Sivakumar Ramadoss

Histone demethylase KDM6B promotes epithelial-mesenchymal transition

Background: TGF-β induces EMT to regulate tumor invasion and metastasis. Results: TGF-β induces KDM6B and promotes EMT by activating SNAI1 via removing H3K27me3 marks. Conclusion: KDM6B is required for TGF-β-induced EMT and breast cancer cell invasion. Significance: Our findings highlight a novel epigenetic mechanism regulating EMT and cancer cell invasion and have potential implications in targeting metastatic breast cancer. Epithelial-mesenchymal transition (EMT) is a critical event that occurs in embryonic development, tissue repair control, organ fibrosis, and carcinoma invasion and metastasis. Although significant progress has been made in understanding the molecular regulation of EMT, little is known about how chromatin is modified in EMT. Chromatin modifications through histone acetylation and methylation determine the precise control of gene expression. Recently, histone demethylases were found to play important roles in gene expression through demethylating mono-, di-, or trimethylated lysines. KDM6B (also known as JMJD3) is a histone demethylase that might activate gene expression by removing repressive histone H3 lysine 27 trimethylation marks from chromatin. Here we report that KDM6B played a permissive role in TGF-β-induced EMT in mammary epithelial cells by stimulating SNAI1 expression. KDM6B was induced by TGF-β, and the knockdown of KDM6B inhibited EMT induced by TGF-β. Conversely, overexpression of KDM6B induced the expression of mesenchymal genes and promoted EMT. Chromatin immunoprecipitation (ChIP) assays revealed that KDM6B promoted SNAI1 expression by removing histone H3 lysine trimethylation marks. Consistently, our analysis of the Oncomine database found that KDM6B expression was significantly increased in invasive breast carcinoma compared with normal breast tissues. The knockdown of KDM6B significantly inhibited breast cancer cell invasion. Collectively, our study uncovers a novel epigenetic mechanism regulating EMT and tumor cell invasion, and has important implication in targeting cancer metastasis.

Targeting BMI1+ Cancer Stem Cells Overcomes Chemoresistance and Inhibits Metastases in Squamous Cell Carcinoma

Squamous cell carcinoma in the head and neck (HNSCC) is a common yet poorly understood cancer, with adverse clinical outcomes due to treatment resistance, recurrence, and metastasis. Putative cancer stem cells (CSCs) have been identified in HNSCC, and BMI1 expression has been linked to these phenotypes, but optimal treatment strategies to overcome chemotherapeutic resistance and eliminate metastases have not yet been identified. Here we show through lineage tracing and genetic ablation that BMI1+ CSCs mediate invasive growth and cervical lymph node metastasis in a mouse model of HNSCC. This model and primary human HNSCC samples contain highly tumorigenic, invasive, and cisplatin-resistant BMI1+ CSCs, which exhibit increased AP-1 activity that drives invasive growth and metastasis of HNSCC. Inhibiting AP-1 or BMI1 sensitized tumors to cisplatin-based chemotherapy, and it eliminated lymph node metastases by targeting CSCs and the tumor bulk, suggesting potential regimens to overcome resistance to treatments and eradicate HNSCC metastasis.

Transducin β-like protein 1 recruits nuclear factor κB to the target gene promoter for transcriptional activation.

ABSTRACT Nuclear factor κB (NF-κB) signaling controls a wide range of cellular functions such as tumor progression and invasion by inducing gene expression. Upon stimulation, NF-κB is translocated to the nucleus and binds to its target gene promoters to activate transcription by recruiting transcription coactivators. Although significant progress has been made in understanding NF-κB-mediated transactivation, little is known about how NF-κB is recruited to its target gene promoters. Here, we report that transducin β-like protein 1 (TBL1) controls the expression of NF-κB target genes by directly binding with NF-κB and facilitating its recruitment to target gene promoters. Tumor necrosis factor alpha stimulation triggered the formation of an NF-κB and TBL1 complex and subsequent target gene promoter binding. Knockdown of TBL1 impaired the recruitment of NF-κB to its target gene promoters. Interestingly, analysis of the Oncomine database revealed that TBL1 mRNA levels were significantly higher in invasive breast cancer tissues than in breast adenocarcinoma tissue. Consistently, TBL1 knockdown significantly reduced the invasive potential of breast cancer cells by inhibiting NF-κB. Our results reveal a new mechanism for the regulation of NF-κB activation, with important implications for the development of novel strategies for cancer therapy by targeting NF-κB.

Gestational Diabetes, Preeclampsia and Cytokine Release: Similarities and Differences in Endothelial Cell Function

Gestational diabetes, pre-eclampsia as well as intra-uterine infection during pregnancy affects the function of the endothelium both in the mother and the fetus leading to endothelial dysfunction. Gestational diabetes is also associated with an increased incidence of pre-eclampsia and it is likely that both the hyperglycemia as well as the release of cytokines especially TNFα during hyperglycemia may play an important role in the pathogenesis of endothelial dysfunction leading to preeclampsia. Similarly, some but not all studies have suggested that infection of the mother under certain circumstances can also lead to preeclampsia as women with either a bacterial or viral infection were at a higher risk of developing preeclampsia, compared to women without infection and infection also leads to a release in TNFα. Endothelial cells exposed to either high glucose or TNFα leads to an increase in the production of H2O2 and to a decrease in endothelial cell proliferation. The cellular and molecular mechanisms involved in this phenomenon are discussed.Gestational diabetes, pre-eclampsia as well as intra-uterine infection during pregnancy has profound effects on the fetus and long term effects on the neonate. All three conditions affect the function of the endothelium both in the mother and the fetus leading to endothelial dysfunction. Gestational diabetes is also associated with an increased incidence of pre-eclampsia and it is likely that both the hyperglycemia as well as the release of cytokines especially TNFα during hyperglycemia may play an important role in the pathogenesis of endothelial dysfunction leading to preeclampsia. It has also been suggested although not universally accepted that under certain circumstances maternal infection may also predispose to pre-eclampsia. Pre-eclampsia is also associated with the release of TNFα and endothelial dysfunction. However, the cellular and molecular mechanism(s) leading to the endothelial dysfunction by either hyperglycemia or by the cytokine TNFα appear to be different. In this chapter, we explore some of the similarities and differences leading to endothelial dysfunction by both hyperglycemia and by the inflammatory cytokine TNFα and the cellular and molecular mechanism(s) involved.

KDM3 epigenetically controls tumorigenic potentials of human colorectal cancer stem cells through Wnt/β-catenin signalling

Human colorectal cancer stem cells (CSCs) are tumour initiating cells that can self-renew and are highly tumorigenic and chemoresistant. While genetic mutations associated with human colorectal cancer development are well-known, little is known about how and whether epigenetic factors specifically contribute to the functional properties of human colorectal CSCs. Here we report that the KDM3 family of histone demethylases plays an important role in tumorigenic potential and survival of human colorectal CSCs by epigenetically activating Wnt target gene transcription. The depletion of KDM3 inhibits tumorigenic growth and chemoresistance of human colorectal CSCs. Mechanistically, KDM3 not only directly erases repressive H3K9me2 marks, but also helps to recruit histone methyltransferase MLL1 to promote H3K4 methylation, thereby promoting Wnt target gene transcription. Our results suggest that KDM3 is a critical epigenetic factor in Wnt signalling that orchestrates chromatin changes and transcription in human colorectal CSCs, identifying potential therapeutic targets for effective elimination of CSCs.

γ-Aminobutyric Acid Is Synthesized and Released by the Endothelium Potential Implications

RATIONALE Gamma aminobutyric acid (GABA), a neurotransmitter of the central nervous system, is found in the systemic circulation of humans at a concentration between 0.5 and 3 μmol/L. However, the potential source of circulating GABA and its significance on the vascular system remains unknown. We hypothesized that endothelial cells (ECs) may synthesize and release GABA to modulate some functions in the EC and after its release into the circulation. OBJECTIVE To assess whether GABA is synthesized and released by the EC and its potential functions. METHODS AND RESULTS Utilizing the human umbilical vein ECs and aortic ECs, we demonstrated for the first time that ECs synthesize and release GABA from [1-(14)C]glutamate. Localization of GABA and the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase in EC were confirmed by immunostaining and immunoblot analysis, respectively. The presence of GABA was further confirmed by immunohistochemistry in the EC lining the human coronary vessel. EC-derived GABA regulated the key mechanisms of ATP synthesis, fatty acid, and pyruvate oxidation in EC. GABA protected EC by inhibiting the reactive oxygen species generation and prevented monocyte adhesion by attenuating vascular cell adhesion molecule -1 and monocyte chemoattractant protein-1 expressions. GABA had no relaxing effect on rat aortic rings. GABA exhibited a dose-dependent fall in blood pressure. However, the fall in BP was abolished after pretreatment with pentolinium. CONCLUSIONS Our findings indicate novel potential functions of endothelium-derived GABA.

γ-Aminobutyric Acid Is Synthesized and Released by the EndotheliumNovelty and Significance

RATIONALE Gamma aminobutyric acid (GABA), a neurotransmitter of the central nervous system, is found in the systemic circulation of humans at a concentration between 0.5 and 3 μmol/L. However, the potential source of circulating GABA and its significance on the vascular system remains unknown. We hypothesized that endothelial cells (ECs) may synthesize and release GABA to modulate some functions in the EC and after its release into the circulation. OBJECTIVE To assess whether GABA is synthesized and released by the EC and its potential functions. METHODS AND RESULTS Utilizing the human umbilical vein ECs and aortic ECs, we demonstrated for the first time that ECs synthesize and release GABA from [1-(14)C]glutamate. Localization of GABA and the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase in EC were confirmed by immunostaining and immunoblot analysis, respectively. The presence of GABA was further confirmed by immunohistochemistry in the EC lining the human coronary vessel. EC-derived GABA regulated the key mechanisms of ATP synthesis, fatty acid, and pyruvate oxidation in EC. GABA protected EC by inhibiting the reactive oxygen species generation and prevented monocyte adhesion by attenuating vascular cell adhesion molecule -1 and monocyte chemoattractant protein-1 expressions. GABA had no relaxing effect on rat aortic rings. GABA exhibited a dose-dependent fall in blood pressure. However, the fall in BP was abolished after pretreatment with pentolinium. CONCLUSIONS Our findings indicate novel potential functions of endothelium-derived GABA.

Abstract 5143: KDM3A promotes cell growth and cisplatin-resistance in ovarian cancer cells

Background: Ovarian cancer is the most lethal gynecological cancer affecting the women worldwide. One of the main challenging hurdles in ovarian cancer treatment is the high recurrence rate due to therapeutic resistance. Recent evidences showed that JmjC-domain (JMJD) containing histone demethylases plays a critical role in controlling gene expression during normal development and cancer progression. Aims: To determine whether histone demethylase plays a role in ovarian cancer progression and therapeutic resistance. Methods: We developed cisplatin-resistance ovarian cancer cell lines SKOV-3 and OVCAR-5 and isolated total RNA to screen the expression pattern of JMJD family genes by real-time RT-PCR. We performed stable knock-down of KDM3A by lentiviral mediated ShRNA transfection and studied its effects on cell proliferation and apoptosis by MTT assay and western blot analysis (PARP, caspase-3), respectively. We also determined the effect of KDM3A knock-down on cell cycle and apoptosis regulators by western blot. Results: Our observations indicated the differential expression of various JMJD family proteins between cisplatin-resistance and sensitive cell lines. To further evaluate our results, we also tested the expression of those differentially expressed JMJD proteins in a widely used cisplatin-resistance and sensitive A2780 cell lines. We observed that KDM3A/JMJD1A is consistently overexpressed in all the cisplatin-resistance cell lines studied. Next, we stably knock-down KDM3A in cisplatin-resistance OVCAR-5 and A2780 cell lines. More interestingly, we found that KDM3A depletion inhibited cell proliferation, induced apoptosis and enhanced the sensitivity to cisplatin treatment. Further, we confirmed that the important regulators of cell cycle and apoptosis including p21 and Bcl-2 were modulated by KDM3A knock-down. Conclusions: Our results indicate that KDM3A is a critical epigenetic factor required for the ovarian cancer growth by promoting cell proliferation and cisplatin-resistance. Our findings might have important clinical implications in targeting ovarian cancer cells by using specific histone demethylase inhibitors. Grant support: This work was supported by Carl and Roberta Deutsch, and Kelly Day Foundations. Citation Format: Sivakumar Ramadoss, Suvajit Sen, Gautam Chaudhuri, Robin Farias-Eisner. KDM3A promotes cell growth and cisplatin-resistance in ovarian cancer cells. [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 5143. doi:10.1158/1538-7445.AM2014-5143

Chemoprotective and Chemosensitizing Effects of Nitric Oxide and Other Biologically Active Gases in Breast Cancer Chemotherapy: Potential Implications

Abstract Since the discovery in 1987 that vascular endothelial cells are able to synthesize nitric oxide (NO) from l -arginine, the existence of this biochemical pathway in many other cell types has been thoroughly documented, and its relevance in biology has become apparent. NO, a free-radical gas, synthesized by a family of isoenzymes called NO synthases, was shown to play a key role as a cell-signaling molecule in the vascular nervous and immune systems. It also exhibited a cytostatic/cytotoxic property at higher concentrations as generated by activated macrophages and endothelial cells. The role of NO in cancer biology and more so in breast cancer, however, started to be elucidated only in 1995 by Thomsen et al. (1995). In this chapter, we will review some of the key aspects of endogenous NO as produced in breast cancer cells and breast tumors. We will allude to the early studies that described NO production in breast tumor tissues and breast cancer cells and point to some of its proposed functions in breast cancer homeostasis. We will discuss how these findings could be of therapeutic benefit and finally end with some information on how a mechanistic cross talk between endogenously produced NO and another gaseous signaling molecule, hydrogen sulfide (H2S), in breast cancer cells may open up new opportunities for intervention. We will also discuss one potential mechanism of infiltration of tumor-associated macrophages in breast cancer and its clinical significance.

γ-Aminobutyric Acid Is Synthesized and Released by the EndotheliumNovelty and Significance: Potential Implications

RATIONALE Gamma aminobutyric acid (GABA), a neurotransmitter of the central nervous system, is found in the systemic circulation of humans at a concentration between 0.5 and 3 μmol/L. However, the potential source of circulating GABA and its significance on the vascular system remains unknown. We hypothesized that endothelial cells (ECs) may synthesize and release GABA to modulate some functions in the EC and after its release into the circulation. OBJECTIVE To assess whether GABA is synthesized and released by the EC and its potential functions. METHODS AND RESULTS Utilizing the human umbilical vein ECs and aortic ECs, we demonstrated for the first time that ECs synthesize and release GABA from [1-(14)C]glutamate. Localization of GABA and the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase in EC were confirmed by immunostaining and immunoblot analysis, respectively. The presence of GABA was further confirmed by immunohistochemistry in the EC lining the human coronary vessel. EC-derived GABA regulated the key mechanisms of ATP synthesis, fatty acid, and pyruvate oxidation in EC. GABA protected EC by inhibiting the reactive oxygen species generation and prevented monocyte adhesion by attenuating vascular cell adhesion molecule -1 and monocyte chemoattractant protein-1 expressions. GABA had no relaxing effect on rat aortic rings. GABA exhibited a dose-dependent fall in blood pressure. However, the fall in BP was abolished after pretreatment with pentolinium. CONCLUSIONS Our findings indicate novel potential functions of endothelium-derived GABA.