Abstract 218: GSTP1 expression in human cancers decreases p53 protein levels and markedly attenuates p53-dependent responses to therapy-induced DNA damage

Abstract

Abstract The antioxidant enzyme glutathione transferase-pi (GSTP1) is expressed at high levels in most human cancers and this has been well linked to increased malignancy, enhanced anticancer drug resistance, and poor prognosis. While the enzymic inactivation anticancer drugs by GSTP1 can explain the tumor drug resistance, at least in part, it cannot fully account for the impact of this protein on aggressive tumor growth and patient's poor response to therapy. Relevant to this notion and analogous to the known negative regulation of JNK and other stress-activated kinases by GST-pi, here, we report for the first time a physical association between p53 and GSTP1 proteins both in vitro and in tumor cells, decreased levels of basal p53, and a significant attenuation in the biological responses of p53 to DNA damage in GSTP1 expressing cancer cell lines. Enforced expression of GSTP1 in GSTP1-deficient MCF-7 breast cancer and UW228 medulloblastoma cell lines induced a reproducible decrease (approx. 40%) in the basal and DNA-damage inducible p53 protein levels. In nuclear extracts from camptothecin-treated MCF-7 and UW228 cells, p53 binding to DNA was 35-45% lower after enforced expression of GST-pi. In contrast, inhibiting GSTP1 by ethacrynic acid or its silencing by specific siRNA in the pi-proficient A549 and H460 lung cancer cells resulted in the increase of basal p53 levels. In all these experiments, the greater levels of basal p53 found in the GSTP1 knock-out cells translated into enhanced binding of the tumor suppressor to DNA in EMSA or DNA-affinity immunoblotting procedures. These changes were also reflected in the activity of p53 as a transcriptional regulator. Semi-quantitative RT-PCR showed that several targets of p53 transcriptional activation viz., p21cip1, MDM2, IGFBP3 and Bax were upregulated in H460 cells transfected with GSTP1 siRNA, while their mRNA levels were significantly lower in MCF-7 cells stably expressing GSTP1 as compared to the untransfected parent cells. Contrary to our expectation, the presence of GSTP1 in MCF-7 cells actually increased the half-life and stability of p53 protein, albeit slightly, indicating that other mechanisms, possibly involving the JNK, may mediate the decrease of basal p53 protein. Significant differences in the cell cycle progression were also evident in this setting; treatment of GSTP1-silenced H460 cells with vincristine or camptothecin resulted in a marked increase of sub G1 DNA content and apoptotic cells. The findings highlight a novel role for GSTP1 in diminishing the DNA damage response of p53 in human cancers. Such observations have been made in chemically-induced hepatic preneoplastic foci that express GSTP1 de novo. Our results bear huge significance for increasing the efficacy of anticancer drugs, not only the alkylating agents, but also of other classes by restoring p53 responsiveness through GSTP1 inhibition. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 218. doi:10.1158/1538-7445.AM2011-218