<i>NTRK3</i> Is a Potential Tumor Suppressor Gene Commonly Inactivated by Epigenetic Mechanisms in Colorectal Cancer

<div><p><i>NTRK3</i> is a member of the neurotrophin receptor family and regulates cell survival. It appears to be a dependence receptor, and thus has the potential to act as an oncogene or as a tumor suppressor gene. NTRK3 is a receptor for NT-3 and when bound to NT-3 it induces cell survival, but when NT-3 free, it induces apoptosis. We identified aberrantly methylated <i>NTRK3</i> in colorectal cancers through a genome-wide screen for hypermethylated genes. This discovery led us to assess whether <i>NTRK3</i> could be a tumor suppressor gene in the colon. <i>NTRK3</i> is methylated in 60% of colon adenomas and 67% of colon adenocarcinomas. <i>NTRK3</i> methylation suppresses <i>NTRK3</i> expression. Reconstitution of NTRK3 induces apoptosis in colorectal cancers, if NT-3 is absent. Furthermore, the loss of <i>NTRK3</i> expression associates with neoplastic transformation <i>in vitro</i> and <i>in vivo</i>. We also found that a naturally occurring mutant <i>NTRK3</i> found in human colorectal cancer inhibits the tumor suppressor activity of <i>NTRK3</i>. In summary, our findings suggest <i>NTRK3</i> is a conditional tumor suppressor gene that is commonly inactivated in colorectal cancer by both epigenetic and genetic mechanisms whose function in the pathogenesis of colorectal cancer depends on the expression status of its ligand, NT-3.</p></div

Assessment of normalized caspase 3 and 7 activity after reconstitution of <i>NTRK3</i> in HCT116 (A), RKO (B) and HT29 (C) cells.

<p>NTRK3 induces caspase activity in HCT116 (MSI), RKO (CIMP) and HT29 (MSS) cells, and NT-3 (100 ng/mL) inhibits this effect in all three cell lines. DMSO is the vehicle control for NT-3, and GUS is the control vector (pDEST27-GUS) used to normalize for nonspecific effects of the transfection on caspase activity. The caspase activity in the GUS transfected cells treated with DMSO was used to normalize the results from the other experimental groups. The asterisks indicate statistically significant differences, <i>p</i><0.05 as determined by a 2-sided Mann-Whitney rank sum test.</p

Assessment of caspase activity and colony formation after reconstitution with mutant <i>NTRK3</i> in RKO.

<p>The three mutant <i>NTRK3</i> constructs contain <i>NTRK3</i> mutants found in primary human CRC. <b>A.</b> The expression of reconstituted wild-type <i>NTRK3</i> (<i>WT</i>), <i>NTRK3-G608S (G608S)</i>, <i>NTRK3-I695V (I695V)</i> and <i>NTRK3-L760I (L760I)</i> was confirmed by western blotting. <b>B.</b> Apoptosis was assessed by normalized caspase 3 and 7 activity in the RKO cell line 48 hours after transfection with <i>WT</i>, <i>G608S</i>, <i>I695V</i> and <i>L760I NTRK3</i> constructs. <b>C.</b> Soft agar colony formation was assessed in stably-transfected RKO cells after 2 weeks. Results are plotted as the mean colony numbers from three independent experiments. <i>L760I</i> did not induce apoptosis (<b>B</b>) or suppress colony formation (<b>C</b>), whereas <i>WT</i>, <i>G608S</i> and <i>I695V</i> did. Thus, the <i>G608S</i> and <i>I695V</i> mutations appear to be passenger mutations. GUS was used as the control vector to normalize for nonspecific effects of the transfection on apoptosis. The asterisks indicate statistically significant differences, p<0.05 as determined by a 2-sided Mann-Whitney rank sum test.</p

Clinical, pathological, and molecular characteristics of CRCs that carry methylated <i>NTRK3</i> and unmethylated <i>NTRK3</i>.

<p>Clinical, pathological, and molecular characteristics of CRCs that carry methylated <i>NTRK3</i> and unmethylated <i>NTRK3</i>.</p

<i>NT3</i> expression and the relationship of <i>NTRK3</i> and <i>NT3</i> mRNA expression in primary colon tissues.

<p><b>A. </b><i>NT3</i> mRNA expression is significantly lower in both the adenoma and adenocarcinoma samples compared to the matched normal colon epithelium (both <i>p</i><0.01) (Please see methods for units on Y axis). <b>B.</b> There is a direct correlation between <i>NT3</i> and <i>NTRK3</i> expression in normal colon and adenocarcinomas (r² = 0.81, Pearson's correlation <i>P</i><0.0001). The units on the Y-axis are relative expression units and each bar on the X-axis represents one sample. The bar graphs are aligned so that the samples correspond in the upper and lower graphs.</p

NTRK3 suppresses <i>in vitro</i> soft agar colony formation (A) and tumor xenograft growth (B–D) of colon cancer cell lines.

<p><b>A.</b> The <i>GUS</i> and <i>NTRK3</i> stably transfected HCT116 cells were grown in soft agar for 2 weeks. Results are plotted as the mean colony numbers from three independent experiments. The asterisks indicate statistically significant differences (<i>p</i><0.05; two-sided student <i>t</i> test). Representative fields depicting colonies of HCT116 cells grown in soft agar are shown. <b>B.</b> The growth of the HCT116-<i>NTRK3</i> and HCT116-<i>GUS</i> xenograft tumors in the <i>nu/nu</i> mice was measured over 3 weeks. The mean tumor volume is indicated, and the asterisks indicate statistically significant differences between the mean tumor volume of the <i>NTRK3</i>-reconstituted and control tumors (two-sided student <i>t</i> test). <b>C.</b> Tumors were removed 21 days after subcutaneous injection. The final tumor weight in NTRK3-expressing tumors was significantly less than the weight of the control tumors (<i>p</i> = 0.0021, n = 10). <b>D.</b> Representative tumors are shown (scale bar: 5 mm) along with confirmatory NTRK3 immunostaining.</p

Bisulfite sequencing results of representative methylated and unmethylated samples as determined by the <i>NTRK3</i> MethyLight (qMSP) assay.

<p>The black boxes indicate methylated CpG dinucleotides, and the white boxes represent unmethylated CpG dinucleotides. Each row depicts the sequencing results for a single clone. <b>A.</b> Schematic diagram of the 5′ end of the <i>NTRK3</i> gene showing the location of the qMSP and bisulfite sequencing primers (TSS: transcriptional start site). <b>B.</b> Results of bisulfite sequencing of DNA from colon cancer cell lines representative of different molecular subtypes: RKO is a CpG island methylator phenotype (CIMP) cell line, HCT116 is microsatellite unstable (MSI) line, and HT29 is a microsatellite stable (MSS) line. These cell lines have dense methylation of <i>NTRK3</i>. <b>C.</b> Representative case of normal colon, which carries unmethylated <i>NTRK3</i>. <b>D. and E.</b> Representative cases of adenomas and adenocarcinomas that carry either methylated or unmethylated <i>NTRK3</i> are shown.</p

Knock-down of <i>Ntrk3</i> expression contributes to neoplastic transformation in normal colon epithelial cells.

<p><b>A. </b><i>Ntrk3</i> mRNA expression was efficiently knocked down by 3 different shRNA constructs (309053, 35392 and 189598) in the immortalized mouse colon epithelial cell line, YAMC. <b>B.</b> Suppression of <i>Ntrk3</i> promotes soft agar colony formation in the YAMC cells. The non-silencing control (NSC) is a control short-hairpin construct for the knockdown experiments. The asterisks indicate statistically significant differences. (<i>p</i><0.05; two-sided student <i>t</i> test).</p