Regulation of cadherin-11 by GSK3 inhibition and TGFbeta1 treatment in cancer
      cells

Farina, Anne Kata.

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Farina, Anne Kata.

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Thesis (Ph.D.)--Georgetown University, 2008.; Includes bibliographical references. Cadherin-11 is a cell-cell adhesion molecule important during embryogenesis as well as bone morphogenesis. Exogenous cadherin-11 expression has been shown to increase cell invasion and metastasis in breast cancer cells. Here I show that cell density, GSK3 inhibition, and TGFβ1 pathway activation regulate cadherin-11 expression in breast and prostate cancer cells. Density has been shown to affect cadherin expression in various cell types. I found that cadherin-11 RNA and protein expression increase with increasing cell density. Further, treatment with GSK3 inhibitors, LiCl or BIO, greatly reduces both the basal level and density-dependent increase in cadherin-11 expression. Using anti-β-catenin RNAi, I found that GSK3 inhibition has two mechanisms of regulating cadherin-11 expression. The first is β-catenin-independent and is important to the regulation of both cadherin-11 RNA and protein. The second mechanism is β-catenin-dependent and regulates cadherin-11 protein expression. Actinomycin D and RNA polymerase II chromatin immunoprecipitation (ChIP) analysis reveal a post-transcriptional mechanism of regulation. Investigation of the cadherin-11 3'-untranslated region (UTR) uncovers several consensus sequences important to RNA stability or translational regulation. GSK3 inhibition utilizes several mechanisms to regulate cadherin-11 expression. TGFβ1 treatment also acts to regulate cadherin-11 expression by repression. TGFβ1 reduces basal levels of cadherin-11 and attenuates the effect of density. The mechanism of TGFβ1 regulation is Smad-independent, although the signal is undoubtedly transmitted through the TGFβ type I receptor (TβRI). A TβRI kinase inhibitor alleviates TGFβ1-dependent repression of cadherin-11 and has a subtle effect on endogenous cadherin-11. Investigation into other Smad-independent pathways activated by TGFβ1 binding suggests that p38 MAPK most likely plays a role in cadherin-11 regulation. ChIP analysis of samples treated with TGFβ1 reveals that cadherin-11 is not transcriptionally downregulated again pointing to RNA stability as the primary mechanism of cadherin-11 regulation. To conclude, cadherin-11 is regulated by GSK3 inhibition and TGFβ1, but not through the well-known mechanisms of these pathways. Further investigation is needed to determine how exactly these pathways affect cadherin-11 expression through RNA stability. Considering the possible interacting proteins, I suggest the exploration of RNA binding proteins such as HuR, β-catenin, and Puf proteins.