Histone lysine demethylase 4B regulates general and unique gene expression signatures in hypoxic cancer cells.

The hypoxic tumor microenvironment promotes tumor survival by inducing the expression of genes involved in angiogenesis and metastasis. As a direct target of hypoxia-inducible factor, lysine demethylase 4B (KDM4B) is overexpressed in multiple cancers, suggesting that a general KDM4B regulatory mechanism may exist in these cancer types. In this study, we sought to further investigate the general and unique roles of KDM4B in ovarian, colon, and renal cancer cells. We first identified a set of potential KDM4B targets shared by SKOV3ip.1, HCT116, and RCC4 cell lines, as well as numerous genes specifically regulated in each cell line. Through Gene Ontology, KEGG, and Oncobox pathway analyses, we found that KDM4B primarily regulated biosynthetic and cell cycle pathways in normoxia, whereas in hypoxia, it regulated pathways associated with inflammatory response and migration. TCGA data analyses reveal high expression of KDM4B in multiple cancer types and differential expression across cancer stages. Kaplan-Meier plots suggest that elevated KDM4B expression may contribute to a better or worse prognosis in a manner specific to each cancer type. Overall, our findings suggest that KDM4B plays complex roles in regulating multiple cancer processes, providing a useful resource for the future development of cancer therapies that target KDM4B expression.

The HIF target MAFF promotes tumor invasion and metastasis through IL11 and STAT3 signaling.

Hypoxia plays a critical role in tumor progression including invasion and metastasis. To determine critical genes regulated by hypoxia that promote invasion and metastasis, we screen fifty hypoxia inducible genes for their effects on invasion. In this study, we identify v-maf musculoaponeurotic fibrosarcoma oncogene homolog F (MAFF) as a potent regulator of tumor invasion without affecting cell viability. MAFF expression is elevated in metastatic breast cancer patients and is specifically correlated with hypoxic tumors. Combined ChIP- and RNA-sequencing identifies IL11 as a direct transcriptional target of the heterodimer between MAFF and BACH1, which leads to activation of STAT3 signaling. Inhibition of IL11 results in similar levels of metastatic suppression as inhibition of MAFF. This study demonstrates the oncogenic role of MAFF as an activator of the IL11/STAT3 pathways in breast cancer.

Collagen Remodeling in the Hypoxic Tumor-Mesothelial Niche Promotes Ovarian Cancer Metastasis.

Peritoneal metastases are the leading cause of morbidity and mortality in high-grade serous ovarian cancer (HGSOC). Accumulating evidence suggests that mesothelial cells are an important component of the metastatic microenvironment in HGSOC. However, the mechanisms by which mesothelial cells promote metastasis are unclear. Here, we report that the HGSOC tumor-mesothelial niche was hypoxic, and hypoxic signaling enhanced collagen I deposition by mesothelial cells. Specifically, hypoxic signaling increased expression of lysyl oxidase (LOX) in mesothelial and ovarian cancer cells to promote collagen crosslinking and tumor cell invasion. The mesothelial niche was enriched with fibrillar collagen in human and murine omental metastases. Pharmacologic inhibition of LOX reduced tumor burden and collagen remodeling in murine omental metastases. These findings highlight an important role for hypoxia and mesothelial cells in the modification of the extracellular matrix and tumor invasion in HGSOC. SIGNIFICANCE: This study identifies HIF/LOX signaling as a potential therapeutic target to inhibit collagen remodeling and tumor progression in HGSOC.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/9/2271/F1.large.jpg.

The dilemma of early preventive oophorectomy in familial small cell carcinoma of the ovary of hypercalcemic type.

•New pathogenic familial SMARCA4 variant, c.3081+1G>T.•Prophylactic surgery in healthy carrier of germline SMARCA4 mutation.•Long term hormone therapy in a 13-year-old girl.

KDM4B: A Nail for Every Hammer?

Epigenetic changes are well-established contributors to cancer progression and normal developmental processes. The reversible modification of histones plays a central role in regulating the nuclear processes of gene transcription, DNA replication, and DNA repair. The KDM4 family of Jumonj domain histone demethylases specifically target di- and tri-methylated lysine 9 on histone H3 (H3K9me3), removing a modification central to defining heterochromatin and gene repression. KDM4 enzymes are generally over-expressed in cancers, making them compelling targets for study and therapeutic inhibition. One of these family members, KDM4B, is especially interesting due to its regulation by multiple cellular stimuli, including DNA damage, steroid hormones, and hypoxia. In this review, we discuss what is known about the regulation of KDM4B in response to the cellular environment, and how this context-dependent expression may be translated into specific biological consequences in cancer and reproductive biology.

Licofelone Enhances the Efficacy of Paclitaxel in Ovarian Cancer by Reversing Drug Resistance and Tumor Stem-like Properties.

Drug development for first-line treatment of epithelial ovarian cancer (EOC) has been stagnant for almost three decades. Traditional cell culture methods for primary drug screening do not always accurately reflect clinical disease. To overcome this barrier, we grew a panel of EOC cell lines in three-dimensional (3D) cell cultures to form multicellular tumor spheroids (MCTS). We characterized these MCTS for molecular and cellular features of EOC and performed a comparative screen with cells grown using two-dimensional (2D) cell culture to identify previously unappreciated anticancer drugs. MCTS exhibited greater resistance to chemotherapeutic agents, showed signs of senescence and hypoxia, and expressed a number of stem cell-associated transcripts including ALDH1A and CD133, also known as PROM1 Using a library of clinically repurposed drugs, we identified candidates with preferential activity in MCTS over 2D cultured cells. One of the lead compounds, the dual COX/LOX inhibitor licofelone, reversed the stem-like properties of ovarian MCTS. Licofelone also synergized with paclitaxel in ovarian MCTS models and in a patient-derived tumor xenograft model. Importantly, the combination of licofelone with paclitaxel prolonged the median survival of mice (>141 days) relative to paclitaxel (115 days), licofelone (37 days), or vehicle (30 days). Increased efficacy was confirmed by Mantel-Haenszel HR compared with vehicle (HR = 0.037) and paclitaxel (HR = 0.017). These results identify for the first time an unappreciated, anti-inflammatory drug that can reverse chemotherapeutic resistance in ovarian cancer, highlighting the need to clinically evaluate licofelone in combination with first-line chemotherapy in primary and chemotherapy-refractory EOC.Significance: This study highlights the use of an in vitro spheroid 3D drug screening model to identify new therapeutic approaches to reverse chemotherapy resistance in ovarian cancer. Cancer Res; 78(15); 4370-85. ©2018 AACR.

KDM4A regulates HIF-1 levels through H3K9me3.

Regions of hypoxia (low oxygen) occur in most solid tumours and cells in these areas are the most aggressive and therapy resistant. In response to decreased oxygen, extensive changes in gene expression mediated by Hypoxia-Inducible Factors (HIFs) contribute significantly to the aggressive hypoxic tumour phenotype. In addition to HIFs, multiple histone demethylases are altered in their expression and activity, providing a secondary mechanism to extend the hypoxic signalling response. In this study, we demonstrate that the levels of HIF-1α are directly controlled by the repressive chromatin mark, H3K9me3. In conditions where the histone demethylase KDM4A is depleted or inactive, H3K9me3 accumulates at the HIF-1α locus, leading to a decrease in HIF-1α mRNA and a reduction in HIF-1α stabilisation. Loss of KDM4A in hypoxic conditions leads to a decreased HIF-1α mediated transcriptional response and correlates with a reduction in the characteristics associated with tumour aggressiveness, including invasion, migration, and oxygen consumption. The contribution of KDM4A to the regulation of HIF-1α is most robust in conditions of mild hypoxia. This suggests that KDM4A can enhance the function of HIF-1α by increasing the total available protein to counteract any residual activity of prolyl hydroxylases.

Genome-independent hypoxic repression of estrogen receptor alpha in breast cancer cells.

BACKGROUND: About 75-80% of breast tumors express the estrogen receptor alpha (ER-α) and are treated with endocrine-target therapeutics, making this the premier therapeutic modality in the breast cancer clinic. However, acquired resistance is common and about 20% of resistant tumors loose ER-α expression via unknown mechanisms. Inhibition of ER-α loss could improve endocrine therapeutic efficacy, benefiting a significant number of patients. Here we test whether tumor hypoxia might commonly produce ER-α loss. METHODS: Using standard molecular and cellular biological assays and a work station/incubator with controllable oxygen levels, we analyze the effects of hypoxia on ER-α protein, mRNA, and transcriptional activity in a panel of independently-derived ER-α positive cell lines. These lines were chosen to represent the diverse genetic backgrounds and mutations commonly present in ER-α positive tumors. Using shRNA-mediated knockdown and overexpression studies we also elucidate the role of hypoxia-inducible factor 1-alpha (HIF-1α) in the hypoxia-induced decrease in ER-α abundance. RESULTS: We present the first comprehensive overview of the effects of bona fide low environmental oxygen (hypoxia) and HIF-1α activity on ER-α abundance and transcriptional activity. We find that stabilized HIF-1α induces rapid loss of ER-α protein in all members of our diverse panel of breast cancer cell lines, which involves proteolysis rather than transcriptional repression. Reduced ER-α severely attenuates ER-α directed transcription, and inhibits cell proliferation without overt signs of cell death in the cell lines tested, despite their varying genomic backgrounds. CONCLUSIONS: These studies reveal a common hypoxia response that produces reduced ER-α expression and cell cycle stalling, and demonstrate a common role for HIF-1α in ER-α loss. We hypothesize that inhibitors of HIF-1α or the proteasome might stabilize ER-α expression in breast tumors in vivo, and work in combination with endocrine therapies to reduce resistance. Our data also suggests that disease re-occurrence in patients with ER-α positive tumors may arise from tumor cells chronically resident in hypoxic environments. We hypothesize that these non-proliferating cells may survive undetected until conditions change to oxygenate the environment, or cells eventually switch to proliferation via other signaling pathways.

KDM4B/JMJD2B is a p53 target gene that modulates the amplitude of p53 response after DNA damage.

The p53 tumor suppressor protein plays a critical role in orchestrating the genomic response to various stress signals by acting as a master transcriptional regulator. Differential gene activity is controlled by transcription factors but also dependent on the underlying chromatin structure, especially on covalent histone modifications. After screening different histone lysine methyltransferases and demethylases, we identified JMJD2B/KDM4B as a p53-inducible gene in response to DNA damage. p53 directly regulates JMJD2B gene expression by binding to a canonical p53-consensus motif in the JMJD2B promoter. JMJD2B induction attenuates the transcription of key p53 transcriptional targets including p21, PIG3 and PUMA, and this modulation is dependent on the catalytic capacity of JMJD2B. Conversely, JMJD2B silencing led to an enhancement of the DNA-damage driven induction of p21 and PIG3. These findings indicate that JMJD2B acts in an auto-regulatory loop by which p53, through JMJD2B activation, is able to influence its own transcriptional program. Functionally, exogenous expression of JMJD2B enhanced subcutaneous tumor growth of colon cancer cells in a p53-dependent manner, and genetic inhibition of JMJD2B impaired tumor growth in vivo. These studies provide new insights into the regulatory effect exerted by JMJD2B on tumor growth through the modulation of p53 target genes.

Epigenetic Therapy for Solid Tumors: Highlighting the Impact of Tumor Hypoxia.

In the last few decades, epigenetics has emerged as an exciting new field in development and disease, with a more recent focus towards cancer. Epigenetics has classically referred to heritable patterns of gene expression, primarily mediated through DNA methylation patterns. More recently, it has come to include the reversible chemical modification of histones and DNA that dictate gene expression patterns. Both the epigenetic up-regulation of oncogenes and downregulation of tumor suppressors have been shown to drive tumor development. Current clinical trials for cancer therapy include pharmacological inhibition of DNA methylation and histone deacetylation, with the aim of reversing these cancer-promoting epigenetic changes. However, the DNA methyltransferase and histone deacetylase inhibitors have met with less than promising results in the treatment of solid tumors. Regions of hypoxia are a common occurrence in solid tumors. Tumor hypoxia is associated with increased aggressiveness and therapy resistance, and importantly, hypoxic tumor cells have a distinct epigenetic profile. In this review, we provide a summary of the recent clinical trials using epigenetic drugs in solid tumors, discuss the hypoxia-induced epigenetic changes and highlight the importance of testing the epigenetic drugs for efficacy against the most aggressive hypoxic fraction of the tumor in future preclinical testing.

Hypoxic induction of AKAP12 variant 2 shifts PKA-mediated protein phosphorylation to enhance migration and metastasis of melanoma cells.

Scaffold proteins are critical hubs within cells that have the ability to modulate upstream signaling molecules and their downstream effectors to fine-tune biological responses. Although they can serve as focal points for association of signaling molecules and downstream pathways that regulate tumorigenesis, little is known about how the tumor microenvironment affects the expression and activity of scaffold proteins. This study demonstrates that hypoxia, a common element of solid tumors harboring low oxygen levels, regulates expression of a specific variant of the scaffold protein AKAP12 (A-kinase anchor protein 12), AKAP12v2, in metastatic melanoma. In turn, through a kinome-wide phosphoproteomic and MS study, we demonstrate that this scaffolding protein regulates a shift in protein kinase A (PKA)-mediated phosphorylation events under hypoxia, causing alterations in tumor cell invasion and migration in vitro, as well as metastasis in an in vivo orthotopic model of melanoma. Mechanistically, the shift in AKAP12-dependent PKA-mediated phosphorylations under hypoxia is due to changes in AKAP12 localization vs. structural differences between its two variants. Importantly, our work defines a mechanism through which a scaffold protein can be regulated by the tumor microenvironment and further explains how a tumor cell can coordinate many critical signaling pathways that are essential for tumor growth through one individual scaffolding protein.

Direct regulation of GAS6/AXL signaling by HIF promotes renal metastasis through SRC and MET.

Dysregulation of the von Hippel-Lindau/hypoxia-inducible transcription factor (HIF) signaling pathway promotes clear cell renal cell carcinoma (ccRCC) progression and metastasis. The protein kinase GAS6/AXL signaling pathway has recently been implicated as an essential mediator of metastasis and receptor tyrosine kinase crosstalk in cancer. Here we establish a molecular link between HIF stabilization and induction of AXL receptor expression in metastatic ccRCC. We found that HIF-1 and HIF-2 directly activate the expression of AXL by binding to the hypoxia-response element in the AXL proximal promoter. Importantly, genetic and therapeutic inactivation of AXL signaling in metastatic ccRCC cells reversed the invasive and metastatic phenotype in vivo. Furthermore, we define a pathway by which GAS6/AXL signaling uses lateral activation of the met proto-oncogene (MET) through SRC proto-oncogene nonreceptor tyrosine kinase to maximize cellular invasion. Clinically, AXL expression in primary tumors of ccRCC patients correlates with aggressive tumor behavior and patient lethality. These findings provide an alternative model for SRC and MET activation by growth arrest-specific 6 in ccRCC and identify AXL as a therapeutic target driving the aggressive phenotype in renal clear cell carcinoma.

AXL is an essential factor and therapeutic target for metastatic ovarian cancer.

The receptor tyrosine kinase AXL is thought to play a role in metastasis; however, the therapeutic efficacy of an AXL-targeting agent remains largely untested in metastatic disease. In this study, we defined AXL as a therapeutic target for metastatic ovarian cancer. AXL is primarily expressed in metastases and advanced-stage human ovarian tumors but not in normal ovarian epithelium. Genetic inhibition of AXL in human metastatic ovarian tumor cells is sufficient to prevent the initiation of metastatic disease in vivo. Mechanistically, inhibition of AXL signaling in animals with metastatic disease results in decreased invasion and matrix metalloproteinase activity. Most importantly, soluble human AXL receptors that imposed a specific blockade of the GAS6/AXL pathway had a profound inhibitory effect on progression of established metastatic ovarian cancer without normal tissue toxicity. These results offer the first genetic validation of GAS6/AXL targeting as an effective strategy for inhibition of metastatic tumor progression in vivo. Furthermore, this study defines the soluble AXL receptor as a therapeutic candidate agent for treatment of metastatic ovarian cancer, for which current therapies are ineffective.

Regulation of the histone demethylase JMJD1A by hypoxia-inducible factor 1 alpha enhances hypoxic gene expression and tumor growth.

The hypoxia-inducible transcription factors (HIFs) directly and indirectly mediate cellular adaptation to reduced oxygen tensions. Recent studies have shown that the histone demethylase genes JMJD1A, JMJD2B, and JARID1B are HIF targets, suggesting that HIFs indirectly influence gene expression at the level of histone methylation under hypoxia. In this study, we identify a subset of hypoxia-inducible genes that are dependent on JMJD1A in both renal cell and colon carcinoma cell lines. JMJD1A regulates the expression of adrenomedullin (ADM) and growth and differentiation factor 15 (GDF15) under hypoxia by decreasing promoter histone methylation. In addition, we demonstrate that loss of JMJD1A is sufficient to reduce tumor growth in vivo, demonstrating that histone demethylation plays a significant role in modulating growth within the tumor microenvironment. Thus, hypoxic regulation of JMJD1A acts as a signal amplifier to facilitate hypoxic gene expression, ultimately enhancing tumor growth.

The metastasis-associated gene Prl-3 is a p53 target involved in cell-cycle regulation.

The p53 tumor suppressor restricts tumorigenesis through the transcriptional activation of target genes involved in cell-cycle arrest and apoptosis. Here, we identify Prl-3 (phosphatase of regenerating liver-3) as a p53-inducible gene. Whereas previous studies implicated Prl-3 in metastasis because of its overexpression in metastatic human colorectal cancer and its ability to promote invasiveness and motility, we demonstrate here that Prl-3 is an important cell-cycle regulator. Consistent with a role in DNA damage-induced cell-cycle arrest, Prl-3 overexpression induces G(1) arrest downstream of p53 by triggering a PI3K-Akt-activated negative feedback loop. Surprisingly, attenuation of Prl-3 expression also elicits an arrest response, suggesting that basal level Prl-3 expression is pivotal for normal cell-cycle progression. Our findings highlight key dose-dependent functions of Prl-3 in both positive and negative regulation of cell-cycle progression and provide insight into Prl-3's role in cancer progression.

HIF gene expression in cancer therapy.

Tumor hypoxia is a feature common to almost all solid tumors due to malformed vasculature and inadequate perfusion. Tumor cells have evolved mechanisms that allow them to respond and adapt to a hypoxic microenvironment. The hypoxia-inducible transcription factor (HIF) family is comprised of oxygen-sensitive alpha (alpha) subunits that respond rapidly to decreased oxygen levels and oxygen-insensitive beta (beta) subunits. HIF binds to specific recognition sequences in the genome and increases the transcription of genes involved in a variety of metabolic and enzymatic pathways that are necessary for cells to respond to an oxygen-poor environment. The critical role of this family of transcriptional regulators in maintaining oxygen homeostasis is supported by multiple regulatory mechanisms that allow the cell to control the levels of HIF as well as its transcriptional activity. This review will focus on how the transcriptional activity of HIF is studied and how it can be exploited for cancer therapy.

Targeting the loss of the von Hippel-Lindau tumor suppressor gene in renal cell carcinoma cells.

Late-stage clear cell renal carcinoma poses a formidable clinical challenge due to the high mortality rate associated with this disease. Molecular and genetic studies have identified functional loss of the von Hippel-Lindau (VHL) gene as a frequent and crucial event in the development of the malignant phenotype of clear cell renal carcinomas. Loss of VHL function thus represents a pathognomonic molecular defect for therapeutic exploitation. The objective of this study was to evaluate the possibility of targeting VHL loss through pharmacologic means. Chromomycin A3 (ChA3) was identified through in silico analysis of existing publicly available drug profiles from the National Cancer Institute as an agent that seemed to selectively target VHL-deficient clear cell renal carcinoma cells. Genotype-selective toxicity was first determined through short-term viability assays and then confirmed with clonogenic studies. Coculture of fluorescently labeled VHL-deficient and VHL-positive cells showed discriminate killing of the VHL-deficient cells with ChA3. Mechanistically, overexpression of hypoxia-inducible factor (HIF)-2alpha in VHL-positive clear cell renal carcinoma cells phenocopied loss of VHL with respect to ChA3 toxicity, establishing ChA3 as a HIF-dependent cytotoxin. This study shows the feasibility of selectively targeting the loss of the VHL tumor suppressor gene in clear cell renal carcinoma for potential clinical benefit and may have greater ramifications in the development of new targeted therapies for the treatment of cancer and other genetic diseases.

Functional analysis of p53 binding under differential stresses.

Hypoxia and DNA damage stabilize the p53 protein, but the subsequent effect that each stress has on transcriptional regulation of known p53 target genes is variable. We have used chromatin immunoprecipitation followed by CpG island (CGI) microarray hybridization to identify promoters bound by p53 under both DNA-damaging and non-DNA-damaging conditions in HCT116 cells. Using gene-specific PCR analysis, we have verified an association with CGIs of the highest enrichment (> 2.5-fold) (REV3L, XPMC2H, HNRPUL1, TOR1AIP1, glutathione peroxidase 1, and SCFD2), with CGIs of intermediate enrichment (> 2.2-fold) (COX7A2L, SYVN1, and JAG2), and with CGIs of low enrichment (> 2.0-fold) (MYC and PCNA). We found little difference in promoter binding when p53 is stabilized by these two distinctly different stresses. However, expression of these genes varies a great deal: while a few genes exhibit classical induction with adriamycin, the majority of the genes are unchanged or are mildly repressed by either hypoxia or adriamycin. Further analysis using p53 mutated in the core DNA binding domain revealed that the interaction of p53 with CGIs may be occurring through both sequence-dependent and -independent mechanisms. Taken together, these experiments describe the identification of novel p53 target genes and the subsequent discovery of distinctly different expression phenomena for p53 target genes under different stress scenarios.

Checking in on hypoxia/reoxygenation.

Hypoxia/reoxygenation is a physiological stress that activates the DNA damage pathway. Significantly, this pathway is initiated during hypoxia, in the absence of detectable DNA damage. Our most recent study determined that during hypoxia, Chk 2 is phosphorylated in an ATM-dependent manner. In addition to this finding, we found that components of the MRN complex were not required for Chk 2 phosphorylation during hypoxia/reoxygenation. Once activated, Chk 2 initiates a signaling cascade, which induces a cell cycle arrest in the G2 phase. Loss of the Chk 2-mediated arrest correlated with an increase in sensitivity to hypoxia/reoxygenation. In contrast, loss of a p53-mediated reoxygenation-induced G1 arrest does not correlate with increased sensitivity to hypoxia/reoxygenation.

Genome-wide analysis of p53 under hypoxic conditions.

Hypoxia is an important nongenotoxic stress that modulates the tumor suppressor activity of p53 during malignant progression. In this study, we investigated how genotoxic and nongenotoxic stresses regulate p53 association with chromatin, p53 transcriptional activity, and p53-dependent apoptosis. We found that genotoxic and nongenotoxic stresses result in the accumulation and binding of the p53 tumor suppressor protein to the same cognate binding sites in chromatin. However, it is the stress that determines whether downstream signaling is mediated by association with transcriptional coactivators. In contrast to p53 induced by DNA-damaging agents, hypoxia-induced p53 has primarily transrepression activity. Using extensive microarray analysis, we identified families of repressed targets of p53 that are involved in cell signaling, DNA repair, cell cycle control, and differentiation. Following our previous study on the contribution of residues 25 and 26 to p53-dependent hypoxia-induced apoptosis, we found that residues 25-26 and 53-54 and the polyproline- and DNA-binding regions are also required for both gene repression and the induction of apoptosis by p53 during hypoxia. This study defines a new role for residues 53 and 54 of p53 in regulating transrepression and demonstrates that 25-26 and 53-54 work in the same pathway to induce apoptosis through gene repression.