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

Averages and standard deviations of band intensities calculated for all repeats of each western blot in Fig. 2a. Specific band intensities normalized to the loading control bands (β-actin). Calculations derived from at least three independent experiments. (DOCX 17 kb

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

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

63 Chemical inhibition of SWI/SNF induces transformed growth in non-mutant cells, mimicking the effects of a SMARCA4 mutation associated with small cell carcinoma of the ovary, hypercalcemic type

Objectives: Using a chemical inhibitor of the SWI/SNF complex (BRM014), we sought to determine if non-cancerous cells harboring wild-type SMARCA4 would spontaneously transform during in vitro culture, thereby providing a new model system for studying the formation of SCCOHT. Methods: Ovarian cells derived from a benign ovarian cyst removal (P583) were serially passaged in media containing BRM014, an inhibitor of the ATPase domains of SMARCA4 and SMARCA2. Cells were interrogated for their ability to acquire characteristics of cellular transformation. Cells were grown for multiple passages to determine if cells would spontaneously immortalize. Loss of contact-dependent inhibition was interrogated with focus-forming assays of confluent monolayers. The ability of cells to escape anoikis and grow independently as spheroids was interrogated in attachment-free growth conditions. All experiments were compared to experiments conducted with an age-matched cell population derived from a familial carrier of a SCCOHT-associated mutation in the SMARCA4 gene (P590, SMARCA4. c.3081+1G>T, prophylactic oophorectomy). Results: P583 cells that were treated with BRM014 formed markedly larger spheroids in attachment-free conditions compared to vehicle control-treated cells. BRM014-treated cells also formed foci in confluent monolayers, while none formed in vehicle control-treated cells. These results phenocopied the growth properties of P590 cells, indicating that inhibition of SMARCA4 and SMARCA2 could mimic the effects of SMARCA4 mutation. Of particular interest was the persistence of treatment: After as little as 48 hours of treatment, cells retained the ability to grow as spheroids for at least 7 days following removal of BRM014. Conclusions: Through the use of a chemical inhibitor of SMARCA4 and SMARCA2, non-mutant ovarian cells were induced to grow in a manner consistent with early stages of transformation, similar to cells harboring a SMARCA4 mutation associated with familial SCCOHT. Our data suggest that through the use of BRM014, multiple pseudo-transformed cell populations can be formed from diverse, non-mutant patient samples, providing robust cellular tool for studying the early stages of SCCOHT formation

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

Regulation of the Histone Demethylase JMJD1A by Hypoxia-Inducible Factor 1α 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

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

KDM4A regulates HIF-1 levels through H3K9me3

Abstract 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