.2019 Dec 5;38(1):483.

doi: 10.1186/s13046-019-1448-9.

NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2

Pengfei Xu¹, Shuangyan Ye¹, Keyi Li¹, Mengqiu Huang¹, Qianli Wang¹, Sisi Zeng¹, Xi Chen¹, Wenwen Gao¹, Jianping Chen¹, Qianbing Zhang¹, Zhuo Zhong², Ying Lin¹, Zhili Rong¹, Yang Xu¹, Bingtao Hao¹, Anghui Peng¹, Manzhao Ouyang³, Qiuzhen Liu^{4 5}

Affiliations

¹ Cancer Research Institute, Guangdong Provincial Key Laboratory of Cancer Immunotherapy, Guangzhou key laboratory of tumor immunology research, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.
² Department of Oncology, Guangzhou Hospital of Integrated Traditional and Western Medicine, Guangzhou, 510800, China.
³ Center for medical transformation, Shunde Hospital, Southern Medical University, Foshan, 528308, China.
⁴ Cancer Research Institute, Guangdong Provincial Key Laboratory of Cancer Immunotherapy, Guangzhou key laboratory of tumor immunology research, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China. liuqiuzhen@126.com.
⁵ Center for medical transformation, Shunde Hospital, Southern Medical University, Foshan, 528308, China. liuqiuzhen@126.com.

PMID:31805977
PMCID: PMC6896289
DOI: 10.1186/s13046-019-1448-9

NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2

Pengfei Xu et al. J Exp Clin Cancer Res.2019.

.2019 Dec 5;38(1):483.

doi: 10.1186/s13046-019-1448-9.

Authors

Affiliations

¹ Cancer Research Institute, Guangdong Provincial Key Laboratory of Cancer Immunotherapy, Guangzhou key laboratory of tumor immunology research, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China.
² Department of Oncology, Guangzhou Hospital of Integrated Traditional and Western Medicine, Guangzhou, 510800, China.
³ Center for medical transformation, Shunde Hospital, Southern Medical University, Foshan, 528308, China.
⁴ Cancer Research Institute, Guangdong Provincial Key Laboratory of Cancer Immunotherapy, Guangzhou key laboratory of tumor immunology research, School of Basic Medical Sciences, Southern Medical University, Guangzhou, 510515, China. liuqiuzhen@126.com.
⁵ Center for medical transformation, Shunde Hospital, Southern Medical University, Foshan, 528308, China. liuqiuzhen@126.com.

PMID:31805977
PMCID: PMC6896289
DOI: 10.1186/s13046-019-1448-9

Abstract

Background: The dysfunction of type I interferon (IFN) signaling is an important mechanism of immune escape and metastasis in tumors. Increased NOS1 expression has been detected in melanoma, which correlated with dysfunctional IFN signaling and poor response to immunotherapy, but the specific mechanism has not been determined. In this study, we investigated the regulation of NOS1 on the interferon response and clarified the relevant molecular mechanisms.

Methods: After stable transfection of A375 cells with NOS1 expression plasmids, the transcription and expression of IFNα-stimulated genes (ISGs) were assessed using pISRE luciferase reporter gene analysis, RT-PCR, and western blotting, respectively. The effect of NOS1 on lung metastasis was assessed in melanoma mouse models. A biotin-switch assay was performed to detect the S-nitrosylation of HDAC2 by NOS1. ChIP-qPCR was conducted to measure the binding of HDAC2, H4K16ac, H4K5ac, H3ac, and RNA polymerase II in the promoters of ISGs after IFNα stimulation. This effect was further evaluated by altering the expression level of HDAC2 or by transfecting the HDAC2-C262A/C274A site mutant plasmids into cells. The coimmunoprecipitation assay was performed to detect the interaction of HDAC2 with STAT1 and STAT2. Loss-of-function and gain-of-function approaches were used to examine the effect of HDAC2-C262A/C274A on lung metastasis. Tumor infiltrating lymphocytes were analyzed by flow cytometry.

Results: HDAC2 is recruited to the promoter of ISGs and deacetylates H4K16 for the optimal expression of ISGs in response to IFNα treatment. Overexpression of NOS1 in melanoma cells decreases IFNα-responsiveness and induces the S-nitrosylation of HDAC2-C262/C274. This modification decreases the binding of HDAC2 with STAT1, thereby reducing the recruitment of HDAC2 to the ISG promoter and the deacetylation of H4K16. Moreover, expression of a mutant form of HDAC2, which cannot be nitrosylated, reverses the inhibition of ISG expression by NOS1 in vitro and decreases NOS1-induced lung metastasis and inhibition of tumor infiltrating lymphocytes in a melanoma mouse model.

Conclusions: This study provides evidence that NOS1 induces dysfunctional IFN signaling to promote lung metastasis in melanoma, highlighting NOS1-induced S-nitrosylation of HDAC2 in the regulation of IFN signaling via histone modification.

Keywords: H4K16ac; HDAC2; IFNα; Melanoma; Metastasis; NOS1; S-nitrosylation.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

**Fig. 1**
NOS1 blocks IFNα-stimulated gene induction and promotes lung metastasis of melanoma.a A375 cells were stimulated with IFNα (1000 U/ml) for 6 h in the presence or absence of simultaneous GSNO (100 μM). The mRNA expression of ISGs was analyzed by RT-PCR.b Control/NOS1 (A375) cells were treated with IFNα (1000 U/ml) for 6 h, followed by RT-PCR analysis.c, d Similar toa, butc N-PLA (100 μM),d L-NAME (1 mM) was used.e Control/NOS1 (A375) cells were cotransfected with pISRE-luc and Renilla-luc reporter plasmids for 24 h, treated with IFNα (1000 U/ml) for 6 h, and analyzed by luciferase assay.f Control/NOS1 (A375) cells were incubated with IFNα (1000 U/ml) for the indicated times, and western blotting was used to detect the protein expression of IRF7.g, h The effect of Over-NOS1 on the expression of IFNα-ISGs ing B16 cells andh tumor tissues was detected by RT-PCR.i Representative images of lung tissue and lung sections stained with H&E from each group are shown.j, k Thej tumor nodules andk lung weight of each group (n = 4).l Mice were sacrificed individually upon signs of metastatic distress and lung metastasis confirmed via histology.m Long rank analysis of mouse survival rates (n = 8). ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001

**Fig. 2**
NOS1 inhibits interferon-induced recruitment of HDAC2.a Negative control siRNA (si-NC) or an siRNA specific for HDAC2 (si-HDAC2) was used to transfect A375 cells for 48 h, and then the knockdown efficiency was determined by RT-PCR and an immunoblotting assay.b, c siRNA was used to transfectb A375 cells andc SW480 cells for 24 h, followed by stimulation with IFNα (1000 U/ml) for 6 h and RT-PCR analysis.d HDAC2 expression plasmids were used to transfect A375 cells for 24 h, and the transfection efficiency was determined by immunoblotting assay.e Similar tob, HDAC2 expression vectors were used to transfect A375 cells for 24 h before IFNα stimulation.f pISRE-luc and Renilla-luc reporter plasmids were used to cotransfect A375 cells in the presence of si-NC (Con), si-HDAC2 (HD2), or HDAC2 expression vectors as indicated. Luciferase activity was measured 24 h after transfection in untreated cells and cells treated with IFNα (1000 U/ml) for 6 h.g A375 cells were incubated with or without IFNα (1000 U/ml, 6 h). ChIP-qPCR was used to detect the binding of HDAC2 to the ISG promoters.h A375 cells were stimulated with (+) or without (−) IFNα (1000 U/ml) for 6 h, and the lysates were precipitated with HDAC2 or IgG antibodies. Western blotting was performed using the indicated antibody.i Similar toh, but Co-IP assays were performed in Control/NOS1 (A375) cells.j Similar tog, ChIP assays were performed in Control/NOS1 (A375) cells by stimulation with IFNα (1000 U/ml) for 6 h.k,l Control/NOS1 (A375) cells were treated with IFNα (1000 U/ml) for 6 h and 12 h, the expression of HDAC2 was detected byk western blotting andl RT-PCR.m The subcellular localization of HDAC2 was visualized by immunofluorescence staining in A375 cells after treatment with or without GSNO for 6 h. Nuclei were stained with DAPI (blue), original magnification: 100×.n A375 cells were treated with or without IFNα (1000 U/ml) for 12 h, and the expression of nuclear and cytoplasmic proteins of HDAC2 was detected by western blotting. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; and ****p < 0.0001. Flag-HD2, Flag-tagged HDAC2 expression vectors; End-HD2, endogenous HDAC2

**Fig. 3**
NOS1 induces S-nitrosylation of HDAC2-C262/274.a Control/NOS1 (A375) cells were treated with or without IFNα (1000 U/ml) for 6 h. Protein extracts were subjected to the biotin-switch assay.b Similar toa, but A375 cells were treated with or without L-NAME (1 mM), N-PLA (100 μM), 1400 W (100 μM) for 24 h.c Biotin-switch assay of A375 cells transfected with HDAC2-WT, HDAC2-C262A/C274A or empty vector (Control) for 24 h and then treated with GSNO (100 μM, 30 min).d Similar toc, but Control/NOS1 (A375) cells were used.e Control/NOS1(A375) cells were immunoprecipitated HDAC2 was subjected to HDAC activity assay.f Over-NOS1 (A375) cells were transfected with the indicated HDAC2 vectors. Flag antibody immunoprecipitates were subjected to HDAC assay. Shown are the averages and SEM (n = 3).g HDAC2 vectors were transfected into Over-NOS1 (A375) cells, followed by stimulation with IFNα (1000 U/ml) for 6 h and RT-PCR analysis.h HDAC2 vectors were transfected into Over-NOS1 (A375) cells and then treated with IFNα (1000 U/ml) for 6 h. HDAC2 immunoprecipitation was followed by ChIP-qPCR analysis. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Flag-HD2, Flag-tagged HDAC2 expression vectors; End-HD2, endogenous HDAC2

**Fig. 4**
NOS1 inhibits the deacetylation of H4K16 by S-nitrosylation of HDAC2.a-d A375 cells were treated with or without IFNα (1000 U/ml) for 1 h. ChIP was performed using specifica anti-H4K16ac,b anti-H4K5ac,c anti-H3ac andd anti-Rbp1 antibodies.e HDAC2 siRNA or the control siRNA were transfected into A375 cells for 24 h, followed by stimulation with IFNα (1000 U/ml) for 1 h. ChIP was performed using H4K16ac antibodies.f Similar toe, but HDAC2 expression vectors were used to transfect A375 cells for 24 h before IFNα stimulation.g HDAC2 siRNA or control siRNA was used to transfect A375 cells for 24 h and then stimulated with or without IFNα (1000 U/ml) for 6 h. Western blotting was performed using the indicated antibodies.h, i Using the same protocol as ine, ChIP assays were performed using specifich anti-Rpb1 andi anti-H4K5ac antibodies.j, k Control/NOS1 (A375) cells were treated with IFNα (1000 U/ml) for 1 h. ChIP assays were performed using specificj anti-H4K16ac andk anti-Rpb1 antibodies.l Over-NOS1 (A375) cells were transfected with HDAC2-WT or HDAC2-MUT vectors for 24 h and then treated with IFNα (1000 U/ml) for 1 h. ChIP assays were performed using specific anti-H4K16ac antibodies. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

**Fig. 5**
NOS1 promotes melanoma lung metastasis by S-nitrosylation of HDAC2-C262/274.a The mRNA expression of HDACs (1, 2, 3) and protein expression of HDAC2 in HDAC2-KO cells were determined by RT-PCR and western blotting.b The same method asa was used to detect the mRNA and protein expression levels of HDAC2 of stably transfected HDAC2-WT and HDAC2-MUT plasmids in HDAC2-KO cells.c-e 3 × 10⁶ B16-WT, B16-HDAC2-KO, B16-HDAC2-WT, B16-HDAC2-MUT cells were intravenously injected into C57BL/6 mice. The mice were sacrificed on the 11th day after tumor cell inoculation. Tumor growth was monitored byc gross morphology,d lung weight (n = 4), ande survival rate (n = 8). ns, not significant; **p < 0.01; ***p < 0.001; ****p < 0.0001

**Fig. 6**
NOS1 inhibits tumor lymphocyte infiltration by S-nitrosylation of HDAC2-C262/274.a, b B16-HDAC2-WT, B16-HDAC2-MUT cells were intravenously inoculated into BALB/c-nu mice (3 × 10⁶/mouse). The mice were sacrificed on the 11th day after inoculation. Tumor growth was monitored byagross morphology andb lung weight (n = 4)**. c-e** Analysis of lymphocyte cell subsets and activation status by flow cytometry. B16-HDAC2-WT or B16-HDAC2-MUT cells were intravenously injected into each C57BL/6 mouse (3 × 10⁶/mouse). The mice were sacrificed on the 11th day, and the lungs were isolated. The infiltration of various immune cell populations into the tumors was evaluated. Tumor cells from lung were stained forc CD45, CD3, CD8, F4/80, CD11b andd CD25 followed by flow cytometry analysis. Representative flow cytometry plots are shown, ande quantification is of data from 3 animals in each group. ns, not significant; *P < 0.05

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NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2

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NOS1 inhibits the interferon response of cancer cells by S-nitrosylation of HDAC2

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