The aberrant methylation of CpG islands in the promoter regions of genes is a common event in many cancers 
. The average colon cancer genome contains 1,000–3,000 abnormally methylated genes 
. In many cases, the aberrant methylation of these genes can silence the expression of tumor suppressor genes and consequently promote tumor formation. However, it is also apparent that the hypermethylation of many genes in cancer has no effect on the expression of the methylated gene and does not influence tumor formation. This later class of methylated genes is felt to represent passenger events in tumorigenesis 
. Through a genome-wide screen for methylated genes in colon cancers, we identified methylated NTRK3
in colon adenomas and adenocarcinomas. Methylated NTRK3
was found in 67% of colorectal adenocarcinomas and 60% of adenomas. With regards to the functional significance of this epigenetic alteration, we found that the aberrant methylation of NTRK3
expression, which suggested NTRK3
might act as a tumor suppressor gene in colon cancer. Our findings are in contrast to other studies in breast cancer that have demonstrated that NTRK3
is oncogenic 
. These opposing results appear to be a consequence of NTRK3 being a dependence receptor, which means that it can induce proliferation when it binds its ligand, NT-3, but induces apoptosis when NT-3 is not available 
. Because NT-3 is expressed in the colon epithelium but not in colon neoplasms, our findings suggest that silencing of NTRK3 releases colon cancer cells from NTRK3-mediated apoptosis. These findings suggest that NTRK3
might function as a novel conditional tumor suppressor gene in CRC.
Although somatic mutations of NTRK3
that are predicted to inactivate function have been observed in CRC, NTRK3
's role as a tumor suppressor gene in CRC has not been clearly demonstrated to date 
. In the present study, we have provided evidence that NTRK3
can have conditional tumor suppressor activities in CRC. A similar role for NTRK3
in neuroblastomas has recently been shown 
. Reconstitution of NTRK3
in the absence of NT-3, the ligand for NTRK3, induced caspase-related apoptosis and cell death in the colon cancer cell lines RKO, HT29 and HCT116. We found that the effects on apoptosis could be suppressed by the treatment of the NTRK3 expressing cell lines with NT-3. Perhaps most importantly, NTRK3 inhibited colony formation in soft agar colony formation assays and suppressed the growth of tumor xenografts, which are hallmark in vitro
effects of tumor suppressor genes. In addition, we have shown that the naturally occurring NTRK3-L760I
mutation impairs NTRK3's ability to induce apoptosis and suppress anchorage independent growth. These findings suggest that NTRK3
is a CRC tumor suppressor gene that is inactivated by both genetic and epigenetic mechanisms.
The demonstration of NTRK3
as a potential conditional tumor suppressor gene in the colon suggests NTRK3 may be the latest member of a class of dependence receptors that suppress colon cancer formation. Other conditional tumor suppressor genes identified in CRC and other cancers, include DCC
. The dependence receptor model purports that some receptors induce different biological effects on cells depending on whether they are in a ligand-bound or ligand-free state. These receptors can induce caspase-mediated apoptosis in the absence of ligand, but induce proliferation when bound by their ligands. Therefore, one of the critical aspects of this study is the assessment of the expression of the NTRK3 ligand NT-3 in the colon. NTRK3's preferred ligand, NT-3, was found to be substantially suppressed in both colorectal adenomas and adenocarcinomas, presumably secondary to hypermethylation of the NT3
promoter region. It is plausible that the loss of NT-3 expression precedes the loss of NTRK3, which would create a clonal survival advantage for those CRC cells that silence NTRK3
. Our studies suggest that inactivation of NTRK3
occurs early in the polyp→cancer sequence and that it contributes to the transformation of colon epithelial cells.
With regards to the results of our studies, it is also important to consider the effects of loss of NT-3 and NTRK3 in the context of the entire neurotrophin receptor and ligand families because cross-talk between the ligand and receptor family members can occur. It has been shown that a precursor of NT-3, proNT-3, can activate p75NTR
and that NT-3 can activate NTRK1 or NTRK2, although this happens with low efficiency 
. However, despite the potential for cross-talk, we did not observe any effects on colon cancer cells that lacked NTRK3 after being treated with NT-3. Therefore, our findings suggest that NTRK3 is the primary and perhaps only receptor for NT-3 in the colon and in colon neoplasms.
When bound to NT-3, NTRK3 functions as a typical receptor tyrosine kinase. Its activation is stimulated by neurotrophin-mediated dimerization and transphosphorylation of an activation loop tyrosine 
. The major pathways activated by the NTRKs are MAPK, PI3K and PLC-γ1, among others 
. Activation of the NTRKs and p75NTR
promote activation of NF-κB, and p75NTR
can activate the JNK pathway 
. Previous studies have demonstrated that the activation of the MAPK and PI3K pathways by NTRK3 promotes cell differentiation, which in turn affects tumor progression 
. In this study, we also found that NTRK3 expression can activate the MAPK pathway. However, in this context the activation of ERK1/2 appears to be involved in the apoptotic response in colon cancer cells. There is a possibility that the MAPK activation we observed in this setting is an indirect effect of NTRK3 and a consequence of unopposed activation of p75NTR
. Our studies do not allow us to exclude this possibility, although even if such a mechanism was present, it would not change the interpretation of NTRK3
as being a colorectal cancer tumor suppressor gene.
In summary, we have identified NTRK3 as a novel conditional tumor suppressor gene in the colon that is inactivated by epigenetic and genetic mechanisms. We have provided evidence that NTRK3 can trigger apoptosis and inhibit tumor growth in the absence of its ligand NT-3 and that these effects are reversed by the addition of NT-3. We also showed that suppression of NTRK3 can induce transformed behavior in immortalized colon epithelial cells. Our studies provide further insight into the complex relationship between NTRK3 and NT-3 in cancers as well as into dependence receptor biology in the colon. This class of tumor suppressor genes may offer new therapeutic strategies in the colon.