Abstract
Histone deacetylase (HDAC) inhibitors (HDACi) show potent and selective antitumor activity despite the fact that they induce histone hyperacetylation in both normal and tumor cells. In this study, we showed that the inducible expression of kRasV12 in nontransformed intestinal epithelial cells significantly lowered the mitochondrial membrane potential (MMP) and sensitized cells to HDACi-induced apoptosis. Consistent with our finding that colon cancer cell lines with mutant Ras have reduced expression of signal transducers and activators of transcription 1 (STAT1), we showed that inducible expression of mutant Ras markedly decreased both basal and inducible expression of STAT1, a transcription factor with tumor suppressor activity. To investigate whether reduced expression of STAT1 in cells that harbor mutant Ras contributes to their increased sensitivity to HDACi, we silenced the expression of STAT1 in HKe-3 cells with small interfering RNA. Despite the fact that silencing of STAT1 was not sufficient to alter the MMP, STAT1 deficiency, like Ras mutations, sensitized cells to apoptosis induced by HDACi. We showed that the induction of p21 by HDACi was significantly impaired in HKe-3 cells with silenced STAT1 expression and showed that the ability of butyrate to activate p21 transcription was diminished in STAT1-deficient HKe-3 cells. Finally, we used cells with targeted deletion of p21 to confirm that p21 protects cells from butyrate-induced apoptosis, strongly suggesting that in these cells STAT1 deficiency promotes butyrate-induced apoptosis through impaired induction of p21. Our data therefore establish that Ras mutations, and consequent reduction in the expression of STAT1, underlie the increased susceptibility of transformed cells to undergo apoptosis in response to treatment with inhibitors of HDAC activity.
Highlights
Treatment of tumor cells with histone deacetylase (HDAC) inhibitors (HDACi), drugs that remodel chromatin and thereby modulate the expression of several genes, has been shown to result in growth arrest, differentiation, and/or apoptosis of many cancer cell lines [1, 2]
We recently reported that the extent of apoptosis in response to butyrate, an inhibitor of HDAC activity, is increased in colon cancer cell lines that carry mutant Ras [3]
To determine whether oncogenic Ras plays a general role in apoptosis induced by HDAC activity (HDACi), we used nontransformed intestinal epithelial cells (IEC)-iKRas intestinal cells with inducible expression of oncogenic k-Ras [21]
Summary
Treatment of tumor cells with histone deacetylase (HDAC) inhibitors (HDACi), drugs that remodel chromatin and thereby modulate the expression of several genes, has been shown to result in growth arrest, differentiation, and/or apoptosis of many cancer cell lines [1, 2]. We previously found that mutations in k-Ras, a common genetic change in human tumors, sensitize colon cancer cells to butyrate, an inhibitor of HDAC activity [3]. Oncogenic k-Ras has been shown recently to be phosphorylated by protein kinase C, which promotes its translocation to mitochondria, a central organelle in apoptosis, and its association with BCL-x, a potent modulator of programmed cell death [4]. We addressed the significance of constitutive Ras signaling, and Ras downstream target genes, in the responsiveness of colon cancer cells to HDACi
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