RET is a potential tumor suppressor gene in colorectal cancer

Abstract

Cancer arises as the consequence of mutations and epigenetic alterations that activate oncogenes and inactivate tumor suppressor genes. Through a genome-wide screen for methylated genes in colon neoplasms, we identified aberrantly methylated RET in colorectal cancer. RET, a transmembrane receptor tyrosine kinase and a receptor for the glial cell-derived neurotrophic factor family ligands, was one of the first oncogenes to be identified, and has been shown to be an oncogene in thyroid cancer and pheochromocytoma. However, unexpectedly, we found RET is methylated in 27% of colon adenomas and in 63% of colorectal cancers, and now provide evidence that RET has tumor suppressor activity in colon cancer. The aberrant methylation of RET correlates with decreased RET expression, whereas the restoration of RET in colorectal cancer cell lines results in apoptosis. Furthermore, in support of a tumor suppressor function of RET, mutant RET has also been found in primary colorectal cancer. We now show that these mutations inactivate RET, which is consistent with RET being a tumor suppressor gene in the colon. These findings suggest that the aberrant methylation of RET and the mutational inactivation of RET promote colorectal cancer formation, and that RET can serve as a tumor suppressor gene in the colon. Moreover, the increased frequency of methylated RET in colon cancers compared with adenomas suggests RET inactivation is involved in the progression of colon adenomas to cancer.

Figure 1

Assessment of RET methylation status in representative normal colon, colorectal adenomas, and colorectal cancers. End-point RET MSP results from representative cases are shown. M=methylated, U=unmethylated. The case numbers are designated across the top of each gel photo.

Figure 2

A. RET expression after treatment with 5-aza-2’-deoxycytidine (5-AZA). Colon cancer cell lines that carry methylated RET (SW48, RKO, AAC1/SB10), methylated and unmethylated RET (HCT116), and unmethylated RET (V411 and SW480) were treated with 5-AZA or vehicle alone and then assessed for RET expression using qRT-PCR. RET is minimally expressed at baseline in all the cell lines except HCT116. The expression of RET is increased >5X after 5-AZA treatment in the SW48 and RKO cell lines. B. RET mRNA expression levels were significantly lower in colorectal tumors (either cancer or adenoma) with methylated RET as compared to those carried unmethylated RET (mean expression:1.04 ± 0.30 and 2.51 ± 0.56, respectively, P = 0.0279, 2-sided student t test). C. Expression of RET mRNA in primary normal colon mucosa, colon adenomas, and colon adenocarcinomas. When compared to normal colon mucosa, RET is significantly reduced in the colon cancers but not in the colon adenomas (p= 0.0057, ANOVA). D. Absolute quantitative measurement of RET mRNA in primary normal colon mucosa and adenocarcinomas. Consistent with the relative mRNA expreesion levels measured in 2C, RET is more highly expressed in normal colonic epithelia cells as compared to the adenocarcinomas. (P = 0.014, 2-sided student t test).

Figure 3

Expression of RET ligands in colorectal cancer cell lines and primary tissues. GDNF was not expressed in any of the colorectal cancer cell lines. (This data is shown in Supplemental Data, Figure S2.) A. Expression of ARTN in colon cancer cell lines. B. Expression of NRTN in colon cancer cell lines. The expression of these ligands is highly variable between cell lines with the highest expression being present in the cell lines HCT116 and SW480, which express RET. C. Expression of the GDNF ligands in normal colon mucosa and colon neoplasms. GDNF is expressed at higher levels in the normal colon compared to colon cancer (P = 0.0263), which is in contrast to ARTN, which is expressed at the same level or higher in colorectal cancers (P = 0.854), and NRTN, which is expressed at the same level in normal colon and colorectal cancers (P = 0.2936). All expression levels were determined using qRT-PCR. The units are relative units calculated after normalization with GUSB, which is a loading control. (A 2-sided student t test was used for the statistical analysis of these results.).

Figure 4

Expression of RET and GDNF in normal colon mucosa, adenomas and adenocarcinomas, assessed by immunohistochemistry. Representative cases assessed for both RET and GDNF expression are shown in the upper panel and lower panels separately. RET and GDNF are expressed in the normal colon epithelium cells, however, decreased or absent RET and no GDNF is detectable in the adenocarcinomas. The representative adenocarcinoma cases shown all carry methylated RET. Each image is from a different tissue sample (200X).

Figure 5

Expression of GFRα1 and GFRα3 mRNA in (A) colorectal cancer cell lines and (B) primary normal colon mucosa and colon adenocarcinomas. A. GFRα3 is highly expressed in SW480, which also expresses RET, compared to SW48 and RKO, which do not express RET. None of the cell lines expressed GFRα1. B. Expression levels were determined by quantitative RT-PCR and show decreased GFRα1 and GFRα3 expression in colorectal cancers compared to normal colon mucosa, although the difference is not statistically significant between normal colon and colorectal cancer (P = 0.1620 for GFRα1 and P = 0.340 for GFRα3, 2-sided student t test). The units are relative units calculated after normalization with GUSB expression, which is a loading control.

Figure 6

Assessment of apoptosis after reconstitution with RET in SW48, (A) RKO (B), and SW480 (C). RET induces caspase activity in SW48 and RKO, and GDNF inhibits this effect in both cell lines. Both SW48 and RKO carry methylated RET. Apoptosis was assessed in the cell lines 48 hours after transfection with RET51. GDNF (100 ng/mL), but not ARTN (100 ng/mL) decreased the amount of apoptosis in SW48 (A) and RKO (B). C. RET51 transfection did not induce apoptosis in SW480, which carries unmethylated RET. All these experiments were performed in triplicate and carried out using the Caspase-Glo 3/7 assay (Promega; top panel) or Cell Death Detection ELISA assay (Roche; bottom panel). The Caspase activity and ELISA assay results are shown as fold changes compared to the vector only group. pcDNA3 was used as the control vector to normalize for nonspecific effects of the transfection on apoptosis. RET51-K758R, which lacks kinase activity, was also used as a second control for these studies. The asterisks indicate statistically significant differences, P < 0.05 as determined by a 2-sided Mann-Whitney rank sum test.

Figure 7

Assessment of apoptosis after reconstitution with human somatic mutated RET in SW48. Apoptosis was assessed in the SW48 cells 48 hours after transfection with parental RET51, RET51-V145G (V145G), RET51-R360W (R360W) or RET51-G593E (G593E). The last 3 mutations were found in human colon cancer samples in previous studies (10). V145G and G593E did not induce apoptosis after transduction, which was consistently caused by the reintroduction of RET51 expression. pcDNA3 was used as the control vector to normalize for nonspecific effects of the transfection on apoptosis. Bax was used as a positive control for apoptosis assays. RET51-K758R (K758R), which lacks kinase activity, was also used as a control for these studies. The asterisks indicate statistically significant differences, P < 0.05 as determined by a 2-sided Mann-Whitney rank sum test.

Figure 8

Soft agar colony formation with RKO (A; carries methylated RET) and SW480 (B; carries unmethylated RET) cells after transfection with pcDNA3, RET K758R or RET51. RET inhibits colony formation in RKO but not SW480. Treatment of the RET transduced RKO with GDNF (100ng/ml) but not ARTN (100ng/ml) blocks the effect of RET on colony formation. (C) Knock-down of RET expression by RET-targeted siRNA promotes the colony formation ability of HCT116 cells, whereas, it suppresses the colony formation ability of the breast cancer cell line MCF7, which is consistent with RET acting as a tumor suppressor gene in colorectal cancer but as an oncogene in other types of cancer. Results are shown as the mean colony numbers from three independent experiments. The asterisks indicate statistically significant differences, P < 0.05 as determined by 2-sided student t test.

Figure 9

Western blot analysis of phosphorylated ERK1/2 (Thr202/Tyr204) and total ERK1/2 in colorectal cancer cells after transfection with RET51 and the control vectors pcDNA 3.0 and pcDNA3-RET K758R, which is an inactive form of RET. A. RET51 transfection induces the phosphorylation of ERK in cell line SW48, which carries methylated RET. RET51-induced phosphorylation of ERK is suppressed in cells treated with U0126 (10 μM). B. RET51 transfection does not affect ERK phophorylation in SW480, which carries unmethylated RET and expresses RET. The empty vector pcDNA3 and a vector containing RET51-K758R, which is a kinase-inactive receptor, do not affect ERK phosphorylation. Protein loading was normalized using actin. Each experiment was done in triplicate and a representative immunoblot is shown. C. Inhibition of MAPK activity after RET reconstitution in SW48 decreases RET induced apoptosis. Cell death induction in the CRC cell line SW48 was quantified 48 hours after transfection with RET51 using the Caspase-Glo 3/7 assay. The selective MAPK/ERK pathway inhibitor, U0126 (10 μM), significantly decreases apoptosis in the SW48 cell line transfected with RET51. 16% FBS was used to stimulate the cells before protein harvest for 30 minutes as an extracellular stimuli control for the activation of Erk1/2. All experiments were performed triplicate, and the results shown are fold changes compared to the empty vector control. The asterisks indicate statistically significant differences, P < 0.05 as determined by a 2-sided Mann-Whitney rank sum test.