Abstract 1635: Short hairpin RNA-mediated transcriptional suppression of glutathione S-transferase P1 suppresses growth and induces apoptosis through altered MAP kinase signaling in melanoma cells

Abstract

Abstract Introduction: The glutathione-S-transferase P1 (GSTP1) protein is highly expressed in melanoma and other human cancers, and is associated with resistance to chemotherapy, aggressive tumor growth, and poor overall patient survival. In addition to its function in phase II metabolism, GSTP1 is a major regulator of downstream signaling pathways that regulate cell survival and apoptosis. In this study, we investigated the role of GSTP1 and of altered MAP kinase signaling in apoptotic signaling and survival of melanoma cells. Methods: Human melanoma cell lines, A375 and DM6, were infected with a lentiviral vector carrying short hairpin RNA (shRNA) targeting GSTP1 or eGFP (vector controls). The infected and uninfected (control) cells were subjected to puromycin selection pressure, and GSTP1 expression in the heterogeneous surviving cells monitored by PCR and western blotting. Population growth kinetics was performed in real time by automated electronic cell sensing. Apoptosis was measured by flow cytometry (FCM) of propidium iodide stained cells and by assaying caspase 3/7 activation levels. Alterations in MAP kinase (P38, ERK, and JNK) levels and phosphorylation state were determined by western blotting, and apoptosis and MAP kinase related gene expression by focused array PCR. Results: GSTP1 transcripts and protein were significantly (up to 90%) suppressed in both cell lines, following GSTP1-shRNA-lentiviral infection and antibiotic selection, with the effect greater in A375 than in DM6. In both cell lines, however, GSTP1 suppression induced a significant decrease in growth rate, R (from 0.058 Hr−1 to 0.007 Hr−1 in A375 and 0.0395 Hr−1 to 0.021 Hr−1 in DM6). Similarly, population doubling times, td, increased from 12.0 hrs to 100.2 hrs in A375 and 17.6 hrs to 33.2 hrs in DM6, and baseline apoptosis increased significantly in both GSTP1-ve cell lines. While MAP kinase (ERK) phosphorylation increased, absolute levels remained unchanged. P38 was unaffected. Focused PCR analysis showed increased transcript levels of ERK regulated cell-cycle genes, including, CDKN2A and CDKN2D in GSTP1-ve A375 cells and CDKN1A, CDKN2C, and CDKN2D in GSTP1-ve DM6 cells. Levels of transcripts of apoptotic signaling genes were also significantly altered by GSTP1 suppression, albeit, with differences between the cell lines; caspase 1 and 10 increased ∼3-fold in A375 while BCL2 and BCL2L1 decreased >2-fold in DM6. Conclusions: Our data indicate that GSTP1 is required for growth of GSTP1-expressing melanoma cells and that in these cells; GSTP1 suppression slows growth and cell-cycle progression, and increases spontaneous apoptosis through altered MAP kinase and pro-apoptotic signaling. These observations provide a rationale for therapeutic targeting of GSTP1 in these tumors. 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 1635. doi:10.1158/1538-7445.AM2011-1635