No abstract

The controlled and specific re-activation of endogenous tumor suppressors in cancer cells represents an important therapeutic strategy to block tumor growth and subsequent progression. Other than ectopic delivery of tumor suppressor-encoded cDNA, there are no therapeutic tools able to specifically re-activate tumor suppressor genes that are silenced in tumor cells. Herein, we describe a novel approach to specifically regulate dormant tumor suppressors in aggressive cancer cells. We have targeted the Mammary Serine Protease Inhibitor (maspin) (SERPINB5) tumor suppressor, which is silenced by transcriptional and aberrant promoter methylation in aggressive epithelial tumors. Maspin is a multifaceted protein, regulating tumor cell homeostasis through inhibition of cell growth, motility and invasion. We have constructed artificial transcription factors (ATFs) made of six zinc-finger (ZF) domains targeted against 18-base pair (bp) unique sequences in the maspin promoter. The ZFs were linked to the activator domain VP64 and delivered in breast tumor cells. We found that the designed ATFs specifically interact with their cognate targets in vitro with high affinity and selectivity. One ATF was able to re-activate maspin in cell lines that comprise a maspin promoter silenced by epigenetic mechanisms. Consistently, we found that this ATF was a powerful inducer of apoptosis and was able to knock down tumor cell invasion in vitro. Moreover, this ATF was able to suppress MDA-MB-231 growth in a xenograft breast cancer model in nude mice. Our work suggests that ATFs could be used in cancer therapeutics as novel molecular switches to re-activate dormant tumor suppressors.

Mitochondrial outer membrane permeabilization (MOMP) is a critical control point during apoptosis that results in the release of pro-apoptotic mitochondrial contents such as cytochrome c. MOMP is largely controlled by Bcl-2 family proteins such as Bax, which under various apoptotic stresses becomes activated and oligomerizes on the outer mitochondrial membrane. Bax oligomerization helps promote the diffusion of the mitochondrial contents into the cytoplasm activating the caspase cascade. In turn, Bax is regulated primarily by anti-apoptotic Bcl-2 proteins including Bcl-xL, which was recently shown to prevent Bax from accumulating at the mitochondria. However, the exact mechanisms by which Bcl-xL regulates Bax and thereby MOMP remain partially understood. In this study, we show that the small CHCH-domain-containing protein CHCHD2 binds to Bcl-xL and inhibits the mitochondrial accumulation and oligomerization of Bax. Our data show that in response to apoptotic stimuli, mitochondrial CHCHD2 decreases prior to MOMP. Furthermore, when CHCHD2 is absent from the mitochondria, the ability of Bcl-xL to inhibit Bax activation and to prevent apoptosis is attenuated, which results in increases in Bax oligomerization, MOMP and apoptosis. Collectively, our findings establish CHCHD2, a previously uncharacterized small mitochondrial protein with no known homology to the Bcl-2 family, as one of the negative regulators of mitochondria-mediated apoptosis.

Activation of androgen receptor (AR) may play a role in the development of castration resistant prostate cancer. Two intracellular tyrosine kinases, Ack1 (activated cdc42-associated kinase) and Src, phosphorylate and enhance AR activity and promote prostate xenograft tumor growth in castrated animals. However, the upstream signals that activate these kinases and lead to AR activation are incompletely characterized. In this study, we investigated AR phosphorylation in response to non-androgen ligand stimulation using phospho-specific antibodies. Treatment of LNCaP and LAPC-4 cells with epidermal growth factor (EGF), heregulin, Gas6 (ligand binding to Mer receptor tyrosine kinase and activating Ack1 downstream), interleukin (IL)-6 or bombesin stimulated cell proliferation in the absence of androgen. Treatment of LNCaP and LAPC-4 cells with EGF, heregulin, or Gas6 induced AR phosphorylation at Tyr-267; IL-6 or bombesin treatment did not. AR phosphorylation at Tyr-534 was induced by treatment with EGF, IL-6 or bombesin, but not by heregulin or Gas6. siRNA-mediated knockdown of Ack1 or Src showed that Ack1 mediates heregulin- and Gas6-induced AR Tyr-267 phosphorylation whereas Src mediates Tyr-534 phosphorylation induced by EGF, IL-6, and bombesin. Dasatinib, a Src inhibitor, blocked EGF-induced Tyr-534 phosphorylation. In addition, we show dasatinib also inhibited Ack1 kinase. Dasatinib inhibited heregulin-induced Ack1 kinase activity and AR Tyr-267 phosphorylation. Dasatinib inhibited heregulin-induced AR-dependent reporter activity. Dasatinib also inhibited heregulin-induced expression of endogenous AR target genes. Dasatinib inhibited Ack1-dependent colony formation and prostate xenograft tumor growth in castrated mice. Interestingly, Ack1 or Src knockdown or dasatinib did not inhibit EGF-induced AR Tyr-267 phosphorylation or EGF-stimulated AR activity, suggesting the existence of an additional tyrosine kinase that phosphorylates AR at Tyr-267. These data suggest that specific tyrosine kinases phosphorylate AR at distinct sites and that dasatinib may exert anti-tumor activity in prostate cancer through inhibition of Ack1.