Changhui Xue

HDAC4 Protein Regulates HIF1α Protein Lysine Acetylation and Cancer Cell Response to Hypoxia

Background: HIF1α is a target of anticancer therapy. Results: Lysines within the HIF1α N terminus are targets of HDAC4 deacetylation. HDAC4 inhibition causes the increase of HIF1α protein acetylation and decrease of protein stability, which lead to the reduction of HIF-1-mediated target gene expressions and activities in cancer cells. Conclusion: HDAC4 provides a novel HIF1α regulatory mechanism. Significance: HIF-1 can be targeted by HDAC4 inhibition. Hypoxia-inducible factor 1 α (HIF1α) is an essential part of the HIF-1 transcriptional complex that regulates angiogenesis, cellular metabolism, and cancer development. In von Hippel-Lindau (VHL)-null kidney cancer cell lines, we reported previously that HIF1α proteins can be acetylated and inhibited by histone deacetylase (HDAC) inhibitors or specific siRNA against HDAC4. To investigate the mechanism and biological consequence of the inhibition, we have generated stable HDAC4 knockdown via shRNA in VHL-positive normal and cancer cell lines. We report that HDAC4 regulates HIF1α protein acetylation and stability. Specifically, the HIF1α protein acetylation can be increased by HDAC4 shRNA and decreased by HDAC4 overexpression. HDAC4 shRNA inhibits HIF1α protein stability. In contrast, HDAC1 or HDAC3 shRNA has no such inhibitory effect. Mutations of the first five lysine residues (lysine 10, 11, 12, 19, and 21) to arginine within the HIF1α N terminus reduce protein acetylation but render the mutant HIF1α protein resistant to HDAC4 and HDACi-mediated inhibition. Functionally, in VHL-positive cancer cell lines, stable inhibition of HDAC4 decreases both the HIF-1 transcriptional activity and a subset of HIF-1 hypoxia target gene expression. On the cellular level, HDAC4 inhibition reduces the hypoxia-related increase of glycolysis and resistance to docetaxel chemotherapy. Taken together, the novel biological relationship between HDAC4 and HIF1α presented here suggests a potential role for the deacetylase enzyme in regulating HIF-1 cancer cell response to hypoxia and presents a more specific molecular target of inhibition.

HIF1α Protein Stability Is Increased by Acetylation at Lysine 709

Background: HIF1α and p300 are key components of HIF-1 transcription complex. Results: Lysine 709 of HIF1α is acetylated by p300, which increases protein stability and HIF-1 activity. Conclusion: p300 has a novel function in stabilizing HIF1α by Lys-709 acetylation. Significance: New insights in how HIF1α is post-translationally regulated by its cofactor to ensure HIF-1 activity. Lysine acetylation regulates protein stability and function. p300 is a component of the HIF-1 transcriptional complex and positively regulates the transactivation of HIF-1. Here, we show a novel molecular mechanism by which p300 facilitates HIF-1 activity. p300 increases HIF-1α (HIF1α) protein acetylation and stability. The regulation can be opposed by HDAC1, but not by HDAC3, and is abrogated by disrupting HIF1α-p300 interaction. Mechanistically, p300 specifically acetylates HIF1α at Lys-709, which increases the protein stability and decreases polyubiquitination in both normoxia and hypoxia. Compared with the wild-type protein, a HIF1α K709A mutant protein is more stable, less polyubiquitinated, and less dependent on p300. Overexpression of the HIF1α wild-type or K709A mutant in cancer cells lacking the endogenous HIF1α shows that the K709A mutant is transcriptionally more active toward the HIF-1 reporter and some endogenous target genes. Cancer cells containing the K709A mutant are less sensitive to hypoxia-induced growth arrest than the cells containing the HIF1α wild-type. Taken together, these data demonstrate a novel biological consequence upon HIF1α-p300 interaction, in which HIF1α can be stabilized by p300 via Lys-709 acetylation.

Malate dehydrogenase 2 confers docetaxel resistance via regulations of JNK signaling and oxidative metabolism

Resistance to chemotherapy represents a significant obstacle in prostate cancer therapeutics. Novel mechanistic understandings in cancer cell chemotherapeutic sensitivity and resistance can optimize treatment and improve patient outcome. Molecular alterations in the metabolic pathways are associated with cancer development; however, the role of these alterations in chemotherapy efficacy is largely unknown.

Identification of a Potent Inhibitor of CREB-Mediated Gene Transcription with Efficacious in Vivo Anticancer Activity.

Recent studies have shown that nuclear transcription factor cyclic adenosine monophosphate response element binding protein (CREB) is overexpressed in many different types of cancers. Therefore, CREB has been pursued as a novel cancer therapeutic target. Naphthol AS-E and its closely related derivatives have been shown to inhibit CREB-mediated gene transcription and cancer cell growth. Previously, we identified naphthamide 3a as a different chemotype to inhibit CREB’s transcription activity. In a continuing effort to discover more potent CREB inhibitors, a series of structural congeners of 3a was designed and synthesized. Biological evaluations of these compounds uncovered compound 3i (666-15) as a potent and selective inhibitor of CREB-mediated gene transcription (IC50 = 0.081 ± 0.04 μM). 666-15 also potently inhibited cancer cell growth without harming normal cells. In an in vivo MDA-MB-468 xenograft model, 666-15 completely suppressed the tumor growth without overt toxicity. These results further support the potential of CREB as a valuable cancer drug target.

Src and STAT3 inhibitors synergize to promote tumor inhibition in renal cell carcinoma

The intracytoplasmic tyrosine kinase Src serves both as a conduit and a regulator for multiple processes required for the proliferation and survival cancer cells. In some cancers, Src engages with receptor tyrosine kinases to mediate downstream signaling and in other cancers, it regulates gene expression. Src therefore represents a viable oncologic target. However, clinical responses to Src inhibitors, such as dasatinib have been disappointing to date. We identified Stat3 signaling as a potential bypass mechanism that enables renal cell carcinoma (RCC) cells to escape dasatinib treatment. Combined Src-Stat3 inhibition using dasatinib and CYT387 (a JAK/STAT inhibitor) synergistically reduced cell proliferation and increased apoptosis in RCC cells. Moreover, dasatinib and CYT387 combine to suppress YAP1, a transcriptional co-activator that promotes cell proliferation, survival and organ size. Importantly, this combination was well tolerated, and caused marked tumor inhibition in RCC xenografts. These results suggest that combination therapy with inhibitors of Stat3 signaling may be a useful therapeutic approach to increase the efficacy of Src inhibitors.

Functional regulation of hypoxia inducible factor-1α by SET9 lysine methyltransferase

HIF-1α is degraded by oxygen-dependent mechanisms but stabilized in hypoxia to form transcriptional complex HIF-1, which transactivates genes promoting cancer hallmarks. However, how HIF-1α is specifically regulated in hypoxia is poorly understood. Here, we report that the histone methyltransferase SET9 promotes HIF-1α protein stability in hypoxia and enhances HIF-1 mediated glycolytic gene transcription, thereby playing an important role in mediating cancer cell adaptation and survival to hypoxic stress. Specifically, SET9 interacts with HIF-1α and promotes HIF-1α protein stability in hypoxia. Silencing SET9 by siRNA reduces HIF-1α protein stability in hypoxia, and attenuates the hypoxic induction of HIF-1 target genes mediating hypoxic glycolysis. Mechanistically, we find that SET9 is enriched at the hypoxia response elements (HRE) within promoters of the HIF-1-responsive glycolytic genes. Silencing SET9 reduces HIF-1α levels at these HREs in hypoxia, thereby attenuating HIF-1-mediated gene transcription. Further, silencing SET9 by siRNA reduces hypoxia-induced glycolysis and inhibits cell viability of hypoxic cancer cells. Our findings suggest that SET9 enriches at HRE sites of HIF-1 responsive glycolytic genes and stabilizes HIF-1α at these sites in hypoxia, thus establishes an epigenetic mechanism of the metabolic adaptation in hypoxic cancer cells.

Systemic Inhibition of CREB is Well-tolerated in vivo

cAMP-response element binding protein (CREB) is a nuclear transcription factor activated by multiple extracellular signals including growth factors and hormones. These extracellular cues activate CREB through phosphorylation at Ser133 by various protein serine/threonine kinases. Once phosphorylated, it promotes its association with transcription coactivators CREB-binding protein (CBP) and its paralog p300 to activate CREB-dependent gene transcription. Tumor tissues of different origins have been shown to present overexpression and/or overactivation of CREB, indicating CREB as a potential cancer drug target. We previously identified 666-15 as a potent inhibitor of CREB with efficacious anti-cancer activity both in vitro and in vivo. Herein, we investigated the specificity of 666-15 and evaluated its potential in vivo toxicity. We found that 666-15 was fairly selective in inhibiting CREB. 666-15 was also found to be readily bioavailable to achieve pharmacologically relevant concentrations for CREB inhibition. Furthermore, the mice treated with 666-15 showed no evidence of changes in body weight, complete blood count, blood chemistry profile, cardiac contractility and tissue histologies from liver, kidney and heart. For the first time, these results demonstrate that pharmacological inhibition of CREB is well-tolerated in vivo and indicate that such inhibitors should be promising cancer therapeutics.

Metabolic reprogramming ensures cancer cell survival despite oncogenic signaling blockade

There is limited knowledge about the metabolic reprogramming induced by cancer therapies and how this contributes to therapeutic resistance. Here we show that although inhibition of PI3K-AKT-mTOR signaling markedly decreased glycolysis and restrained tumor growth, these signaling and metabolic restrictions triggered autophagy, which supplied the metabolites required for the maintenance of mitochondrial respiration and redox homeostasis. Specifically, we found that survival of cancer cells was critically dependent on phospholipase A2 (PLA2) to mobilize lysophospholipids and free fatty acids to sustain fatty acid oxidation and oxidative phosphorylation. Consistent with this, we observed significantly increased lipid droplets, with subsequent mobilization to mitochondria. These changes were abrogated in cells deficient for the essential autophagy gene ATG5 Accordingly, inhibition of PLA2 significantly decreased lipid droplets, decreased oxidative phosphorylation, and increased apoptosis. Together, these results describe how treatment-induced autophagy provides nutrients for cancer cell survival and identifies novel cotreatment strategies to override this survival advantage.

Abstract 458: Regulation of HIF1a by Set9 lysine methyltransferase

HIF-1 is consisted of an oxygen-sensitive HIF-1α and a constitutive HIF-1β subunit. The activity of HIF-1 is implicated in human cancer development. In normoxia, the HIF-1α subunit is constantly synthesized, but rapidly degraded by the oxygen-dependent mechanism. In hypoxia due to tumor growth and anti-angiogenic therapy, HIF-1α is stabilized and dimerizes with HIF-1β to form HIF-1, which transactivates genes promoting cancer hallmarks, including angiogenesis, metabolic reprogramming, migration and invasion. Notably, HIF-1 increases multiple genes on the glycolytic pathway, which mediates the metabolic adaptation of hypoxic stress. Therefore, HIF-1α is an intriguing anticancer target. However, how HIF-1α is regulated in hypoxia is poorly understood. Here, we report that the histone methyltransferase Set9 promotes HIF-1α protein stability in hypoxia and plays an important role in cancer cell glycolytic adaptation. Specifically, Set9 interacts with HIF-1α to maintain its stability in hypoxia. Silencing of Set9 by siRNA in hypoxia significantly reduces HIF-1α protein level, reduces HIF-1 transcriptional activity, and attenuates expressions of HIF-1-responsive glycolytic genes as well as hypoxia-induced glycolysis. Further, we have found that Set9 binds to the chromatins of hypoxia response elements within HIF-1-responsive glycolytic genes. The results suggest that Set9 epigenetically contributes to the glycolytic phenotype of cancer through HIF-1. Citation Format: Qiong Liu, Hao Geng, Changhui Xue, Tomasz M. Beer, David Z. Qian. Regulation of HIF1a by Set9 lysine methyltransferase. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 458. doi:10.1158/1538-7445.AM2014-458

Abstract P4-15-08: Discovery of a potent inhibitor of CREB-mediated gene transcription that completely suppresses the growth of triple-negative breast cancer cells

Background: Breast cancers are a heterogeneous group of diseases with distinct and complex mechanisms of pathogenesis. Triple-negative breast cancers (TNBC) form a subgroup of breast cancers with poor prognosis. TNBCs lack the expression of estrogen receptor (ER), progesterone receptor (PR) or HER2 and no targeted therapies exist. The cyclic-AMP (cAMP) response element binding protein (CREB) is a stimulus-induced transcription factor activated by multiple extracellular signals through phosphorylation. The transcription activity of CREB depends on its phosphorylation at Ser133 by mitogen- and stress-activated protein serine/threonine kinases, which are often dysregulated in TNBCs. The phosphorylated CREB (p-CREB) can then bind the mammalian transcription co-activator, CREB-binding protein (CBP), via the kinase-inducible domain (KID) in CREB and KID-interacting (KIX) domain in CBP. This binding event will further recruit other transcriptional machinery to the gene promoter to initiate CREB-dependent gene transcription. CREB is overexpressed in breast cancer tissues compared to normal mammary tissues and the level of expression inversely correlates with disease-free survival. Genetic studies have shown that inhibition of CREB9s activity leads to decreased breast cancer cell proliferation. We describe here the discovery of a potent small molecule inhibitor of CREB-mediated gene transcription with in vitro and in vivo activity in TNBCs. Methods: The small molecule inhibitor (compound 1) was reporter earlier by us (Li and Xiao ChemBioChem 2009). Starting from this lead compound, we designed and synthesized a more potent compound 2. The in vitro antiproliferative activity and apoptosis induction were evaluated in MDA-MB-231 and MDA-MB-468 cells with MTT assays and flow cytometry, respectively. In vivo activity was investigated in a human xenograft model of MDA-MB-468. The CREB target gene expression was investigated by Western blot and qRT-PCR analysis. Results: Starting from compound 1, which is a low micromolar and cell-permeable inhibitor of CREB-mediated gene transcription, we designed and synthesized compound 2. Compound 2 potently inhibited CREB-mediated gene transcription with IC50 ∼80 nM. In vitro, this compound inhibited proliferation of MDA-MB-231 and MDA-MB-468 cells with concomitant activation of apoptosis and necrosis. In contrast, compound 2 was not toxic to normal human mammary epithelial cells. In vivo, compound 2 completely suppressed the growth of MDA-MB-468 cells at a dose not toxic to the mice. Conclusion: Compound 2 potently inhibited CREB-mediated gene transcription and TNBC cell growth in vitro and in vivo. These results suggest that CREB is a promising druggable target for TNBCs. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-15-08.