ShcA tyrosine phosphorylation sites can replace ShcA binding in signalling by middle T-antigen.

ShcA and Grb2 are crucial components in signalling by most tyrosine kinase-associated receptors. How ever, it is not clear whether Grb2 bound directly to the receptor is equivalent to Grb2 associated via ShcA. We have used signalling stimulated by the middle T-antigen (MT) of polyoma virus to address this question. The two known Grb2-binding sites from murine ShcA, 313Y and 239/240YY, could functionally replace the MT ShcA-interacting region in transformation assays using Rat2 fibroblasts. This demonstrates that signal output from membrane-bound ShcA requires only these two sequences and the ShcA-binding site in MT does not recruit other signalling molecules. Two standard Grb2-interacting sequences, either from the EGF receptor or the ShcA 313Y region, could not replace the requirement for ShcA binding to MT, indicating an enhanced role for the ShcA 239/240YY motif. Sos1 and the docking protein Gab1 are brought into the MT complex through Grb2 association and this may be more effective using the 239/240YY sequence.

Progression of familial adenomatous polyposis (FAP) colonic cells after transfer of the src or polyoma middle T oncogenes: cooperation between src and HGF/Met in invasion.

Little is known about the the signalling pathways driving the adenoma-to-carcinoma sequence in human colonic epithelial cells. Accumulation and activation of the src tyrosine kinase in colon cancer suggest a potential role of this oncogene in this early progression. Therefore, we introduced either activated src (m-src), polyoma-MT alone or combined with normal c-src in the adenoma PC/AA/C1 cell line (PC) to define the function and phenotypic transformations induced by these oncogenes in familial adenomatous polyposis (FAP) colonic epithelial cells. Functional expression of these oncoproteins induced the adenoma-to-carcinoma conversion, overexpression of the hepatocyte growth factor (HGF) receptor Met, but failed to confer invasiveness in vivo and in vitro, or to produce alterations in cell proliferation and differentiation. In contrast, PC-msrc cells became susceptible to the HGF-induced invasion of collagen gels and exhibited sustained activation of the pp60src tyrosine kinase and Tyr phosphorylation of the 120-kDa E-cadherin, which was further increased by HGF Transcripts of HGF were clearly identified by reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot in the parental and transformed PC cells, suggesting an autocrine mechanism. Taken together, the data indicate that: (1) experimental activation of src and PyMT pathways directly induces tumorigenicity and Met upregulation in a colon adenoma cell line; (2) HGF-activated Met and src cooperate in inducing invasion; (3) in view of the molecular associations between catenins and cadherin or the tumour-suppressor gene product APC, the cell adhesion molecule E-cadherin may constitute a downstream effector of src and Met.

Neoplastic progression of human colorectal cancer is associated with overexpression of the stromelysin-3 and BM-40/SPARC genes.

The interaction of neoplastic cells with the extracellular matrix is a critical event for the initiation of cancer invasion and metastasis. This study was designed to evaluate the potential implication of stromelysin-3 (ST3), a newly identified member of the matrix-degrading metalloproteinase family, and of BM-40/SPARC, a glycoprotein associated with the extracellular matrix, during the progression of human colorectal cancers. We analyzed the relative abundance of ST3 and BM-40/SPARC transcripts by Northern blot, and their distribution by in situ hybridization, in normal mucosa, benign adenomas, and primary colorectal adenocarcinomas and their liver metastases. The ST3 and BM-40/SPARC transcripts were overexpressed in primary colorectal cancers and their liver metastases compared to non-neoplastic mucosa. These transcripts were localized in stromal fibroblasts adjacent to the neoplastic foci. Overexpression of ST3 correlated with the progression of human colorectal tumors toward local invasion and liver metastasis. Induction of these genes also occurred in diverticulitis and digestive neoplasms such as gastric and esophageal carcinomas.

The arotinoid Ro 40-8757 has antiproliferative effects in drug-resistant human colon and breast cancer cell lines in vitro.

We have examined the antiproliferative effects of the arotinoid Ro 40-8757 in 3 drug-resistant human adenocarcinoma cell lines: the colonic cells HT29-5FU and CaCo2, and the mammary cells MCF-7mdr1. Whereas all-trans retinoic acid had no effect at the concentration of 10(-6) M, Ro 40-8757 was found to exert a high antiproliferative action with similar inhibitory potency (IC50) in drug-resistant and parental cell lines (range, 0.06 x 10(-6) to 0.57 x 10(-6) M). We conclude that: (1) thymidylate synthase is not involved in the mechanism of action of Ro 40-8757; (2) the mdr1 gene product does not recognize this retinoic derivative, and (3) Ro 40-8757, alone or in combinations with other cytotoxic drugs, can be very useful in patients with progressive disease after conventional chemotherapy.

Neoplastic progression of human and rat intestinal cell lines after transfer of the ras and polyoma middle T oncogenes.

BACKGROUND: Activation of the p21ras and pp60c-src oncoproteins occurred at high incidence in the early stage of human colorectal carcinogenesis. Our study aimed to investigate the role of these signal-transduction pathways in the process of initiation and promotion of the malignant phenotype in intestinal cells. METHODS: The human Ha-ras and the polyoma middle T (Py-MT) viral oncogenes were transferred into large T oncogene of simian virus 40 immortalized rat intestinal epithelial SLC-44 cells and human colonic adenocarcinoma Caco-2 cells. RESULTS: These transfers conferred the tumorigenic and invasive phenotypes on immortalized SLC-44 cells and potentiated the tumorigenicity of Caco-2 cells and markedly repressed the terminal differentiation of this cell line. In SLC-44T cells, induction of the invasive phenotype by the activated Ha-ras oncogene correlated with weak expression of E-cadherin and reduced accumulation of the transcripts encoding the basement membrane components alpha 1 (IV) collagen, nidogen, and BM40, which might result partly from the inactivation of the transforming growth factor beta signaling pathway. The down-regulation of the alpha 1 (IV) collagen messenger RNA in SLC-44T cells was not due to the protein kinase C-dependent pathways or the secretion of autocrine factor(s). CONCLUSIONS: These results suggest that the activation of the p21ras and Py-MT/pp60c-src oncogenic pathways are critical effectors at different stages of colorectal carcinogenesis and in Caco-2 cells interferes with the program of enterocyte differentiation.

Increased protein kinase C alpha expression in human colonic Caco-2 cells after insertion of human Ha-ras or polyoma virus middle T oncogenes.

The proteins encoded by ras and src protooncogenes are frequently activated in a constitutive state in human colorectal cancers. To investigate the mechanism(s) whereby oncogenic p21ras and pp60c-src contribute to malignant transformation of intestine, human colonic Caco-2 cells transfected with an activated (Val 12) human Ha-ras gene (Caco-2-T cells) or Py-MT oncogene, a constitutive activator of pp60c-src tyrosine kinase activity (Caco-2-MT cells), were analyzed for tumorigenicity, protein kinase C (PKC) isoform expression, and PKC activity. As compared with control vector Caco-2-H cells, Caco-2-T and Caco-2-MT cells displayed: (a) an enhanced tumorigenicity in nude mice; (b) a 4-fold increase in the level of PKC-alpha mRNA which was not due to enhanced mRNA stability and was mediated through a PKC-independent pathway since it persisted after PKC depletion; (c) increased PKC-alpha immunoreactive protein content (3-fold), total PKC catalytic activity (3.5-fold), and total cell number of [3H]phorbol-12,13-dibutyrate binding sites (4-fold); and (d) a 1.7-fold higher membrane-bound/total PKC activity ratio together with 1.8- and 1.5-fold increases in [3H]arachidonate- and [3H]myristate-labeled diacylglycerol levels. In conclusion, the tumorigenic progression induced by oncogenic p21ras or the Py-MT/pp60c-src complex in Caco-2 cells is associated with increased PKC-alpha gene transcription and PKC-alpha expression as well as with constitutive PKC activation. These results provide the first evidence that the PKC-alpha gene is a target for the signaling pathways of oncogenically activated p21ras and pp60c-src in human colonic cells. They raise the possibility that PKC-alpha is an effector of these oncoproteins for activation of Caco-2 cell tumorigenic potential.

Cyclic amp-responsive gene-transcription in cellular proliferation and transformation.

The proteins binding the cAMP responsive elements constitute a family of transcription factors (e.g. CREB 327/341, ATF, HB16, CREM) which operate as dimers and regulate positively and negatively gene expression upon interaction with the cAMP responsive motifs CRE/ATF. These proteins regulate transcription after cAMP- dependent phosphorylation (CREB), or interaction with the adenovirus E1A oncoprotein (ATF-2). Subtle modulation might be further attained by the cross-talk between the different signal-transduction pathways and the heterodimerization of different classes of transcription factors. The CREB/ATF transcription factors therefore exert a pleiotropic action during the integration of extra/intra cellular signals in the resulting adaptation of cellular responses. Dysregulation might be associated with pathological situations related to the uncontrolled cell proliferation, oncogenic progression and metastasis.