Defense Date

2009

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Human Genetics

First Advisor

Shawn Holt

Second Advisor

Lynne Elmore

Third Advisor

Joy Ware

Fourth Advisor

Jolene Windle

Fifth Advisor

Michael Idowu

Abstract

Breast cancer is one of the most prevalent and deadly forms of cancer in women and is not restricted by race or ethnicity. Although a wealth of knowledge has been amassed on the biology of breast cancer, including its risk factors, diagnosis, prognosis, prevention, and treatment, it remains a serious health concern and active area of research. Initial response to standard chemotherapeutic and radiotherapeutic regimens is generally strong for many patients, yet breast tumors often recur, leading to more aggressive and resistant tumors. Because recurrence is such a clinical issue, more effective therapeutic approaches are needed to eliminate partial clinical responses and undesirable side effects. Molecular chaperones like the heat shock protein 90 (Hsp90) family are regarded as ubiquitous, highly conserved proteins that mainly respond upon induction of stress or disruption in cellular homeostasis. Chaperones are critically involved in controlling the conformation, stability, function, and degradation of many oncogenic client proteins by assisting in trafficking, remodeling of improperly folded client proteins, and suppression of protein aggregation. Hsp90-mediated folding events are an ATP-dependent process that involves the association with various co-chaperones and stimulators, including Hsp70, Hsp40, HOP, p23, and Aha1 for client stabilization and modification. Notably, Hsp90 seems to be particularly associated with cell signaling clientele, such as nuclear hormone receptors, protein kinases, and many other oncogenic proteins that directly influence the hallmarks of cancer. In order to define the role of chaperones in breast cancer progression, we assessed chaperone expression levels in normal and tumor lines. Based on our initial finding of increased expression of Hsp90 and p23 in immortal and cancer cell lines, it is possible that this phenomenon may be reflected in normal breast tissue as well as breast carcinoma specimens. Indeed, we observed high Hsp90 expression in invasive carcinomas, such that high nuclear expression correlates with a greater TNM stage, while high cytoplasmic Hsp90 correlates with ER negativity, suggesting that elevated Hsp90 may be an indicator or marker of advanced disease. p23 expression also increases appreciably in established breast cancer cell lines with invasive breast tissue specimens displaying an opposite trend. Although p23 does not appear to have any relationship with TNM stage, significant relationships with ER and PR emerged, with higher nuclear p23 correlating to ER positivity and higher total p23 being positively associated with PR presence. Due to the differential expression of Hsp90 in normal, DCIS, and invasive breast carcinomas, we determined the impact on Hsp90 function, finding that total Hsp90 in tumor cells is associated with an increase in both complexed and uncomplexed Hsp90, which is in contrast to a previously reported study. The intrinsic nature of heat shock proteins makes them especially relevant to a cell’s defense against cancer initiation. The preferential accumulation of Hsp90 in cancer cells also forms the basis for the unique sensitivity of tumor cells to Hsp90 inhibition. The Hsp90 specific inhibitor, radicicol, is more potent in terms of blocking ATPase activity than other Hsp90-related compounds like geldanamycin, which is much more toxic. All Hsp90 inhibitors prevent the association of the co-chaperone p23 with Hsp90, resulting in destabilization of the client protein. For these reasons, it may be possible that Hsp90 inhibition would sensitize breast cancer cells to be more responsive to standard chemotherapeutics. We determined that radicicol negatively alters cellular proliferation, and in combination with Adriamycin, elicits a more robust decline in growth and the expression of Hsp90 client proteins. This finding was associated with an increase in senescent cells without a detectable affect on apoptosis. Radicicol in combination with cisplatin or Taxol contributed to an increase in cell death (apoptosis) and differentially altered the expression of client proteins. Finally, ER negative breast cancer cells do not display altered p53 expression upon radicicol and Adriamycin treatment. Blockade of ER activity in ER positive cells with tamoxifen induced significant reductions in proliferation and decreased p53 expression without a corresponding decrease in p21 levels. In conclusion, these results point to the utility of Hsp90 inhibition as a valid form of targeted therapy for breast cancer, and the value of radicicol as a potential adjuvant treatment option in combination with standard chemotherapeutics.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

August 2009

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