMPs which are known to induce the tumor progression (9,10). Recently, a study has shown that in the lung tissue from COPD patients at early stage of the disease and far away from the tumor, a pro-tumoral program was activated in fibroblasts (11,12). This program depended on mTOR and lead to the secretion of IL-6 known to be involved in the invasion of cancer cells. These data suggest that senescent lung fibroblasts could participate to the lung progression of COPD patients via the SASP. However, the composition of SASP of senescent lung fibroblasts are not completely characterized. sPLA2 XIIA belongs to a family of Ca2+ -dependent low-molecular weight enzymes composed by 10 catalytically active isoforms whose role in tumor development is unclear (13). Indeed, their effect can be pro or anti tumoral and depends on microenvironment and on the type of cancers (14). sPLA2 XIIA is an atypical form of sPLA2, expressed ubiquitously and increased in various lung inflammatory diseases such as asthma and pneumonia (15,16). This isoform is characterized by a low catalytic activity compared with the others sPLA2, suggesting that sPLA2 XIIA acts mainly by binding to its receptors (PLA2R1 and heparan sulfate proteoglycans -syndecans-) (17). However, the role of sPLA2 XIIA is unknown in the tumor progression, especially in the lung tumor.
SUMMARY OF THE INVENTION:
The present invention relates to methods of diagnosis, prognosis and treatment of patients suffering from cancer.
In a first aspect, the invention relates to an in vitro method for diagnosing cancer in a subject, comprising the steps of (i) determining the expression level of the secreted PLA2 XIIA in a sample obtained from said patient, and (ii) comparing the expression level determined at step i) with its predetermined reference value, and iii) concluding that the subject suffers from cancer when the expression level of the secreted sPLA2 XIIA is higher than its predetermined reference value.
In a second aspect, the invention relates to an in vitro method for predicting the survival time of a patient suffering from cancer in a subject, comprising the steps of (i) determining the expression level of the secreted PLA2 XIIA in a sample obtained from said subject, and (ii) comparing the expression level determined at step i) with its predetermined reference value, and iii) concluding that the subject that the patient will have a short survival time when the level determined at step i) is higher than the predetermined reference value or concluding that the patient will have a long survival time when the level determined at step i) is lower than the predetermined reference value.
In a third aspect, the invention relates to a method for preventing or treating cancer in a subject, comprising the administering to said subject a therapeutically effective amount of a sPLA2 XIIA inhibitor and/or at least one inhibitor of sPLA2 XIIA receptors .
In particular, the present invention is defined by the claims.
DETAILED DESCRIPTION OF THE INVENTION:
The inventors show that secreted PLA2 XIIA (sPLA2 XIIA) increases the proliferation, the migration and organoid growth of both lung cancer cell lines (NCI and A549). They shows that sPLA2 XIIA induces notably the epithelial mesenchymal transition (EMT), which is involved in the invasion of cancer cells and in the formation of metastasis. They also demonstrate that the effect of sPLA2 XIIA is mediated in particular by its receptor syndecan 1 and 4.
Taken altogether, these data define sPLA2 XIIA as a circulating biomarker of poor prognosis in lung cancer and establish a requirement for sPLA2 XIIA inhibition for the treatment or prevention of lung cancer, especially in the COPD patients.
Methods for diagnosis and predicting the survival time
Accordingly, in a fist aspect, invention relates to an in vitro method for diagnosing cancer in a subject, comprising the steps of (i) determining the expression level of the secreted PLA2 XIIA (sPLA2 XIIA) and/or at least one sPLA2 XIIA receptors in a biological sample obtained from said subject, and (ii) comparing the expression level(s) for each marker determined at step i) with a predetermined reference value, and iii) concluding that the subject suffers from cancer when the expression level of the secreted sPLA2 XIIA and/or at least one sPLA2 XIIA receptors is higher than a predetermined reference value for each marker.
In particular embodiment the at least one sPLA2 XIIA receptor is selected among PLA2R1, syndecan and glypican.
In particular embodiment the at least one sPLA2 XIIA receptor is selected among syndecan and glypican.
In particular embodiment the at least one sPLA2 XIIA receptor is syndecan. As used herein, the term “secreted phospholipase A2 group XIIA” or “sPLA2 XIIA” refers to one of catalytically active isoforms of secreted phospholipase A2 (sPLA2) which catalyzes the calcium-dependent hydrolysis of the 2-acyl groups in 3-sn phosphoglycerides. The sequence of said protein can be found under the Uniprot accession number Q9BZM1. sPLA2s are released in plasma and biological fluids of patients with various inflammatory diseases. Indeed, sPLA2 has been shown to promote inflammation in mammals by catalyzing the first step of the arachidonic acid pathway by breaking down phospholipids, resulting in the formation of fatty acids including arachidonic acid. sPLA2s act through specific receptors: soluble and membrane-bound M-type sPLA2 receptor (PLA2R1) and heparan sulfate proteoglycans such as syndecans and glypicans. Inventors found also an up-regulation of these specific receptors in cancer cell line.
As used herein, the term “PLA2R1” has its general meaning in the art and refers to the secretory phospholipase A2 receptor. The sequence of said protein can be found under the Uniprot accession number QI 3018.
As used herein, the term “syndecan” has its general meaning in the art and refers to a single transmembrane domain proteins acting as coreceptors for G protein-coupled receptors. The syndecan protein family has four members: syndecan- 1, -2, -3 and -4 whose the amino acid length is 310, 201, 346 and 198 respectively. The subclassification of the family depending on the existence of GAG binding sites either at both ends of the ectodomain (syndecan-1 and -3) or at the distal part only (syndecan-2 and -4) and a relatively long Thr- Ser-Pro-rich area in the middle of syndecan- 3’s ectodomain. The sequence of protein syndecan-1, -2, -3, and -4 can be found under the Uniprot accession number Pl 8827, P34741, 075056 and P31431, respectively.
As used herein, the term “glypican” has its general meaning in the art and refers to heparan sulfate proteoglycans. Glypicans are critically involved in developmental morphogenesis, and have been implicated as regulators in several cell signaling pathways. These include the Wnt and Hedgehog signaling pathways, as well as signaling of fibroblast growth factors and bone morphogenic proteins. The regulating processes performed by glypicans can either stimulate or inhibit specific cellular processes. The glypican protein family has six members: glypican-1, -2, -3 -4, -5 and -6 whose the protein sequence can be found under the Uniprot accession number P35052, Q8N158, P51654, 075487, P78333 and Q9Y625 respectively.
In some embodiments, concerning all the methods of the invention, the at least one sPLA2 XIIA receptor is selected from the group consisting in PLA2R1, syndecan-1, syndecan- 2, syndecan-3, syndecan-4, glypican-1, glypican-2, glypican-3, glypican-4, glypican-5 and glypican-6.
In some embodiments, concerning all the methods of the invention, the at least one sPLA2 XIIA receptor is 1, 2, 3, 4, 5, 6, or 7 sPLA2 XIIA receptor selected from the group consisting in PLA2R1, syndecan-1, syndecan-4, glypican-1, glypican-2, glypican-4 and glypican-6 are determined in step i).
In some embodiments, concerning all the methods of the invention, the at least one sPLA2 XIIA receptor is 1, 2, 3 or 4 receptor selected from the group consisting in syndecan-1, syndecan-4, glypican-1 and glypican-4.
In some embodiments, concerning all the methods of the invention, the at least one sPLA2 XIIA receptor is syndecan-1 and/or syndecan-4.
In some embodiments, concerning all the methods of the invention, the expression of syndecan-1 and/or sPLA2 XIIA are determined in step i).
In some embodiments, concerning all the methods of the invention, the expression of syndecan-4 and/or sPLA2 XIIA are determined in step i).
In some embodiments, concerning all the methods of the invention, the expression of syndecan-4, syndecan-1 and/or sPLA2 XIIA are determined in step i).
As used herein, the term “subject” refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human. In a particular embodiment, the subject is a human who is susceptible to have cancer. In a particular embodiment, the subject is a human who is susceptible to have lung cancer, colorectal cancer, breast cancer or prostate cancer. In a particular embodiment, the subject furthermore suffers from chronic obstructive pulmonary disease (COPD).
As used herein the term "biological sample" in the context of the present invention is a biological sample isolated from a subject and can include, by way of example and not limitation, bodily fluids and/or tissue extracts such as homogenates or solubilized tissue obtained from a subject. Tissue extracts are obtained routinely from tissue biopsy and autopsy material. Bodily fluids useful in the present invention include blood, bone marrow aspirate, urine, saliva, broncho-alveolar lavage (LBA) or any other bodily secretion or derivative thereof. As used herein "blood" includes whole blood, plasma, serum, circulating cells, constituents, or any derivative of blood. In a particular embodiment, the biological sample is a blood sample, serum sample or a tumor tissue sample. In a particular embodiment, the biological sample is previously obtained from the subject.
As used herein, the term "cancer” and "tumors" refer to or describe the pathological condition in mammals that is typically characterized by unregulated cell growth. More precisely, in the use of the invention, diseases, namely tumors that expresses/secretes sPLA2 XIIA are most likely to respond to the inhibitors of the invention.
In particular embodiment, the cancer is selected from the group consisting of breast cancer, melanoma, ovarian cancer, lung cancer, liver cancer, pancreatic cancer, melanoma, squamous cell carcinoma, endometrial cancer, head and neck cancer, bladder cancer, malignant glioma, prostate cancer, colorectal cancer, colon adenocarcinoma or gastric cancer.
In preferred embodiments, regarding all the methods of the invention, the cancer is lung cancer, colorectal cancer, breast cancer or prostate cancer..
As used herein, the term "lung cancer" includes, but is not limited to all types of lung cancers at all stages of progression like lung carcinomas metastatic lung cancer, non-small cell lung carcinomas (NSCLC) or small cell lung carcinomas (SCLC).
In some embodiments, the lung cancer is a non-small cell lung carcinomas (NSCLC).
As used herein, the term “non-small cell lung cancer” or "NSCLC" has its general meaning in the art and includes a disease in which malignant cancer cells form in the tissues of the lung. Examples of non-small cell lung cancers include, but are not limited to, squamous cell carcinoma, large cell carcinoma or lung adenocarcinoma.
In some embodiments, the lung cancer is a lung adenocarcinoma.
As used herein, the term “lung adenocarcinoma” has its general meaning in the art and refers to a subtype of non-small cell lung cancer. Lung adenocarcinoma starts in glandular cells, which secrete substances such as mucus, and tends to develop in smaller airways, such as alveoli. Lung adenocarcinoma is usually located more along the outer edges of the lungs. Lung adenocarcinoma tends to grow more slowly than other lung cancers.
An additional object of the invention relates to an in vitro method for determining whether is at risk of developing or having cancer in a subject, comprising the steps of (i) determining the expression level of sPLA2 XIIA and/or at least one sPLA2XIIA receptors selected among PLA2R1, syndecan and glypican in a biological sample obtained from said subject, (ii) comparing the expression levels for each marker determined at step (i) with a predetermined reference value, and iii) concluding that the subject is at risk of having cancer when the expression levels determined at step (i) is higher than a predetermined reference value for each marker.
In particular embodiment, the cancer is lung cancer and the subject suffers from chronic obstructive pulmonary disease. The method of the present invention is particularly suitable to determine the risk of a COPD subject to develop or have a lung cancer.
As used herein, the term "chronic obstructive pulmonary disease" or "COPD" has its general meaning in the art and refers to a set of physiological symptoms including chronic cough, expectoration, exertional dyspnea and a significant, progressive reduction in airflow that may or may not be partly reversible. COPD is a disease characterized by a progressive airflow limitation caused by an abnormal inflammatory reaction to the chronic inhalation of particles. The Global Initiative for Chronic Obstructive Lung Disease (GOLD) has classified 4 different stages of COPD : stage I, where the forced expiratory volume in one second (FEV1) > 80 % normal, to stage IV where the FEVl<30% normal. This disease has been identified as an independent risk factor for the development of lung cancer, with one-third of COPD patients succumbing to this disease.
As used herein, the term "risk" relates to the probability that an event will occur over a specific time period, and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort, or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of absolute risks of a subject compared either to the absolute risks of low risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of event and (1- p) is the probability of no event) to no conversion. Risk evaluation," or "evaluation of risk" in the context of the present invention, encompasses predicting the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another. Risk evaluation can also comprise the prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of conversion. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk. In some embodiments, the present invention may be used so as to discriminate those at risk from normal.
In some embodiment, the cancer is a resistant cancer, in particular a resistant lung cancer, a resistant colorectal cancer, a resistant breast cancer or a resistant prostate cancer.
In some embodiment, the cancer is a resistant lung cancer.
As used herein, the term “resistant cancer” refers to cancer, which does not respond to classical treatment. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. The resistance to drug leads to rapid progression of metastatic of cancer.
In another words, the invention refers to an in vitro method for predicting the risk of developing or having a resistant cancer in a subject comprising i) determining the level of sPLA2 XIIA and/or at least one sPLA2 XIIA receptors in a sample obtained from the subject ii) comparing the level(s) for each marker determined at step i) with a predetermined reference value and iii) concluding that the subject is at high risk of having a resistant cancer when the level(s) determined at step i) is higher than a predetermined reference value for each marker and concluding that the subject is at low risk of having a resistant cancer when the level(s) determined at step i) is lower than a predetermined reference value for each marker.
A further object of the present invention relates to an in vitro method for predicting the response to treatment in a patient suffering from cancer, comprising the step of determining in a sample obtained from said patient the level of sPLA2 XIIA and/or at least one sPLA2 XIIA receptors, wherein a low level of sPLA2 XIIA and/or at least one sPLA2 XIIA receptors indicate that the subject is at high risk of achieving a response.
Typically, high levels of sPLA2 XIIA and/or at least one sPLA2 XIIA receptors indicate that the subject is at low risk of achieving a response, whereas low levels of sPLA2 XIIA and/or at least one sPLA2 XIIA receptors indicate that the subject is at high risk of achieving a response.
As used herein, the term “high” refers to a measure that is significantly greater than normal, greater than a standard, such as a predetermined reference value or a subgroup measure, or that is relatively greater than another subgroup measure. As used herein, the term “low” refers to a level that is less than normal or less than a standard, such as a predetermined reference value or a subgroup measure that is relatively less than another subgroup level. In some embodiments, the “predetermined reference value” is relative to a number or value derived from population studies, including without limitation, patients of the same or similar age range, patients in the same or similar ethnic group, and patients having the same severity of cancer. Such predetermined reference values can be derived from statistical analyses and/or risk prediction data of populations obtained from mathematical algorithms and computed indices of the disease. Typically, the predetermined reference value is a threshold or cutoff value. Typically, a "threshold value" or "cutoff value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based on the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skill in the art. For example, retrospective measurement in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of in a group of reference, one can use algorithmic analysis to statistically treat the levels determined in samples to be tested and thus obtain a classification standard having significance for sample classification. The full name of the ROC curve is the receiver operator characteristic curve, also known as the receiver operation characteristic curve. It is mainly used for clinical and biochemical diagnostic tests. The ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cutoff values (thresholds or critical values, boundary values between normal and abnormal diagnostic test results) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate, and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point with high sensitivity and specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result improves as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used to draw the ROC curve, such as MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE- ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.
In some embodiments, the predetermined reference value is typically determined by carrying out a method comprising the steps of: a) providing a collection of samples from patients suffering from cancer, and especially lung cancer; b) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding patient (i.e. having or not cancer, the duration of the disease- free survival (DFS) and/or the overall survival (OS)); c) providing a serial of arbitrary quantification values; d) determining the level of the marker of interest (i.e. sPLA2 XIIA and/or sPLA2XIIA receptors) for each sample contained in the collection provided at step a); e) classifying said samples in two groups for one specific arbitrary quantification value provided at step c), respectively: (i) a first group comprising samples that exhibit a quantification value for level that is lower than the said arbitrary quantification value contained in the said serial of quantification values; (ii) a second group comprising samples that exhibit a quantification value for said level that is higher than the said arbitrary quantification value contained in the said serial of quantification values; whereby two groups of samples are obtained for the said specific quantification value, wherein the samples of each group are separately enumerated; f) calculating the statistical significance between (i) the quantification value obtained at step e) and (ii) the actual clinical outcome of the patients from which samples contained in the first and second groups defined at step f) derive; g) reiterating steps f) and g) until every arbitrary quantification value provided at step d) is tested; h) setting the said predetermined reference value as consisting of the arbitrary quantification value for which the highest statistical significance (most significant) has been calculated at step g)-
In one embodiment of the present invention, the threshold value may also be derived from sPLA2 XIIA, PLA2R1, syndecan or glypican expression level (or ratio, or score) determined in a sample derived from one or more cancer patients. Furthermore, retrospective measurement of the sPLA2 XIIA, PLA2R1, syndecan or glypican level (or ratio, or scores) in properly banked historical patients samples may be used in establishing these threshold values. Reference values are easily determinable by the one skilled in the art, by using the same techniques as for determining the level of sSPLA2 XIIA, PLA2R1, syndecan or glypican expression level in samples previously collected from the patient under testing.
The detection and quantification of sPLA2 XIIA and/or sPLA2XIIA receptors in the sample can be detected by any method known in the art.
As used herein, the term "expression level" refers, e.g., to a determined level of expression of gene of interest or protein of interest (i.e sPLA2 XIIA and/or sPLA2XIIA receptors). The expression level of expression indicates the amount of expression product in a sample. The expression product of a gene of interest can be the nucleic acid of interest itself, a nucleic acid transcribed or derived therefrom, or the a polypeptide or protein derived therefrom.
Measuring the expression level of sPLA2 XIIA and/or sPLA2XIIA receptors can be done by measuring the gene expression level of these genes and can be performed by a variety of techniques well known in the art.
Typically, the expression level of a gene may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example, the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR).
Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA).
Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.
Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labelled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labelled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colourless substance into a coloured substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.
Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene- 1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -di ethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthal ene-1 -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDarninofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol -reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).
In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al., Science 281 :20132016, 1998; Chan et al., Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can he produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif.).
Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.
Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.
Alternatively, an enzyme can he used in a metallographic detection scheme. For example, silver in situ hybridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670,113). Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).
In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.
For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.
Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et al., Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al., Am. .1. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.
Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following nonlimiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.
In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.
It will be appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can he envisioned, all of which are suitable in the context of the disclosed probes and assays.
Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are “specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).
The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.
In a particular embodiment, the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi- quantitative RT-PCR.
In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210). In another embodiment, the expression level is determined by metabolic imaging (see for example Yamashita T et al., Hepatology 2014, 60: 1674-1685 or Ueno A et al., Journal of hepatology 2014, 61 : 1080-1087).
Expression level of a gene may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the response of antipsychotic treatment, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1, TFRC, GAPDH, TBP and ABLP This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.
Measuring the expression level of the proteins sPLA2 XIIA and/or sPLA2XIIA receptors can be performed by a variety of techniques well known in the art. Typically protein expression level may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS), flow cytometry, mass cytometry or ELISA performed on the sample.
In the present application, the “level of protein” or the “protein level expression” means the quantity or concentration of said protein. In still another embodiment, the “level of protein” means the quantitative measurement of the protein expression relative to a negative control. Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.
The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresismass spectroscopy technique (CE-MS). etc. The reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.
The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.
Particularly, a mass spectrometry-based quantification methods may be used. Mass spectrometry-based quantification methods may be performed using either labelled or unlabelled approaches [DeSouza and Siu, 2012], Mass spectrometry -based quantification methods may be performed using chemical labeling, metabolic labeling or proteolytic labeling. Mass spectrometry-based quantification methods may be performed using mass spectrometry label free quantification, a quantification based on extracted ion chromatogram (EIC) and then profile alignment to determine differential level of polypeptides.
Particularly, a mass spectrometry-based quantification method particularly useful can be the use of targeted mass spectrometry methods as selected reaction monitoring (SRM), multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), data independent acquisition (DIA) and sequential window acquisition of all theoretical mass spectra (SWATH) [Moving target Zeliadt N 2014 The Scientist;Liebler Zimmerman Biochemistry 2013 targeted quantitation pf proteins by mass spectrometry; Gallien Domon 2015 Detection and quantification of proteins in clinical samples using high resolution mass spectrometry. Methods v81 pl5-23 ; Sajic, Liu, Aebersold, 2015 Using data-independent, high-resolution mass spectrometry in protein biomarker research: perspectives and clinical applications. Proteomics Clin Appl v9 p 307-21],
Particularly, the mass spectrometry-based quantification method can be the mass cytometry also known as cytometry by time of flight (CYTOF) (Bandura DR, Analytical chemistry, 2009).
Particularly, the mass spectrometry-based quantification is used to do peptide and/or protein profiling can be use with matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF), surface-enhanced laser desorption/ionization time of flight (SELDI-TOF; CLINPROT) and MALDI Biotyper apparatus [Solassol, , Lhermitte, Boulle, Maudelonde, Mange 2006 Clinical proteomics and mass spectrometry profiling for cancer detection. Journal: Expert Review of Proteomics V3, 13, p311-320 ; FDA K130831],
Methods of the invention may comprise a step consisting of comparing the proteins and fragments concentration in circulating cells with a control value. As used herein, "concentration of protein" refers to an amount or a concentration of a transcription product, for instance the proteins of the invention. Typically, a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a concentration. In a particular embodiment, "concentration of proteins" may refer to fragments of the proteins of the invention.
As used herein, the term “diagnosing” refers to classifying a disease or a symptom, determining a severity of the disease, monitoring disease progression, forecasting an outcome of a disease and/or prospects of recovery.
The inventors shows that sPLA2 XIIA expression as well as sPLA2XIIA receptors are involved in proliferation and migration of cancer cells, especially lung cancer cells, two markers of cancer progression and poor prognosis (increase tendency to metastasize).
The method of the present invention is thus particularly suitable for predicting the duration of the overall survival (OS), progression-free survival (PFS) and/or the disease-free survival (DFS) of the cancer patient.
Thus, an another object of the invention relates to an in vitro method for predicting the survival time of a subject suffering from cancer comprising the steps of (i) determining the expression level of sPLA2 XIIA and/or at least one sPLA2XIIA receptors selected among PLA2R1, syndecan and glypican in a biological sample obtained from said subject, (ii) comparing the expression levels for each marker determined at step (i) with a predetermined reference value, and iii) providing a good prognosis when the expression level determined at step (i) is lower than a predetermined reference value for each marker, or providing a bad prognosis when the expression level determined at step (i) is higher than a predetermined reference value for each marker.
As used herein, the term “Overall survival (OS)” denotes the percentage of people in a study or treatment group who are still alive for a certain period of time after they were diagnosed with or started treatment for a disease, such as cancer. The overall survival rate is often stated as a five-year survival rate, which is the percentage of people in a study or treatment group who are alive five years after their diagnosis or the start of treatment.
As used herein, the term “Free Survival (FS)” (or Event-Free-Survival) denotes the length of time after primary treatment for a cancer ends that the patient remains free of certain complications or events that the treatment was intended to prevent or delay. These events may include the return of the cancer or the onset of certain symptoms, such as bone pain from cancer that has spread to the bone.
Typically, the expression “short survival time” indicates that the patient will have a survival time that will be lower than the median (or mean) observed in the general population of patients suffering from said cancer. When the patient will have a short survival time, it is meant that the patient will have a “poor prognosis”. Inversely, the expression “long survival time” indicates that the patient will have a survival time that will be higher than the median (or mean) observed in the general population of patients suffering from said cancer. When the patient will have a long survival time, it is meant that the patient will have a “good prognosis”.
In some embodiments, the predictive method of the present invention is performed during the course of treatment, where the quantification of sPLA2 XIIA and/or sPLA2XIIA receptors is carried out before, during and as follow-up to a course of therapy. Desirably, therapy to cancer results in a decrease in the sPLA2 XIIA level and/or sPLA2XIIA receptors level in the subject’s sample.
Accordingly, in another object, the invention relates to an in vitro method for monitoring the treatment of cancer in a subject in need thereof, comprising the steps of i) determining the level of sPLA2 XIIA and/or at least one sPLA2XIIA receptors selected among PLA2R1 syndecan and glypican in a sample obtained from the subject at a first specific time tl and at a second specific time t2, wherein when tl is prior to treatment, t2 is during or following treatment and when tl is during treatment, t2 is later during treatment or following treatment ii) comparing the level(s) determined at tl with the level(s) determined at t2, and iii) concluding that said treatment is efficient when the level(s) determined at tl is lower than the level(s) determined at t2.
In some embodiments, when it is concluded that the treatment is efficient, the treatment is continued.
As used herein, the term “treatment” refers to treatments well known in the art and used to treat cancer, in particular lung cancer, colorectal cancer, breast cancer or prostate cancer. In the context of the invention, treatment refers to classical treatment (such as radiation therapy, chemotherapy immunotherapy, HD AC inhibitor) and sPLA2XIIA inhibitor or an inhibitor of sPLA2XIIA receptor.
As used herein, the term “immunotherapy” has its general meaning in the art and refers to the treatment that consists in administering an immunogenic agent i.e. an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response. In a particular embodiment, the immunotherapy consists of use of an immune check point inhibitor.
As used herein, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins. As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489). Examples of stimulatory checkpoint include CD27 CD28 CD40, CD122, CD137, 0X40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also called CD276, was originally understood to be a co-stimulatory molecule but is now regarded as co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumour escape. B and T Lymphocyte Attenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA. CTLA-4, Cytotoxic T-Lymphocyte-Associated protein 4 and also called CD152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDO, Indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme. A related immune-inhibitory enzymes. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis. KIR, Killer-cell Immunoglobulin-like Receptor, is a receptor for MHC Class I molecules on Natural Killer cells. LAG3, Lymphocyte Activation Gene-3, works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells. PD- 1, Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014. An advantage of targeting PD-1 is that it can restore immune function in the tumor microenvironment. TIM-3, short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines. TIM-3 acts as a negative regulator of Thl/Tcl function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti -turn or T-cell response, an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. The inhibition includes reduction of function and full blockade.
In some embodiments, the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands. In a particular embodiment, the immune checkpoint inhibitors is an PD1 inhibitor. In a particular embodiment, the immune checkpoint inhibitor is an antibody. Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.
In a particular embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302. Examples of anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK). In some embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody such as described in WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897. Examples of anti-PD-Ll antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech/Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS). In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154. In some embodiments, the immune checkpoint inhibitor inhibits Tim-3 or its ligand. In a particular embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO201 1155607, WO2015117002, WO2010117057 and W02013006490. In some embodiments, the immune checkpoint inhibitor is a small organic molecule. Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA. In a particular embodiment, small organic molecules interfere with transduction pathway of PD-1 and Tim-3. For example, they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway. In a particular embodiment, the small organic molecules interfere with Indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor. IDO is involved in the tryptophan catabolism (Liu et al 2010, Vacchelli et al 2014, Zhai et al 2015). Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), P- (3-benzofuranyl)-alanine, P-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5- methoxy-tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9- vinylcarbazole, acemetacin, 5- bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a P- carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1-methyl-tryptophan, P-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and P-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof. In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4-fluorophenyl)-N'- hydroxy-4-{[2-(sulfamoylamino)-ethyl]amino}-l,2,5-oxadiazole-3 carboximidamide. In a particular embodiment, the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-l,2,4-Triazole-3,5-diamine, l-(6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)- 5H-benzocyclohepten-2-yl], In a particular embodiment, the inhibitor is CA-170 (or AUPM- 170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand-1 (PD-L1) and V-domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015). Preclinical data of CA-170 are presented by Curis Collaborator and Aurigene on November at ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics. In some embodiments, the immune checkpoint inhibitor is an aptamer. Typically, the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG- 3, TIM-3 or VISTA. In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration. Thus, aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti-PD-1 aptamer. Particularly, the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015.
As used herein, the term “chemotherapy” refers to use of chemotherapeutic agents to treat a subject. As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth.
Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancrati statin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.
As used herein, the term “radiation therapy” or “radiotherapy” have their general meaning in the art and refers the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow. One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy. The use of machines to focus radiation (such as x-rays) on a cancer site is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, the radiation therapy is external radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction is given but fewer fractions.
As used herein, the term histone “histone deacetylase inhibitor” called also HDACi, refers to a class of compounds that interfere with the function of histone deacetylase. Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Typically, inhibitors of HDACs modulate transcription and induce cell growth arrest, differentiation and apoptosis. HDACis also enhance the cytotoxic effects of therapeutic agents used in cancer treatment, including radiation and chemotherapeutic drugs.
A further object of the present invention relates to an in vitro method of predicting the risk of relapse in a subject suffering from cancer i) comprising determining the level of of sPLA2 XIIA and/or at least one sPLA2XIIA receptors selected among PLA2R1, syndecan and glypican in a sample obtained from the subject ii) comparing the expression levels determined at step (i) with a predetermined reference value for each marker and iii) concluding that the subject is at high risk of relapse when the level of the expression level determined at step (i) is higher than a predetermined reference value for each marker.
Typically, high levels of sPLA2 XIIA and/or sPLA2XIIA receptors indicate that the subject is at high risk of relapse, whereas low levels of of sPLA2 XIIA and/or sPLA2XIIA indicate that the subject is at low risk of relapse.
As used herein, the term "relapse" refers to the return of signs and symptoms of a disease after a subject has enjoyed a remission after treatment. Thus, if initially, the target disease is alleviated or healed, or the progression of the disease is halted or slowed down and subsequently, when the disease or one or more characteristics of the disease resume, the subject is referred to as being "relapsed." Typically, the treatment is surgery, immunotherapy, chemotherapy, radiation therapy.
Method for treating cancer
The inventors demonstrate that the inhibition of sPLA2 XIIA and sPLA2 XIIA receptors decrease the proliferation rate in lung cancer cell lines. Furthermore, the inhibition of sPLA2 XII receptor decrease the migration of cancer cells.
Accordingly, in another aspect, the invention relates to a method of preventing or treating cancer in a subject in need thereof comprising a step of administrating to said subject a therapeutically amount of an inhibitor of sPLA2 XIIA and/or at least one inhibitor of sPLA2 XIIA receptor.
Thus, the invention relates to an inhibitor of sPLA2 XIIA and/or at least one inhibitor of sPLA2 XIIA receptor for use in the treatment of cancer in a subject in need thereof.
In particular embodiment, the inhibitor of sPLA2 XIIA and/or the inhibitor of sPLA2 XIIA receptor prevents migration and proliferation of cancerous cell.
The present invention further contemplates a method of preventing or treating cancer by preventing migration and proliferation of tumor in a patient in need thereof comprising administering to the patient a therapeutically effective amount of a sPLA2 XIIA and/or at least one inhibitor of sPLA2 XIIA receptor.
In preferred embodiment, the cancer is lung cancer. In some embodiments, the cancer is colorectal cancer, breast cancer or prostate cancer.
In preferred embodiment, the cancer is lung cancer and the subject suffers from COPD. As used herein, the term “inhibitor of sPLA2 XIIA” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of sPLA2 XIIA. The term encompasses any sPLA2 XIIA inhibitor that is currently known in the art or that will be identified in the future. By "activity" of sPLA2 XIIA is meant inducing tumor cell migration and proliferation.
Tests for determining the capacity of a compound to be sPLA2 XIIA inhibitor are well known to the person skilled in the art. In a preferred embodiment, the inhibitor specifically binds to sPLA2 XII2 in a sufficient manner to inhibit the biological activity of sPLA2 XII2. Binding to sPLA2 XII2 and inhibition of the biological activity of sPLA2 XII2 may be determined by any competing assays well known in the art. For example, the assay may consist in determining the ability of the agent to be tested as sPLA2 XII2 inhibitor to bind to sPLA2 XII2. The binding ability is reflected by the Kd measurement. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). KD values for binding biomolecules can be determined using methods well established in the art. In specific embodiments, an antagonist that "specifically binds to sPLA2 XII2 is intended to refer to an inhibitor that binds to human TRIM24 polypeptide with a KD of IpM or less, lOOnM or less, lOnM or less, or 3nM or less. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of TRIM24. The functional assays may be envisaged such evaluating the ability to inhibit a) induction of tumor cell migration and/or b) tumor cell proliferation. Such inhibitor inhibits the proliferation and migration of cancerous cells that overexpress sPLA2 XIIA. Such inhibitor inhibits the proliferation and migration induced by sPLA2 XIIA.
In a particular embodiment, the inhibitor of sPLA2 XIIA is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA, endonuclease or antisense oligonucleotide.
The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.
In a particular embodiment, the inhibitor of sPLA2 XIIA is a peptide.
In a particular embodiment, the inhibitor of sPLA2 XIIA is a GAG-binding peptide. As used herein, the term “GAG -binding peptide” refers to a peptide which is able to bind to the GAG-binding site of sPLA2 XIIA,
In a particular embodiment, the inhibitor of sPLA2 XIIA is a heparin-binding peptide
In other words, in a particular embodiment, the inhibitor of sPLA2 XIIA is a peptide which block GAG-sPLA2 XII interaction.
In a particular embodiment, the inhibitor of sPLA2 XIIA is an aptamer.
“Aptamers” are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. In a particular embodiment, the inhibitor of sPLA2 XIIA is a small organic molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.
In some embodiment, the inhibitor of sPLA2 XIIA is a glycosaminoglycan (GAG) or a glycosaminoglycan mimetic.
In some embodiment, the inhibitor of sPLA2 XIIA is a heparin or heparin mimetics such as 2,3-O-desulfated heparin (ODSH) and hybrid heparin-like compound isolated from Litopenaeus vannamei shrimp as described in Wang M. et al. Colloids Surf B Biointerfaces. 2017; and Brito, A. et al. . Int J Biol Macromol. 2018.
As used herein, the term “glycosaminoglycan” or “GAG” has its general meaning in the art and refers to a class of biomolecules expressed virtually on all mammalian cells and usually covalently attached to proteins, forming proteoglycans.
As used herein, the term “glycosaminoglycan mimetics” has its general meaning in the art and refers to compounds that block GAG-protein interaction. The glycosaminoglycan mimetic for use according to the invention can compete with heparan sulfate proteoglycans, i.e syndecan or glypican and block their interaction with sPLA2 XIIA. Examples of GAG mimetics includes heparin, heparin mimetics such as 2,3-O-desulfated heparin (ODSH) and hybrid heparin-like compound isolated from Litopenaeus vannamei shrimp; fucoidane, chitine, chitosane modified polysaccharides, sulfated caffeic acid (CDSO3), synthetically sulfated oligosaccharides, oligosaccharide-aglycone conjugates, non-carbohydrate-bases sulfated mimetics, muparfostat (PI-88); PG545; EP80061; SST0001 (roneparstat); fondaparinux (arixtra); Indraparinux; Idrabiotapariux; SR123781; AV5026 (semuloparin); M402 (necuparanib); GMI-1070 and regenerating agents (RGTAs) such as OTR4120 and OTR4131. As used herein the term “heparin” has its general meaning in the art and refers to glycosaminoglycan of formula C12H19NO20S3 which is used as an anticoagulant (blood thinner). Its CAS Number is 9005-49-6.
In some embodiments, the inhibitor of sPLA2 XIIA is an antibody.
As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMTP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388. In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.
In a particular, the inhibitor of sPLA2 XIIA is an intrabody having specificity for sPLA2 XIIA. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. According to the invention, sdAb can particularly be llama sdAb.
Example of anti- sPLA2 XIIA antibody includes but are not limited to antibody provided by Santa Cruz under reference sc-514423.
In some embodiments, the inhibitor of sPLA2 XIIA is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of sPLA2 XIIA.
In a particular embodiment, the inhibitor of sPLA2 XIIA expression is siRNA. “A short hairpin RNA (shRNA)” is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide- long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.
In some embodiments, the inhibitor of sPLA2 XIIA expression is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention Oin the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR). In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences. In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038/cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534/genetics. H3.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836-843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.
In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).
In some embodiment, the inhibitor of sPLA2XIIA receptor is selected in the group consisting of inhibitor of PLA2R1, inhibitor of syndecan and inhibitor of glypican. As used herein, the term “inhibitor of PLA2R1” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of PLA2R1, including, for example, reduction or blocking the interaction between PLA2R1 and sPLA2 XIIA. Such inhibitor can inhibit the proliferation and migration of cancerous cells that overexpress sPLA2 XIIA. In some embodiments, the inhibitor of PLA2R1 may be a molecule that binds to PLA2R1 selected from the group consisting of small molecule, antibodies, aptamers, proteolyse and polypeptides. In some embodiments, the inhibitor of PLA2R1 is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of PLA2R1.
As used herein, the term “inhibitor of glypican” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of glypican, including, for example, reduction or blocking the interaction between glypican and sPLA2 XIIA.. Such inhibitor can inhibit the proliferation and migration of cancerous cells that overexpress sPLA2 XIIA. In some embodiments, the inhibitor of glypican may be a molecule that binds to glypican selected from the group consisting of small molecule, antibodies, aptamers, proteolyse and polypeptides. In some embodiments, the inhibitor of glypican is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of glypican. In some embodiment, the inhibitor of glypican is selected from the group consisting in glypican-1 inhibitor, glypican-2 inhibitor, glypican-3 inhibitor, glypican-4 inhibitor, glypican-5 inhibitor and glypican-6 inhibitor.
In some embodiment, the inhibitor of glypican is glypican-1 inhibitor and/or glypican- 4 inhibitor.
As used herein, the term “inhibitor of syndecan” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of syndecan and including, for example, reduction or blocking the interaction between syndecan and sPLA2 XIIA. Such inhibitor can inhibit the proliferation and migration of cancerous cells that overexpress sPLA2 XIIA. In some embodiments, the inhibitor of syndecan may be a molecule that binds to syndecan selected from the group consisting of small molecule, antibodies, aptamers, proteolyse and polypeptides. In some embodiments, the inhibitor of syndecan is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of syndecan. In some embodiment, the inhibitor of syndecan is selected from the group consisting in syndecan-1 inhibitor, syndecan-2 inhibitor, syndecan-3 inhibitor, syndecan-4. In a particular embodiment, the inhibitor of glypican or syndecan is a synthetic xyloside such as P-D- xyloside; heparinase; heparan-sulfate lyase; an anti-heparan-sulfate antibody; a sulfotransferase inhibitor, a glycosyltransferase inhibitor; an antibody; an aptamers; siRNA or an antisense oligonucleotide.
In some embodiment, the inhibitor of sPLA2 XIIA receptor is heparin.
In some embodiment, the inhibitor of sPLA2 XIIA receptor is an inhibitor of syndecan- 1 and/or syndecan-4.
In some embodiment, the inhibitor of syndecan is an anti-syndecan- 1 antibody and/or anti-syndecan-4 antibody.
Example of anti- syndecan- 1 antibody includes but are not limited to antibody as disclosed in CN115960231, CN104059151, WO202257289, antibody provided by Santa Cruz under reference DL-10, an human anti-syndecan- 1 antibody as disclosed in Orecchia P, et al. A novel human anti-syndecan- 1 antibody inhibits vascular maturation and tumour growth in melanoma. Eur J Cancer. 2013 (expressly incorporated herein by reference).
Example of anti- syndecan-4 antibody includes but are not limited to antibody provided by LS Bio under reference LS-C 150078, antibody provided by Santa Cruz under reference Sc- 12766, antibody as disclosed in Godmann L, et al.. Antibody-mediated inhibition of syndecan- 4 dimerisation reduces interleukin (IL)-l receptor trafficking and signalling. Ann Rheum Dis. 2020.
Example of anti-heparan-sulfate antibody includes but are not limited to antibody provided by R&D systems under reference HS6ST1, antibody provided by Amsbio under reference 3 G10, 10E4 and JM403.
In some embodiments, the inhibitor of sPLA2 XIIA and the inhibitor of sPLA2XIIA receptor are used as a combined preparation.
In some embodiments, the i) inhibitor of sPLA2 XIIA inhibitor and ii) the inhibitor of syndecan-1 and/or inhibitor of syndecan-4 are as a combined preparation.
As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy. Such administration may be simultaneous, separate or sequential. For simultaneous administration the product of the invention may be administered as one composition or as separate compositions, as appropriate. As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.
In some embodiment, the inhibitor of sPLA2 XIIA and/or the inhibitor of sPLA2XIIA receptor is administered in combination with a classical treatment of cancer.
As used herein, the term “classical treatment” refers to any compound, natural or synthetic (i.e chemotherapy, immune checkpoint inhibitor, radiation therapy HD AC inhibitor), used for the treatment of cancer as described above.
Subject identified with a poor prognosis or having cancer according to the method of the invention can be treated with an inhibitor of sPLA2 XIIA and/or with at least one inhibitor of its receptors (PLA2R1, syndecan and glypican).
Thus, in a particular embodiment, the subject has been identified with a poor prognosis or cancer according to the method of the invention.
More particularly, the invention relates to a method of treating cancer comprising the steps of: i) determining whether a subject suffers from cancer according to the method as described above and ii) administrating to said subject a therapeutically amount of inhibitor of sPLA2 XIIA and/or at least one inhibitor of sPLA2XIIA receptor when it is concluded at step i) that the subject suffers from cancer.
In preferred embodiment, the cancer is lung cancer. In some embodiments, the cancer is colorectal cancer, breast cancer or prostate cancer.
As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).
As used herein, the term "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired therapeutic result. A therapeutically effective amount of the immune checkpoint inhibitor of the present invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the immune checkpoint inhibitor of the present invention to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. The efficient dosages and dosage regimens for the immune checkpoint inhibitor of the present invention depend on the disease or condition to be treated and may be determined by the persons skilled in the art. A physician having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician could start doses of the immune checkpoint inhibitor of the present invention employed in the pharmaceutical composition at levels lower than that required achieving the desired therapeutic effect and gradually increasing the dosage until the desired effect is achieved. In general, a suitable dose of a composition of the present invention will be that amount of the compound, which is the lowest dose effective to produce a therapeutic effect according to a particular dosage regimen. Such an effective dose will generally depend upon the factors described above. For example, a therapeutically effective amount for therapeutic use may be measured by its ability to stabilize the progression of disease. Typically, the ability of a compound to inhibit cancer may, for example, be evaluated in an animal model system predictive of efficacy in human tumors. A therapeutically effective amount of a therapeutic compound may decrease tumor size, or otherwise ameliorate symptoms in a subject. One of ordinary skill in the art would be able to determine such amounts based on such factors as the subject's size, the severity of the subject's symptoms, and the particular composition or route of administration selected. An exemplary, non-limiting range for a therapeutically effective amount of a inhibitor of the present invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as 0.3, about 1, about 3 mg/kg, about 5 mg/kg or about 8 mg/kg. An exemplary, non-limiting range for a therapeutically effective amount of an inhibitor of the present invention is 0.02-100 mg/kg, such as about 0.02-30 mg/kg, such as about 0.05-10 mg/kg or 0.1-3 mg/kg, for example about 0.5-2 mg/kg. Administration may e.g. be intravenous, intramuscular, intraperitoneal, or subcutaneous, and for instance administered proximal to the site of the target. Dosage regimens in the above methods of treatment and uses are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several divided doses may be administered over time or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. In some embodiments, the efficacy of the treatment is monitored during the therapy, e.g. at predefined points in time.
Typically, the sPLA2 XIIA inhibitors and/or its receptor inhibitors of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained- release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
Thus the present invention also relates to a pharmaceutical composition comprising a sPLA2 XIIA inhibitors and/or sPLA2 XII receptor inhibitors according to the invention and a pharmaceutically acceptable carrier for use in the treatment of cancer.
The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:
Figure 1. Up-regulation of PLA2 XIIA’s receptors in cancer cell line Gene expression level of receptors expressed as ratio of expression of the housekeeping gene 36B4 in cancer cell line (A549 and NCI Hl 385). * p<0.05 A549 vs. NCI. GLYP glypicans, SD, syndecans, NCI (NCIH1385).
Figure 2. Conditioned medium induces the proliferation of cancer cell lines via sPLA2 XIIA. A) The rate of cell proliferation was evaluated by using MTT assay. Cells were exposed to conditioned medium (CM) either from fibroblasts stemmed from smokers controls (S-C, n=12) and patients with COPD (COPD, n= 14) or from cancer associated fibroblasts (CAF, n=5) at non senescent stage (passage 3) and senescent stage (passage 7) for 24 hours. B) The rate of cell proliferation was evaluated after treatment with conditioned medium deprived in sPLA2 XIIA. Conditioned medium from fibroblasts at senescent stage have been deprived in sPLA2 XIIA by using immunoprecipitation method. A 549 and NCI have been treated with these mediums. Data are presented as mean ± SEM in the whole Figure. * p<0.05 cells exposed to CM vs. cells without treatment; $ p< 0.05 A549 vs NCI; f p<0.05 cells exposed to CM vs cells treated with anti-sPLA2 XIIA. CM S-C conditioned medium from fibroblasts isolated from smokers controls. CM COPD conditioned medium from fibroblasts isolated from COPD patients. CM CAF conditioned medium from CAF
Figure 3. sPLA2 XIIA induces the proliferation of cancer cell line via SD1 and SD4. A) The rate of cell was measured by using MTT assay. Cells were treated with different concentrations of sPLA2 XIIA for 24 hours. The role of heparan sulfate proteoglycans was evaluated by pre-treatment of cells with either heparinase I and III or antibody against heparin sulfate proteoglycans (anti-HS), and then treated with different doses of sPLA2 XIIA. B) A549, C) NCI. The role of SDC-1 and 4 was analyzed by pre-treatment of cells with antibodies against SDC-1 or SDC-4, respectively and exposed to different doses of sPLA2 XIIA A 549 B NCI C. Data are presented as mean ± SEM in the whole Figure. * p<0.05 cells exposed to sPLA2 XIIA vs. cells without treatment; f p<0.05 cells exposed to sPLA2 XIIA vs cells treated with heparinase or anti-HS or anti-SDl or anti-SD4.
Figure 4: sPLA2 XIIA activates MAPK pathway. A) Representative western blot image of p-ERK, ERK, p-p38 and p38 in cancer cell line (A 549). Cells were exposed to different doses of sPLA2 XIIA for 5, 15, 30, 60 and 120 minutes. The rate of cell proliferation was evaluated by using MTT assay. Cells were pre-treated with either an inhibitor of ERK (U0160, 10 pM) or inhibitor of p38 (SB202190, lOpM) and exposed to different doses of sPLA2 XIIA. * p<0.05 cells exposed to sPLA2 XIIA vs. cells without treatment; f p<0.05 cells exposed to sPLA2 XIIA vs cells treated with inhibitors.
Figure 5 : sPLA2 XIIA induces the migration of cancer cell line. The migration of two cancer cell lines was evaluated by using wound healing assays. Cells were pre-treated with antibody against heparin sulfate proteoglycans (anti-HS) and exposed to different doses of sPLA2 XIIA after the scratch test. A) Representative of wound healing test B, C, D) speed of wound healing, after scratch test. E) Representative western blot image of p-FAK and FAK in cancer cell line (A 549). Cells were exposed to different doses of sPLA2 XIIA for 5, 15, 30, 60 and 120 minutes. * p<0.05 cells exposed to sPLA2 XIIA vs. cells without treatment; f p<0.05 cells exposed to sPLA2 XIIA vs cells treated with anti-HS.
Figure 6: sPLA2 XIIA induces the epithelial mesenchymal transition. Cell were treated with different concentration of sPLA2 XIIA for 6 days. The expression of mesenchymal markers (vimentin, N cadherin and aSMA) and epithelial marker (E cadherin) was evaluated by immunostaining and western blot in A 549 A) and in NCI B).
Figure 7 : sPLA2 XIIA is involved for a part in the lung cancer development. Cancer cell lines and fibroblasts from S-C and COPD patients were co-cultured for 14 days in Matrigel in 24-well Transwell supports. Coculture A549 and senescent fibroblasts. Data are presented as mean ± SEM in the whole Figure. * p<0.05 A549 alone versus co culture A549 and senescent fibroblasts from S-C and COPD. f p<0.05 organoids versus organoids exposed to anti-sPLA2 XIIA.
Figure 8: sPLA2 XIIA induces the proliferation of cancer cell line. The rate of cell proliferation was measured by using MTT assay. Cells were treated with different concentrations of sPLA2 XIIA for 24 hours. Breast cancer line (MDA MB 231), colorectal cancer line (HT29), lung cancer line (A 549), and prostate cancer line (PC3).
EXAMPLE 1:
Results:
Up regulation sPLA2XIIA in fibroblasts and CAF and its receptors in cancer cell line
In order to study the effects of sPLA2 XIIA on lung cancer, we evaluated the gene and protein expression of its receptors in two lung cancer cell lines : A549 and NCI Hl 563 (Figure 1). These two cell lines display mutations which are most represented in lung cancer : mutation of KRAS for A549 cell line whereas NCIH1563 present a lack of expression of FGFR1 and a pl6 inactivation. Expression of syndecan 1 (SDC-1) and glypican 2 (GLY 2) at the mRNA level were significantly increased in A549 cell line whereas the expression of PLA2 Rl, syndecan 4 (SDC-4) and glypican 1 (GLY 1) were more increased in NCI Hl 563 cell line (Figure 1). These data were confirmed by immunostaining (data not shown).
In order to analyze the relevance of the in vitro data, we then evaluated the expression of SDC-1 and -4 in slides of lung cancer tissue from patients treated for chemotherapy and immunotherapy. By using cadherin (a marker of cancer cells), we showed a localization of these SDC1 and 4 in situ in lung cancer (data not shown) suggesting a resistance of these cells to these treatments.
Conditioned medium induces the proliferation of cancer cell lines via sPLA2 XIIA
In order to determine if conditioned medium from senescent fibroblasts could induce the proliferation of cancer cell line, cancer cells were exposed to conditioned medium from fibroblasts isolated from smokers controls (S-C) and COPD patients at non senescent (passage 3) and senescent stages (passage 7) (Figure 2A). The conditioned medium from senescent fibroblasts increased the rate of proliferation in the two cell lines whereas no effect was detected whichever could be the type concerned of cells. Moreover, conditioned medium from senescent COPD fibroblasts raised the proliferation rate with an higher effect than the one from S-C fibroblasts. These date were confirmed by a immunostaining with an antibody directed against Ki67 (a marker of proliferation) (data not shown).
To go further and define if sPLA2 XIIA could be involved in this increase, cells were treated with conditioned medium from senescent fibroblasts deprived to sPLA2 XIIA (Figure 2B). This medium from senescent smokers did not change the proliferation rate whatever could be the cell line concerned whereas the one from senescent COPD fibroblasts significantly decreased the proliferation rate of the two cell lines (Figure 2B). These results confirm a role of sPLA2 XIIA in conditioned medium from senescent COPD fibroblasts.
In parallel, cancer cell lines were treated with conditioned medium from CAF at nonsenescent and senescent stages (figure 2A). The conditioned medium from senescent CAF induced the proliferation of cancer cell lines with a higher effect for NCI Hl 563. In interesting manner, the effect of conditioned medium from senescent CAF was similar of the ones of conditioned medium from senescent fibroblasts from COPD patients. The treatment of cancer cell lines with medium deprived to sPLA2 XIIA decreased the proliferation rate of these cells (data not shown). These results confirm that senescent CAF and fibroblast share a common pathway. sPLA2 XIIA induces the proliferation of cancer cell line via SD1 and SD4 We next questioned whether sPLA2 XIIA could be in turn involved in cancer progression.. Therefore, we analyzed if a single exposure of cancer cells to sPLA2 XIIA at the concentrated measured in supernatant from senescent fibroblasts and CAF could induce the proliferation of cancer cells and whether one of these cell line could be more susceptible to this effect or not .
Cells were exposed to different concentration of sPLA2 XIIA (0.3 to 5 ng/ml). Twenty- four hours incubation of cancer cell with the different concentrations of sPLA2 XIIA increased the proliferation rate whatever the type of cell lines considered. This effect began from 0.3 ng/ml to 2 ng/ml for the NCI- Hl 563 cell line whereas for the A 549 cell line, it started from 0;5 ng/ml to 1.5 ng/ml suggesting a susceptibility more important for NCI-H1563 cell line (Figure 3A). In order to determine the role of sPLA2 XIIA receptors in the induction of proliferation, cancer cells were treated with heparinase I and III to cleave the heparan sulfate chains on proteoglycans. The exposure to heparinase significantly reduced sPLA2 XIIA- induced proliferation in the two cell lines within the three groups of fibroblasts (Figure 3B, 3C). These effects were confirmed by an anti-heparan sulfate antibody, reinforcing the involvement of heparan sulfate proteoglycans in sPLA2XIIA-induced proliferation (Figure 3B and 3C). In compliment with these experiments, we assessed the role of syndecans. The SDC- 1 and 4 antibodies significantly decreased the proliferation rate in two cell lines (data not shown). sPLA2 XIIA activates MAPK pathways
Since syndecans are known to activate several signaling pathways such as MAPK and JAK/STAT (19-22), we analyzed protein expression level of these signaling pathways after treatment with sPLA2 XIIA at different concentrations per extended periods (5 minutes to 1 hours). The protein expression of p-ERK and p-p38 increased after 5 minutes of treatment in the two cell lines (Figure 4A and data not shown). In addition, to confirm if the MAPK were involved in the proliferation of cancer cell line, we treated the cells with U0126 and SB202190 which are inhibitors of ERK and p38 respectively. These treatments abolished the proliferation induced by sPLA2 XIIA (Figure 4B and 4C).
Since sPLA2 XIIA induces the proliferation, we investigated if this isoform could be involved in migration and EMT. sPLA2 XIIA induces the migration of cancer cell line In order to determine if sPLA2 XIIA could be involved in the migration of cancer cells, the wound healing after a scratch assay was followed for fourty height hours. Thirty six hours after the scratch, the different concentrations of sPLA2 XIIA provided a better migratory phenotype to cancer cells, as wound closure was accelerated (Figure 5A). In order to determine the role of sPLA2 XIIA receptors in the induction of migration, cancer cells were treated with an anti-heparan sulfate antibody. This treatment abolished the migration induced by sPLA2 XIIA confirming a role of heparan sulfate proteoglycan in this effect (Figure 5B, 5C, 5D).
Since Focal adhesion kinase is involved in the migration and proliferation process, we investigated the expression of this protein after exposure to different concentrations of sPLA2 XIIA per extended periods (5 minutes to 1 hours). The protein expression of p-FAK increased after 30 minutes of treatment and seem to be maintained after 1 hours of exposure (Figure 5E). sPLA2 XIIA induces the epithelial mesenchymal transition (EMT)
Since, EMT is involved in the invasion of cancer cells and in the formation of metastasis, we treated cell line with different concentrations of sPLA2 XIIA for 6 days. This chronic exposure decreased the expression of cadherin and increased the mesenchymal markers such vimentin, N cadherin and aSMA with a higher effect for NCI Hl 563 cell line (Figure 6A, 6B) sPLA2 XIIA participates for a part to the development of lung cancer
We further tested if sPLA2 XIIA involves the development of lung cancer by using an organoid assay. Both cell lines were co cultured with fibroblasts from S-C and COPD patients at non senescent (passage 3) and senescent stages (passage 7) (data not shown) for 14 days. The co-culture of cancer cells with fibroblasts at senescent stage increased the number of organoids in comparison with the culture of A549 alone whereas no effect was observed for the co culture with fibroblasts at non senescent stage (Figure 7). This effect was more important for the NCI Hl 563 cell line (data not shown). The treatment with an anti-sPLA2 XIIA of the co culture with senescent fibroblasts had no effect on the number of organoids but decreased the size of organoids whatever the type of cell line considered confirming a role of sPLA2 XIIA in cancer development.
EXAMPLE 2:
Materials and Methods
Cells line A 549, MDA-MB-231, PC3 and HT29 cells were purchased from ATCC and cultured in High Glucose DMEM (life technologies) supplemented with 10% FBS (life technologies), l x antibiotic/antimycotic solution (life technologies)
Cell treatments
Fibroblasts were exposed to different doses of sPLA2 XIIA (0.5, 1,5 and 5 ng/ml) for 24 h. the rate of proliferation was analyzed by MTT assay.
Gene expression analysis
Total RNA was extracted with an RNeasy kit (Qiagen, Courtaboeuf, France) according to manufacturer’s instructions. The gene expression level of sPLA2 XIIA, was analyzed by RT- qPCR using the QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems) and expressed as the ratio to a house-keeping gene (36B4).
Results
The expression of sPLA2 XIIA present a low cancer specificity. However, according to the Cancer Genome Atlas (TCGA), its expression is higher in five types of cancers: breast, prostate, renal, colorectal and lung cancer. We have confirmed that A549 and NCI cell lines expressed sPLA2 XIIA by RTQPCR and ELISA assay. The concentration was 70 pg/ml et 184 pg/ml respectively. We have also shown that the exposure of these lung cancer cell lines to sPLA2 XIIA increased the rate of proliferation and migration of these cells, suggesting that the environment could participate to the cancer progression. In order to check if sPLA2 XIIA could induce the same effects on other cancer cell lines, we treated breast cancer line (MDA MB 231), prostate cancer line (PC3) and colorectal cancer line (HT29) with sPLA2 XIIA. A 24-hour incubation of these cancer cells with different concentrations of sPLA2 XIIA increased the proliferation rate for all cell lines considered. However, PC3 and HT29 cell lines were more sensitive to this protein than MDA MB 231 cell lines (Figure 8).
Moreover, a recent article has demonstrated that a decrease of sPLA2 XIIA expression in HT29 cell line by transfection lead to cancer progression (an increase of proliferation and migration), suggesting that a lack of sPLA2 XIIA could be a factor of worse prognostic. Regarding our results, we could suggest that sPLA2 XIIA secreted by the environment could act with the same way on cancer cells by decreasing its own expression and favor the cancer development. REFERENCES:
Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.
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