Abstract
Loss of TP53 and RB1 function have both been linked to poor response to DNA damaging drugs in breast cancer patients. We inactivated TP53 and/or RB1 by siRNA mediated knockdown in breast cancer cell lines varying with respect to ER/PgR and Her-2 status as well as TP53 and RB1 mutation status (MCF-7, T47D, HTB-122 and CRL2324) and determined effects on cell cycle arrest, apoptosis and senescence with or without concomitant treatment with doxorubicin. In T47D cells, we found the cell cycle phase distribution to be altered when inactivating TP53 (P=0.0003) or TP53 and RB1 concomitantly (P≤0.001). No similar changes were observed in MCF-7, HTB-122 or CRL2324 cells. While no significant change was observed for the CRL2324 cells upon doxorubicin treatment, MCF-7, T47D as well as HTB-122 cells responded to knockdown of TP53 and RB1 in concert, with a decrease in the fraction of cells in G1/G0-phase (P=0.042, 0.021 and 0.027, respectively). Inactivation of TP53 and/or RB1 caused no change in induction of apoptosis. Upon doxorubicin treatment, inactivation of TP53 or RB1 separately caused no induction of apoptosis in MCF-7 and HTB-122 cells; however, concomitant inactivation leads to a slightly reduced activation of apoptosis. Interestingly, upon doxorubicin treatment, concomitant inactivation of TP53 and RB1 caused a decrease in senescence in MCF-7 cells (P=0.027). Comparing the effects of concomitant knockdown on apoptosis and senescence, we observed a strong interaction (P=0.001). We found concomitant inactivation of TP53 and RB1 to affect various routes of response to doxorubicin treatment in breast cancer cells.
Highlights
TP53 inactivation lead to a slight upregulation of pRb in chemotherapy treated cells, supporting the hypothesis that the Rb-pathway may act as compensatory pathway in TP53 inactivated tumor cells in response to cellular stress
We assessed the potential impact of knockdown of TP53 or RB1, as well as both genes concomitantly, on three different cellular endpoints potentially induced by doxorubicin treatment: cell cycle arrest, apoptosis and senescence
We aimed at systematically exploring the effect of TP53 and RB1 inactivation, separately and in concert, on the cellular response to doxorubicin treatment using a panel of cell lines representing various breast cancer subtypes, respectively.[23]
Summary
Chemotherapy resistance is the main cause of therapy failure and death among cancer patients; yet the mechanisms behind resistance remains poorly understood.[1]Doxorubicin causes DNA double helical breaks leading to activation of cell cycle arrest, apoptosis or entry of a permanent state of cell cycle arrest, senescence.[2,3,4]In regulation of these processes, the tumor suppressor’s p53 (encoded by the TP53 gene) and the retinoblastoma protein, pRb (encoded by the RB1 gene) are both known to have key roles.[5,6,7]TP53 mutations have been associated with resistance to DNA damaging chemotherapy treatment across different malignancies.[8,9,10,11,12] the finding that anthracyclines may work on some tumors harboring TP53 mutations while other tumors respond to therapy despite lack of TP53 function has lead us to postulate that redundant gene pathways may act in concert.[1,13]pRb, regulating the transition between G1/G0- and S-phase in the cell cycle, has a key role executing cell cycle arrest in response to DNA damage.[14,15] While conflicting evidence has linked RB1 mutations to lack of, as well as an improved response to chemotherapy;[16,17] previously we reported mutations in RB1 to be associated with a poor response to anthracyclines and mitomycin in breast cancer.[18,19]. Doxorubicin causes DNA double helical breaks leading to activation of cell cycle arrest, apoptosis or entry of a permanent state of cell cycle arrest, senescence.[2,3,4]. In regulation of these processes, the tumor suppressor’s p53 (encoded by the TP53 gene) and the retinoblastoma protein, pRb (encoded by the RB1 gene) are both known to have key roles.[5,6,7].
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