K-ras proto-oncogene exhibits tumor suppressor activity as its absence promotes tumorigenesis in murine teratomas.

Ras proteins transduce signals from membrane-bound receptors via multiple downstream effector pathways and thereby affect fundamental cellular processes, including proliferation, apoptosis, and differentiation. K-ras activating mutations play a key role in neoplastic progression and are particularly prevalent in colorectal, pancreatic, and lung cancers. The present study addressed whether the K-ras proto-oncogene displays a tumor suppressor function by comparative analysis of mouse teratomas derived from wild-type embryonic stem (ES) cells, K-ras null (K-ras(-/-)) ES cells, and K-ras(-/-) ES cells that stably reexpress either wild-type K-ras(gly12) or oncogenic K-ras(val12). K-ras(-/-) and K-ras(val12) teratomas were significantly larger than teratomas that expressed wild-type K-ras, contained significantly higher proportions of undifferentiated embryonal carcinoma-like cells, and showed significantly increased mitotic activity. However, K-ras(val12) but not K-ras(-/-) teratomas exhibited significantly higher levels of apoptosis than wild-type teratomas. K-ras(-/-) and K-ras(val12) ES cells showed a higher capacity for stem cell self-renewal in vitro compared with wild-type ES cells, and reexpression of K-ras(gly12) in K-ras(-/-) ES cells restored the K-ras(-/-) phenotype to wild-type values. Thus, in view of evidence that tumors can derive from tissue stem cells and that tumors harbor "cancer stem cells," aberrant K-ras expression could promote neoplastic progression by increasing their capacity for self-renewal.

Functional Smoothened is required for expression of GLI3 in colorectal carcinoma cells.

We investigated a role for Hedgehog signalling in colon cancer by studying transcription of members of the pathway in human colorectal carcinoma cell lines. We determined the methylation status and screened the gene encoding the Hedgehog receptor-associated protein Smoothened (SMO) for putative mutations. In three cell lines lacking SMO expression the SMO promoter was fully methylated and the transcription factor GLI3 was not expressed. Two additional cell lines both having one methylated SMO allele and expressing mutant SMO did not express GLI3. Our results suggest that expression of wild-type SMO is required for expression of GLI3 by a mechanism that is independent of conventional Hedgehog signalling.

Expression of Sonic hedgehog pathway genes is altered in colonic neoplasia.

The Hedgehog (Hh) signalling pathway is crucial for normal development and patterning of numerous human organs including the gut. Hh proteins are also expressed during gastric gland development and gastric epithelial differentiation in adults. Recently, dysregulation of these developmentally important genes has been implicated in cancer, leading to the present study of the expression of Hh signalling proteins in colon cancer. In this study, normal colon and colonic lesions (hyperplastic polyp, adenoma, and colonic adenocarcinoma) were examined by immunohistochemistry using antibodies against Hh signalling molecules: the secreted protein Sonic hedgehog (SHH), its receptor Patched (PTCH), and the PTCH-associated transmembrane protein Smoothened (SMOH). The study shows that Hh signalling pathway members are expressed in normal colonic epithelium. SHH was expressed at the top of the crypts and in a few basally located cells, while PTCH was detected in the neuroendocrine cells and SMOH at the brush border of superficial epithelium. RT-PCR analysis of laser-microdissected crypts from normal human colon confirmed that mRNAs encoding these proteins were expressed in colonic epithelium. Expression of SHH, PTCH, and SMOH was up-regulated in hyperplastic polyps, adenomas, and adenocarcinomas of the colon, and SHH expression correlated with increased expression of the proliferation marker Ki-67 in all lesions examined. To address whether the Hh signalling pathway is functional in the gut, the effect of Shh on epithelial cells in vitro was explored by treating primary murine colonocytes with either Shh peptide or neutralizing anti-Shh antibody. The proportion of cells in the S-phase was assessed by bromodeoxyuridine (BrdU) incorporation. It was found that exogenous Shh promotes cell proliferation in colonocytes, while anti-Shh inhibits proliferation, suggesting that Shh is required during proliferation of epithelial cells in vitro. It is suggested that SHH is required during epithelial proliferation in the colon and that there is a possible role for Hh signalling in epithelial colon tumour progression in vivo.