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The impact of Bcl-3 expression and signaling in cancer

Saamarthy, Karunakar LU (2015) In Lund University, Faculty of Medicine Doctoral Dissertation Series 2015:93.
Abstract (Swedish)
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... (More)
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. (Less)
Abstract
Bcl-3 is a proto-oncogene and belongs to the inhibitor of κB (IκB) family and is known to be upregulated in numerous cancers. Bcl-3 can initiate transcription of different genes involved in cell survival and cell growth. Cell proliferation is achieved through binding of Bcl-3 to cyclin D1 promoter and facilitates the rapid transition from G1 to S phase. The aim of this thesis was to investigate the role of Bcl-3 in different types of cancers including prostate, colon, and melanoma as well as to identify a selective inhibitor against Bcl-3 protein. In prostate cancer (PC) we observed the upregulation of Bcl-3 in cancer cells adjacent to infiltration of inflammatory cells. More precisely in PC cell lines we found out that Bcl-3 was... (More)
Bcl-3 is a proto-oncogene and belongs to the inhibitor of κB (IκB) family and is known to be upregulated in numerous cancers. Bcl-3 can initiate transcription of different genes involved in cell survival and cell growth. Cell proliferation is achieved through binding of Bcl-3 to cyclin D1 promoter and facilitates the rapid transition from G1 to S phase. The aim of this thesis was to investigate the role of Bcl-3 in different types of cancers including prostate, colon, and melanoma as well as to identify a selective inhibitor against Bcl-3 protein. In prostate cancer (PC) we observed the upregulation of Bcl-3 in cancer cells adjacent to infiltration of inflammatory cells. More precisely in PC cell lines we found out that Bcl-3 was upregulated with the stimulation of IL-6 through STAT3 signaling pathway. Silencing of Bcl-3 in PC cell lines sensitize these cells against anticancer drugs treatment and formed small tumors in vivo compared to control treated cells. In colon cancer we found that Bcl-3 localization differed comparing non-cancerous with cancer cells. Bcl-3 was mainly localized in cytoplasm of cancer tissue compared to nucleus in normal colon tissue. Out of six colon cancer lines three of them showed highest expression of Bcl-3. Cell fractionation analysis of these cell lines reiterated that majority of Bcl-3 protein was localized in the cytoplasm. Furthermore, the cytoplasmic localization of Bcl-3 in cancer cells correlated positively with proliferation marker Ki67 but not with the apoptotic marker cleaved caspase 3. Results obtained from this study suggest that cytoplasmic presence of Bcl-3 could be used as a diagnostic marker in certain stages of colon cancer. Since so far no inhibitors against Bcl-3 exist, we decided to identify a specific inhibitor against Bcl-3 protein. For this purpose we screened among 1368 synthetic compounds and could identify a specific Bcl-3 inhibitor. Treatment of melanoma cells with Bcl-3 inhibitor or its related derivate reduced the proliferation and arrested cells at G1/S phase of the cell cycle. Results from this study suggest that our newly identified Bcl-3 inhibitor can be used as anticancer drug to reduce the proliferation in melanoma. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Moustakas, Aristidis, Professor
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Cyclin D1, BCL3ANT, Colorectal cancer, Melanoma, Prostate cancer, ID, IL-6, Bcl-3
categories
Higher Education
in
Lund University, Faculty of Medicine Doctoral Dissertation Series
volume
2015:93
pages
50 pages
publisher
Division of Translational Cancer Research, Molecular Tumor Pathology
defense location
Medicon Village, Building 302 Lecture Hall, Lund
defense date
2015-09-04 13:00
ISSN
1652-8220
ISBN
978-91-7619-172-9
language
English
LU publication?
yes
id
db087898-2b4f-447d-aa61-f82dcff659ac (old id 7760991)
date added to LUP
2015-08-13 10:12:45
date last changed
2016-09-19 08:44:49
@phdthesis{db087898-2b4f-447d-aa61-f82dcff659ac,
  abstract     = {Bcl-3 is a proto-oncogene and belongs to the inhibitor of κB (IκB) family and is known to be upregulated in numerous cancers. Bcl-3 can initiate transcription of different genes involved in cell survival and cell growth. Cell proliferation is achieved through binding of Bcl-3 to cyclin D1 promoter and facilitates the rapid transition from G1 to S phase. The aim of this thesis was to investigate the role of Bcl-3 in different types of cancers including prostate, colon, and melanoma as well as to identify a selective inhibitor against Bcl-3 protein. In prostate cancer (PC) we observed the upregulation of Bcl-3 in cancer cells adjacent to infiltration of inflammatory cells. More precisely in PC cell lines we found out that Bcl-3 was upregulated with the stimulation of IL-6 through STAT3 signaling pathway. Silencing of Bcl-3 in PC cell lines sensitize these cells against anticancer drugs treatment and formed small tumors in vivo compared to control treated cells. In colon cancer we found that Bcl-3 localization differed comparing non-cancerous with cancer cells. Bcl-3 was mainly localized in cytoplasm of cancer tissue compared to nucleus in normal colon tissue. Out of six colon cancer lines three of them showed highest expression of Bcl-3. Cell fractionation analysis of these cell lines reiterated that majority of Bcl-3 protein was localized in the cytoplasm. Furthermore, the cytoplasmic localization of Bcl-3 in cancer cells correlated positively with proliferation marker Ki67 but not with the apoptotic marker cleaved caspase 3. Results obtained from this study suggest that cytoplasmic presence of Bcl-3 could be used as a diagnostic marker in certain stages of colon cancer. Since so far no inhibitors against Bcl-3 exist, we decided to identify a specific inhibitor against Bcl-3 protein. For this purpose we screened among 1368 synthetic compounds and could identify a specific Bcl-3 inhibitor. Treatment of melanoma cells with Bcl-3 inhibitor or its related derivate reduced the proliferation and arrested cells at G1/S phase of the cell cycle. Results from this study suggest that our newly identified Bcl-3 inhibitor can be used as anticancer drug to reduce the proliferation in melanoma.},
  author       = {Saamarthy, Karunakar},
  isbn         = {978-91-7619-172-9},
  issn         = {1652-8220},
  keyword      = {Cyclin D1,BCL3ANT,Colorectal cancer,Melanoma,Prostate cancer,ID,IL-6,Bcl-3},
  language     = {eng},
  pages        = {50},
  publisher    = {Division of Translational Cancer Research, Molecular Tumor Pathology},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine Doctoral Dissertation Series},
  title        = {The impact of Bcl-3 expression and signaling in cancer},
  volume       = {2015:93},
  year         = {2015},
}