Wnt signaling. Ramray Bhat.

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1 Wnt signaling Ramray Bhat

2 Starting with animal biology and viral infections The discovery of certain laboratory murine strains that were highly susceptible to mammary gland cancer. The susceptibility was inherited through transmission to progeny through milk The factor was retroviral in nature: MMTV (Mouse mammary tumor virus).

3 Viral integration The discovery of viral oncogenes/protooncogenes such as Src, Myc, Ras (Bishop and Varmus, 1985). However oncogenic retroviruses in animals generally led to sarcomas and hematomas. Why did MMTV cause an epithelial carcinoma? Especially since there did not seem to be any known retroviral oncogene it bore. The association of retroviral integration with tumors caused seem to be dependent on conserved viral integration in specific sites. Such integration could result in loss of mutations but could it also result in gain of function? (an interesting and novel hypothesis) ALV integrated near c-myc gene brought about its overexpression and therefore, Burkitt s lymphoma.

4 Discovering integration site 1 Strategy: search for common insertion sites in distinct MMTV induced tumors to check for common conserved oncogenes that are activated by the insertion. The search for a tumor with a single viral insertion. Subsequently use probes to search for such integration sites in other tumors and the identification of common integration site

5 Int1 a proto-oncogene

6 History: Wng Nusslein-Volhard and Wieschaus screen, segment polarity genes, Wingless (Sharma and Chopra, 1976), identification of wingless as int1, Capecchi and Smithies knockout target, Swaying classical mouse mutant (CNS defects)

7 The link between development and cancer Capecchi and McMahon s laboratory made knockout mouse: embryonic lethal with severe CNS defects McMahon and Moon, Cell, 1989

8 Back to cancer Hereditary forms of cancer: adenomatous polyposis coli: frameshift mutations in a giant gene APC. Homolog mutated in a strain of mice called Min (Multiple Intestinal Neoplasia) strain. Hopkins colon cancer center

9 And to some cell biology APC interacted with another segment polarity gene called β catenin or Armadillo (Drosophila ortholog) (Eric Weischaus s group) Masatoshi Takeichi showed that β catenin interacted with E cadherin to regulate epithelial cell adhesion. Overexpression of cadherin and underexpression of β catenin also led to loss of dorsal induction. There loss of catenin phenocopies loss of Wnt signaling. (Heasman et al, Cell, 1994) Genetic studies by Norbert Perrimon s group showed that GSK3 is a negative regulator of β catenin. Hopkins colon cancer center

10 Dissecting the pathway APC βcat GSK3 Expression of domain-dead mutants of GSK3 in xenopus embryos results in dorsal axis duplication, phenocopying Wnt (Dominguez et al, PNAS 1995) The domain death led to increased levels of β catenin, not through increased synthesis but through stabilization of the protein. Several putative Ser/Thr phosphorylation sites on the N terminus. Mutation would again stabilize β catenin leading to phenocopy Wnt pathway.

11 Back to cancer APC βcat GSK3 In sporadic forms of colon cancer some individuals did not show mutation of APC. Might β catenin accumulate mutations stabilizing it against degradation? Indeed non- APC affected tumors showed mutations in the N terminus that resulted in GSK-insensitive β catenin (work of Vogelstein and Clevers) 2 more genes found to play a role Dishevelled and Axin. Dishevelled was a negative regulator of GSK3 Axin was shown by Marc Kirschner s group to be a rate limiting factor for β catenin activity and Wnt signaling.

12 Missing components: receptor and transcription factor Receptor serendipitously found in a cdna library for understanding the molecular biology of vision. Frizzled a protein which had earlier been shown to bind to Dishevelled was found to have an ortholog in Drosophila which was a segment polarity gene When expressed in Drosophila cells led to increased levels of β catenin Work on C. elegans by Craig Mello among others showed POP1 a transcription factor was affected by MOM set of proteins (MOM2: Wnt and MOM5: Frizzled) POP1 ortholog is TCF- LEF (T cell specific factor, Lymphoid enhancerbinding factor) shown later to bind to β catenin and mediate mesodermal specification