The impact of Bcl-3 expression and signaling in cancer

University dissertation from Division of Translational Cancer Research, Molecular Tumor Pathology

Abstract: Popular Abstract in English Resistant to drug induced apoptosis and uncontrolled proliferation are biggest hurdles of cancer treatment. Bcl-3 is a proto-oncogene involved in both survival and proliferation in numerous cancers. In this thesis we investigated the molecular mechanisms through which Bcl-3 facilitates the survival and proliferation of cancer cells, furthermore we aimed to identify the Bcl-3 specific inhibitor. In the first project using the tissue microarray (TMA) of prostate cancer patients we could observe the upregulation of Bcl-3 at the same site where infiltrating leukocytes was observed. Next we investigated the expression of Bcl-3 in prostate cancer (PC) cell lines and found that Bcl-3 was highly expressed in androgen independent cell lines compared to androgen dependent cell line. Earlier studies have shown that Bcl-3 expression is induced by IL-6 via STAT3 signaling pathway in multiple myeloma. In PC cell lines also we could observe similar mechanism where IL-6 induced upregulation of Bcl-3 levels. By using the STAT3 specific inhibitors we could abolish the expression of Bcl-3 confirming that expression of Bcl-3 is dependent on STAT3. In androgen dependent cell line LNCap Bcl-3 was upregulated and translocated to the nucleus when stimulated by IL-6 and this mechanism was disrupted in the presence of STAT3 inhibitors. We could also show that Bcl-3 knock down cells are more susceptible to anticancer drug treatment. By this we propose that removal of Bcl-3 lead to the sensitization of cancer cells to chemotherapeutic drug induced apoptosis. Proto-oncogene Bcl-3 is widely recognized as a nuclear protein. In order to understand the role of Bcl-3 in colorectal cancer (CRC) we analysed the expression and localization of Bcl-3. We found that Bcl-3 is localized the cytoplasm in the colon cancer patients compared to the normal cells where it is localized in the nucleus. Further we investigated the expression of Bcl-3 in CRC cell lines and found that out of six cell lines three of them showed highest expression of Bcl-3. Localization of Bcl-3 was analysed in these cell lines by using cell fractionation, and the results validated the CRC patient’s data, by having majority of protein in cytoplasm. Cytoplasmic localization of Bcl-3 correlated with the proliferation marker Ki-67 but not with the apoptotic marker cleaved caspase 3. In conclusion we found that Bcl-3 is localized in the cytoplasm compared to the nucleus in colon cancer patients and this cytoplasmic localization is essential for CRC proliferation. Localization status of Bcl-3 could serve as diagnostic factor in CRC. Based on the previous findings where Bcl-3 forms a complex with NFκB family member’s p50/p52 and binds to the cyclin D1 promoter leading to increased proliferation, we aimed to identify the selective inhibitor for Bcl-3. In order to find the inhibitor for Bcl-3 we screened 1368 synthetic compounds that we received from national cancer institute. Screening was performed in cell based luciferase reporter assay. Initially, we explored the levels of Bcl-3 in melanoma cell lines and selected Mel Juso cells that had lowest levels of Bcl-3 for screening. From the screening we could identify 15 compounds, which could effectively block cyclin D1 promoter activity. Next by using the fluorescence-based thermal shift assays we found one small molecule that could potentially bind to the ankyrin repeat domain of Bcl-3. We termed this small molecule as BCL3ANT and could confirm that BCL3ANT reduces cyclin D1 promoter activity in a concentration dependent manner. To improve the potency of BCL3ANT we synthesized 5 different analogues based on the structure of BCL3ANT. Amongst all the analogues tested one analogue (A27) showed down regulated cyclin D1 expression compared to DMSO control. In two of the melanoma cell lines harbouring high levels of Bcl-3 we could show that BCL3ANT and its analogue A27 down regulate cyclin D1 promoter activity and protein levels of cyclin D1 without affecting the levels of Bcl-3. Cell cycle analysis revealed that treatment of BCL3ANT and A27 in melanoma cells arresting the cells in G1/S phase of the cell cycle. It is very well known that Bcl-3 forms a complex with either p50 or p52 and binds to the cyclinD1 promoter and drives its expression. Based on our findings we hypothesize that BCL3ANT and A27 might inhibit the Bcl-3-p50 or Bcl-3-P52 complex formation and reduce the proliferation in melanoma cells. In conclusion we have identified a selective Bcl-3 inhibitor BCL3ANT and its analogue A27 that inhibit Bcl-3 mediated cyclin D1 upregulation in melanoma cells. BCL3ANT and A27 could be used in treatment of melanoma where Bcl-3 mediated uncontrolled proliferation is observed.