Abstract
Estrogens are required for the proliferation of hormone dependent breast cancer cells, making estrogen receptor (ER) positive tumors amenable to endocrine therapies such as antiestrogens. However, resistance to these agents remains a significant cause of treatment failure. We previously demonstrated that inactivation of the retinoblastoma protein (pRb) family tumor suppressors causes antiestrogen resistance in MCF-7 cells, a widely studied model of estrogen responsive human breast cancers. In this study, we investigate the mechanism by which pRb inactivation leads to antiestrogen resistance. Cdk4 and cdk2 are two key cell cycle regulators that can phosphorylate and inactivate pRb, therefore we tested whether these kinases are required in cells lacking pRb function. pRb family members were inactivated in MCF-7 cells by expressing polyomavirus large tumor antigen (PyLT), and cdk activity was inhibited using the cdk inhibitors p16INK4A and p21Waf1/Cip1. Cdk4 activity was no longer required in cells lacking functional pRb, while cdk2 activity was required for proliferation in both the presence and absence of pRb function. Using inducible PyLT cell lines, we further demonstrated that pRb inactivation leads to increased cyclin A expression, cdk2 activation and proliferation in antiestrogen arrested cells. These results demonstrate that antiestrogens do not inhibit cdk2 activity or proliferation of MCF-7 cells in the absence of pRb family function, and suggest that antiestrogen resistant breast cancer cells resulting from pRb pathway inactivation would be susceptible to therapies that target cdk2.
Highlights
40 percent of human breast tumors depend on estrogens for proliferation [1], and are treated with drugs such as antiestrogens and aromatase inhibitors, which target the estrogen receptor (ER) [2]
Our previous results obtained with SV40 large tumor antigens (LT) indicated that the pRb binding domain of LT was required for conferring antiestrogen resistance to MCF-7 cells [12]
In transient transfection experiments only a certain fraction of all cells take up transfected DNA and express detectable levels of polyomavirus large tumor antigen (PyLT), the percentage of cells that were actively synthesizing DNA was determined in both cells that expressed PyLT (T+) and those that did not (T-) (Figure 1A, upper panel)
Summary
40 percent of human breast tumors depend on estrogens for proliferation [1], and are treated with drugs such as antiestrogens and aromatase inhibitors, which target the estrogen receptor (ER) [2]. Estrogens and antiestrogens control proliferation of breast cancer cells by regulating the expression of multiple components of the cell cycle machinery including cyclins D1 and A, cdc25a and the cyclin dependent kinase inhibitors p21Waf1/Cip (p21), and p27 Kip (p27) [4,5,6]. These molecules regulate the activity of the cyclin dependent kinases (cdks), cdk and cdk, which in turn phosphorylate and inactivate tumor suppressors of the retinoblastoma protein (pRb) family [4]. The pRb family of proteins inhibit the G1 to S phase transition by sequestering the E2F family of transcription factors [7]
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