Inhibition of Heat Shock-Induced H3K9ac Reduction Sensitizes Cancer Cells to Hyperthermia.

Heat stress, clinically known as hyperthermia, is a promising adjunctive modality in cancer treatment. However, the efficacy of hyperthermia as a monotherapy is limited and the underlying mechanism remains poorly understood. Targeting histone modifications is an emerging strategy for cancer therapy, but little is known regarding the role of heat stress in altering these modifications. Here, we report that heat shock inhibits H3K9 acetylation (H3K9ac) via histone deacetylase 6 (HDAC6) regulation. Heat shock inhibits the interaction between HDAC6 and heat shock protein 90 (HSP90), enhances nuclear localization of HDAC6, and promotes HDAC6 phosphorylation, which is regulated by protein phosphatase 2A (PP2A). Combining hyperthermia with HDAC inhibitors vorinostat or panobinostat leads to better anti-cancer effects compared to monotherapy. KEAP1 and DPP7 as genes affected by heat-induced inhibition of H3K9ac, and combining them with hyperthermia can better induce apoptosis in tumor cells. This study reveals previously unknown mechanisms of H3K9ac decreased by heat shock in cancer cells and highlights a potential combinational therapy involving hyperthermia and targeting of these new mechanisms.

O-GlcNAcylation of PFKFB3 is required for tumor cell proliferation under hypoxia.

The protein O-GlcNAcylation catalysed by O-GlcNAc transferase (OGT) is tightly regulated by glucose availability. It is upregulated and essential for tumor cell proliferation under hypoxic conditions. However, the mechanism behind is still unclear. Here, we showed that the glycolytic regulator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3), which also promotes cell cycle progression in the nucleus, was O-GlcNAcylated in response to hypoxia. The O-GlcNAcylation of PFKFB3 could compete phosphorylation by hypoxia-activated ERK at the same modification site Ser172. Phosphorylated PFKFB3 could interact with the protein G3BP2 and retain in the cytosol; this in turn led to the accumulation of hypoxia-induced-P27 in the nucleus resulting in the cell cycle arrest. Such a pathway was compromised by high level of PFKFB3 O-GlcNAcylation in tumor cells contributing to cell cycle progression. Consistently, the PFKFB3-Ser172 phosphorylation level inversely correlated with the OGT level in pancreatic cancer patients. Our findings uncovered an O-GlcNAcylation mediated mechanism to promote tumor cell proliferation under metabolic stress, linking the aberrant OGT activity to tumorigenesis in pancreatic cancer.

Molecular mechanisms of synergistic induction of apoptosis by the combination therapy with hyperthermia and cisplatin in prostate cancer cells.

Prostate Cancer has become the second leading cause of male cancer-related incidence and mortality in United States. Hyperthermia (HT) is known to serve as a powerful tool in treatment of prostate cancer in clinical. The combination treatment with HT and cisplatin has a synergistic effect to inhibit prostate cancer progression and demonstrates better clinical effectiveness than HT or chemotherapy alone. But molecular mechanisms of this phenomenon have not been illuminated clearly. In this study, we used MTS assay to examine cell viabilities of PC-3, LNCaP, DU-145 and RM-1 cells after treated by HT and cisplatin. Then colony formation of PC-3 and DU-145 cells after treated with HT and cisplatin were photographed. To investigate whether the combination therapy would enhance apoptosis of PC-3 and DU-145 cells, we used Western blot analysis to detect expression level of proteins on apoptosis-regulated signaling pathway in PC-3 and DU-145 cells. Our results showed that the combination treatment decreased cell viabilities and colony formation of prostate cancer cells in a dose-dependent manner and this combination therapy enhanced apoptosis of PC-3 and DU-145 cells via activation of Caspase-3 and cleavage of PARP. We also found that the combination therapy could down-regulate the anti-apoptotic Bcl-2 and IAP family proteins. At last, the combination therapy activated AMPKα-JNK signaling pathway and inhibited Akt-mTOR-p70s6k signaling pathway to promote apoptosis of PC-3 and DU-145 cells. In conclusion, this study clearly elucidated how the combination therapy with HT and cisplatin promoted apoptosis of prostate cancer cells in synergy.

Hepatitis B virus X protein reduces starvation-induced cell death through activation of autophagy and inhibition of mitochondrial apoptotic pathway.

The hepatitis B virus X protein (HBx) has been implicated in the development of hepatocellular carcinoma (HCC) associated with chronic infection. As a multifunctional protein, HBx regulates numerous cellular pathways, including autophagy. Although autophagy has been shown to participate in viral DNA replication and envelopment, it remains unclear whether HBx-activated autophagy affects host cell death, which is relevant to both viral pathogenicity and the development of HCC. Here, we showed that enforced expression of HBx can inhibit starvation-induced cell death in hepatic (L02 and Chang) or hepatoma (HepG2 and BEL-7404) cell lines. Starvation-induced cell death was greatly increased in HBX-expressing cell lines treated either with the autophagy inhibitor 3-methyladenine (3-MA) or with an siRNA directed against an autophagy gene, beclin 1. In contrast, treatment of cells with the apoptosis inhibitor Z-Vad-fmk significantly reduced cell death. Our results demonstrate that HBx-mediated cell survival during starvation is dependent on autophagy. We then further investigated the mechanisms of cell death inhibition by HBx. We found that HBx inhibited the activation of caspase-3, an execution caspase, blocked the release of mitochondrial apoptogenic factors, such as cytochrome c and apoptosis-inducing factor (AIF), and inhibited the activation of caspase-9 during starvation. These results demonstrate that HBx reduces cell death through inhibition of mitochondrial apoptotic pathways. Moreover, increased cell viability was also observed in HepG2.2.15 cells that replicate HBV and in cells transfected with HBV genomic DNA. Our findings demonstrate that HBx promotes cell survival during nutrient deprivation through inhibition of apoptosis and activation of autophagy. This highlights an important potential role of autophagy in HBV-infected hepatocytes growing under nutrient-deficient conditions.