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.2009 Feb 24;7(2):e39.
doi: 10.1371/journal.pbio.1000039.

Atonal homolog 1 is a tumor suppressor gene

Affiliations

Atonal homolog 1 is a tumor suppressor gene

Wouter Bossuyt et al. PLoS Biol..

Abstract

Colon cancer accounts for more than 10% of all cancer deaths annually. Our genetic evidence from Drosophila and previous in vitro studies of mammalian Atonal homolog 1 (Atoh1, also called Math1 or Hath1) suggest an anti-oncogenic function for the Atonal group of proneural basic helix-loop-helix transcription factors. We asked whether mouse Atoh1 and human ATOH1 act as tumor suppressor genes in vivo. Genetic knockouts in mouse and molecular analyses in the mouse and in human cancer cell lines support a tumor suppressor function for ATOH1. ATOH1 antagonizes tumor formation and growth by regulating proliferation and apoptosis, likely via activation of the Jun N-terminal kinase signaling pathway. Furthermore, colorectal cancer and Merkel cell carcinoma patients show genetic and epigenetic ATOH1 loss-of-function mutations. Our data indicate that ATOH1 may be an early target for oncogenic mutations in tissues where it instructs cellular differentiation.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Loss ofAtoh1 Enhances Tumor Formation in the Mouse Colon
(A) shows the incidence of polyps in AOM-treated mice. (B) shows the incidence of polyps in theAtoh1wt andAtoh1Δintestine mice in anAPCmin background. In (A) and (B), the two-tailed Fisher exact test was used. (C) Bar graph shows the average number of macroscopically visible polyps (>1 mm) in the colons of AOM-treatedAtoh1wt (WT;n = 9 polyps in 12 mice, white bars) andAtoh1Δintestine (n = 82 polyps in 8 mice, black bars). (D) The average number of polyps in the colons ofAPCmin (n = 27 polyps in 27 mice) andAPCmin; Atoh1Δintestine (n = 123 polyps in 10 mice). (E and F) The average maximum diameter of each polyp is shown as a bar graph forAtoh1wt orAtoh1Δintestine mice. (E) Comparison of polyp size from AOM-treatedAtoh1wt andAtoh1Δintestine mice; and (F) fromAPCmin andAPCmin;Atoh1Δintestine mice. For (C–F), the two-tailed Studentt-test was used to measure significance. Error bars indicate the standard error of the mean. Double asterisks (**) indicatep <0.01; triple asterisks (***) indicatep <0.001.
Figure 2
Figure 2. Increased Proliferation Contributes to Tumorigenesis inAtoh1Δintestine Colon
(A) Hematoxylin and eosin staining of a representative adenoma in AOM-treatedAtoh1Δintestine colon (5× magnification). The section is in the rectal region and is characterized by cystic structures with severe crypt hyperplasia with multifocal embedded adenomas and severe lymphocyte infiltration of the submucosa. (L, lumen; M, muscle). (A)′ and (A)′′ show higher magnification (20×) of two areas of the adenoma, indicating the aberrant branched crypt structures that are imbedded in the submucosa. (B) The colons of AOM-treatedAtoh1wt mice showed large GALT (highlighted by arrows) that were macroscopically counted as polyps. Magnification is 5×. Note the relative size of the GALT compared to adjacent normal-appearing crypts. (C) Histological analysis of colon inAPCmin. (D) Histological analysis inAPCmin;Atoh1Δintestine mice show highly dysplastic adenomas with cystic structures and lymphocyte infiltration. (E) Proliferation was measured by determining the percentage of BrdU-positive epithelial cells in AOM-treatedAtoh1wt andAtoh1Δintestine normal-appearing colonic crypts. The white bar represents crypts fromAtoh1wt mice; the gray bar represents nondeletedAtoh1wt crypts, and the black barAtoh1-null crypts inAtoh1Δintestine mice. (F) Apoptosis was measured by determining the percentage of cleaved caspase-3 (c-Caspase 3)-positive epithelial cells in AOM-treatedAtoh1wt andAtoh1Δintestine normal-appearing colonic crypts. Bar shading indicates crypt genotype as in (E). For each graph in (E and F), the two-tailed Studentt-test was used to measure significance: a single asterisk (*) indicatesp < 0.05; double asterisks (**) indicatep < 0.01. Error bars indicate the standard error of the mean.
Figure 3
Figure 3. Loss ofATOH1 Expression and Genomic Deletions in CRC Patient Samples
(A)ATOH1 expression normalized to GADPH and control colon samples:ATOH1 expression is lower in tumor compared to control samples. Additionally, adenocarcinomas have significantly lowerATOH1 expression than adenomas. Box plot indicating 25–75 percentiles, central line indicates the median, red cross indicates the mean. Error bars indicate next data point. Outliers are represented as dots. Double asterisks (**) indicatep < 0.01 (t-test). (B) Deletions in theATOH1 locus. Upper dashed line indicates the upper limit of single-deletion detection set at 0.70 of the ratio ofATOH1 locus to control locus. White and gray bars indicate different primer sets. (C) EndogenousATOH1 expression in different MCC14.2-derived cell lines and human keratinocytes (HK) in response toAtoh1 expression. (D) Detection of methylation at theATOH1 locus using pull-down assay of methylated DNA. Bands in “M-CpG pulldown” indicate positive forATOH1 methylation. “Input” shows DNA input before the pull-down. Samples were processed blindly. Internal methylated and unmethylated controls are shown in the last four lanes. (E) Inhibition of methyltransferase activity with 5-aza-deoxycytosine for 7 d leads to an increase ofATOH1 expression in the Ht29 cell line. Error bars indicate the standard deviation. (F) Graph representing observations on the genomic and mRNA levels. First column: percentage of patients showing deletions or duplications (black: deletions, gray: two copies, and white: duplications). Second column: percentage of patients showing methylation using the pull-down of methylated DNA assay (black: methylated, and white: not methylated). Third column: mRNA expression of CRC samples versus control colon (black: low expression, grey: normal expression, and white: high expression).
Figure 4
Figure 4. JNK Pathway Influenced by Atoh1
(A) Gene expression analysis in colon crypts fromAtoh1Δintestine andAtoh1wt mice. Quantitative RT-PCR was used to assess gene expression in isolated colon crypts. The ratio of gene expression is shown, in which negative numbers indicate reduced expression inAtoh1Δintestine crypts.t-tests determinedp-values shown for each gene. (B) Western analysis of p27kip, p21waf1, and c-JUN inAPCmin;Atoh1wt, andAPCmin;Atoh1Δintestine colonic tissues and polyps. Representative colonic tissue and polyp lysates ofAPCmin;Atoh1wt, andAPCmin;Atoh1Δintestine were used for western analysis of p27kip, p21waf1, and c-JUN. Actin was used a loading control. p21waf1 was significantly up-regulated in polyps compared to nonneoplastic colon tissue. c-Jun protein levels were significantly reduced in colonic polyps uponAtoh1 loss. p27 is down-regulated in preneoplastic colonic tissue upon loss ofAtoh1. Quantifications are shown in Figure S12A–S12C. (C) Representative tissues were used for Western analysis of phosphorylated JNK1 and JNK2. Total JNK1 and JNK2 are shown as control. Actin loading control is shown below. pJNK1, but not pJNK2, was significantly reduced in colon tissue fromAtoh1Δintestine compared toAtoh1wt mice. Quantifications are shown in Figure S12D1–S12D2′.
Figure 5
Figure 5.ATOH1 Suppresses Growth by Interfering with the Cell Cycle
(A) Constructs used for creating lentiviral and transfection vectors. (B) Western blot analysis for Atoh1 and chromogranin on untransduced MCC14.2 cells (lane 1), GFP-transduced MCC14.2 cells (lane 2), and two independently derived MCC14.2 cell lines transduced withAtoh1-IRES-eGFP (lane 3: MCC14.2-Atoh1.1a, and lane 4: MCC14.2-Atoh1.2a). Quantifications in Figure S12E and S12F. (C) Doubling time in hours for MCC14.2 (lane 1), MCC14.2-GFP (lane 2), and four lines independently transduced withAtoh1-IRES-eGFP (lanes 3–6: MCC14.2-Atoh1.1a, MCC14.2-Atoh1.1b, MCC14.2-Atoh1.2a, and MCC14.2-Atoh1.2b, respectively). (D) Assay for growth in soft agar. Colonies per view with a 10× lens, lane 1: MCC14.2, lane 2: MCC14.2-GFP, lane 3: MCC14.2-Atoh1.1a, and lane 4: MCC14.2-Atoh1.2a. Error bars indicate the 25th and 75th percentiles. The triple asterisks (***) in (C and D) indicate a significant difference from MCC14.2 (t-test:p < 0.001). (E) Percentage of cells positively labeled for BrdU and past S-phase in a BrdU pulse-chase experiment; double asterisks (**) indicatep < 0.01 (t-test).
Figure 6
Figure 6. ATOH1 Leads to Activation of Apoptosis and Expression of p21waf1
(A) Western blot analysis for p21waf1, cleaved caspase-3, cleaved caspase-9, and phosphorylated JNK of lysates of MCC14.2 cells, MCC14.2-GFP, and two MCC14.2 cell lines transduced withAtoh1-IRES-eGFP (MCC14.2-Atoh1.1a and MCC14.2-Atoh1.2a). The corresponding actin loading controls are shown under each blot. Quantifications are shown in Figure S12G–S12J. (B) Western blot analysis for p21waf1, cleaved caspase-3, cleaved caspase-9, and p-JNK of lysates of Ht29 cell line transfected with pCLIG-eGFP (left lane) or pCLIG-Atoh1-IRES-eGFP (right lane); actin loading controls are shown under the respective blots. Quantifications are shown in Figure S12K–S12N. (C) The molecular changes are specific to functionalato: western blot of lysates of Ht29 cells transfected with CMV-ato, CMV-ato1, or empty vector. Actin loading controls are shown below the respective blots. Quantifications are shown in Figure S12O–S12Q. (D) Graph expressing ratio of cell numbers of MCC14.2-derived cell lines transfected with pMSCV-ATOH1ERD-IRES-eGFP versus cell number of cell lines transfected with pMSCV-IRES-eGFP (vector). Statistical analysis was done usingt-test under different conditions compared to MCC14.2. Single asterisk (*) indicatesp < 0.05; double asterisks (**) indicatep < 0.01. (E) MCC1 cells transfected with dominant-negativeATOH1ERD fusion (lanes 1 and 2) and with empty vector control (lanes 3 and 4). Western blot analysis for p21waf1, phosphorylated JNK, and cleaved caspase-3 on lysates of MCC1 cell line. The actin loading control of each blot is shown below. Quantifications are shown in Figure S12R–S12T.
Figure 7
Figure 7. Molecular Mechanism for ATOH1 Function
(A–D) show that ATOH1′s anti-oncogenic function acts through receptor tyrosine kinases. (A) Doubling times in hours of MCC14.2 and MCC14.2 transduced witheGFP (lane 2) orAtoh1-IRES-eGFP (lanes 3 and 4) with increasing concentrations of K252a. (B) AnnexinV-positive signals normalized to transfected MCC14.2 cells (GFP). Cells transduced witheGFP (lanes 1 and 2) andAtoh1-IRES-eGFP (lanes 3 and 4) with 0.33 μM K252a (lanes 2 and 4) or DMSO as a control (lanes 1 and 3). The double asterisks (**) indicate significant difference from GFP transfected without K252a (t-test:p < 0.05). (C) AnnexinV-positive signals normalized to transfected Ht29 cells (GFP). Cells transfected witheGFP (lanes 1 and 2) andAtoh1-IRES-eGFP (lanes 3 and 4) with 0.33 μM K252a (lanes 2 and 4) or DMSO as control (lanes 1 and 3). The double asterisks (**) indicate significant difference from GFP transfected without K252a (t-test:p < 0.05). (D) Inhibition of RTKs inhibits p21waf1 expression and JNK phosphorylation. Western blot analysis for p21waf1 and pJNK of MCC14.2 (lanes 1 and 5), MCC14.2 transduced witheGFP (lanes 2 and 6), and two MCC14.2-derived cell lines transduced withAtoh1-IRES-eGFP (lanes 3, 4, 7, and 8) with 0.3 μM K252a (lanes 1–4) and with DMSO as a control (lanes 5–8). Actin loading controls are shown below each blot. Quantifications are shown in Figure S12U and S12V. (E) JNK regulates apoptosis and p21waf1 expression. Western blot analysis for p21waf1 and cleaved caspase-3 of MCC14.2 (lanes 1 and 5), MCC14.2 transduced witheGFP (lanes 2 and 6), and two MCC14.2-derived cell lines transduced withAtoh1-IRES-eGFP (lanes 3, 4, 7, and 8) with 1 μM JNK inhibitor (lanes 1–4) and with DMSO as a control (lanes 5–8). Actin loading control is shown below the blots. Phospho-JNK (P-JNK) blot shows the effect of the JNK inhibitor on JNK phosphorylation status Quantifications are shown in Figure S12W–S12Y. (F) Relative cell numbers (the number of Tam67-transfected cells divided by the number of mock-transfected cells) for MCC14.2 and MCC14.2 transduced witheGFP (lane 2) orAtoh1-IRES-eGFP (lanes 3 and 4) with dominant-negative c-jun (TAM67).
Figure 8
Figure 8. Schematic Representation of the Potential Mechanism of ATOH1′s Function as a Tumor Suppressor
In preneoplastic tissue,Atoh1 keeps malignant transformation in check in a JNK-dependent mechanism by the induction of apoptosis and the inhibition of cell cycle progression. WhenAtoh1 is lost due to deletion or methylation, these brakes on oncogenesis fail, and malignant transformation can progress.
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