KRCC1, a modulator of the DNA damage response.

The lysine-rich coiled-coil 1 (KRCC1) protein is overexpressed in multiple malignancies, including ovarian cancer, and overexpression correlates with poor overall survival. Despite a potential role in cancer progression, the biology of KRCC1 remains elusive. Here, we characterize the biology of KRCC1 and define its role in the DNA damage response and in cell cycle progression. We demonstrate that KRCC1 associates with the checkpoint kinase 1 (CHK1) upon DNA damage and regulates the CHK1-mediated checkpoint. KRCC1 facilitates RAD51 recombinase foci formation and augments homologous recombination repair. Furthermore, KRCC1 is required for proper S-phase progression and subsequent mitotic entry. Our findings uncover a novel component of the DNA damage response and a potential link between cell cycle, associated damage response and DNA repair.

MicroRNA-195 controls MICU1 expression and tumor growth in ovarian cancer.

MICU1 is a mitochondrial inner membrane protein that inhibits mitochondrial calcium entry; elevated MICU1 expression is characteristic of many cancers, including ovarian cancer. MICU1 induces both glycolysis and chemoresistance and is associated with poor clinical outcomes. However, there are currently no available interventions to normalize aberrant MICU1 expression. Here, we demonstrate that microRNA-195-5p (miR-195) directly targets the 3' UTR of the MICU1 mRNA and represses MICU1 expression. Additionally, miR-195 is under-expressed in ovarian cancer cell lines, and restoring miR-195 expression reestablishes native MICU1 levels and the associated phenotypes. Stable expression of miR-195 in a human xenograft model of ovarian cancer significantly reduces tumor growth, increases tumor doubling times, and enhances overall survival. In conclusion, miR-195 controls MICU1 levels in ovarian cancer and could be exploited to normalize aberrant MICU1 expression, thus reversing both glycolysis and chemoresistance and consequently improving patient outcomes.

Cystathionine beta synthase regulates mitochondrial dynamics and function in endothelial cells.

Mutations in the human cystathionine beta synthase (CBS) gene are known to cause endothelial dysfunction responsible for cardiovascular and neurovascular diseases. CBS is the predominant hydrogen sulfide (H2 S)-producing enzyme in endothelial cells (ECs). Recently, H2 S was shown to attenuate ROS and improve mitochondrial function. Mitochondria are metabolic organelles that actively transform their ultrastructure to mediate their function. Therefore, we questioned whether perturbation of CBS/H2 S activity could drive mitochondrial dysfunction via mitochondrial dynamics in ECs. Here we demonstrate that silencing CBS induces mitochondria fragmentation, attenuates efficient oxidative phosphorylation, and decreases EC function. Mechanistically, CBS silencing significantly elevates ROS production, thereby leading to reduced mitofusin 2 (MFN2) expression, decouple endoplasmic reticulum-mitochondria contacts, increased mitochondria fission, enhanced receptor-mediated mitophagy, and increased EC death. These defects were significantly rescued by the treatment of H2 S donors. Taken together our data highlights a novel signaling axis that mechanistically links CBS with mitochondrial function and ER-mitochondrial tethering and could be considered as a new therapeutic approach for the intervention of EC dysfunction-related pathologies.

KRCC1: A potential therapeutic target in ovarian cancer.

Using a systems biology approach to prioritize potential points of intervention in ovarian cancer, we identified the lysine rich coiled-coil 1 (KRCC1), as a potential target. High-grade serous ovarian cancer patient tumors and cells express significantly higher levels of KRCC1 which correlates with poor overall survival and chemoresistance. We demonstrate that KRCC1 is predominantly present in the chromatin-bound nuclear fraction, interacts with HDAC1, HDAC2, and with the serine-threonine phosphatase PP1CC. Silencing KRCC1 inhibits cellular plasticity, invasive properties, and potentiates apoptosis resulting in reduced tumor growth. These phenotypes are associated with increased acetylation of histones and with increased phosphorylation of H2AX and CHK1, suggesting the modulation of transcription and DNA damage that may be mediated by the action of HDAC and PP1CC, respectively. Hence, we address an urgent need to develop new targets in cancer.

MDR1 mediated chemoresistance: BMI1 and TIP60 in action.

Chemotherapy-induced emergence of drug resistant cells is frequently observed and is exemplified by the expression of family of drug resistance proteins including, multidrug resistance protein 1 (MDR1). However, a concise mechanism for chemotherapy-induced MDR1 expression is unclear. Mechanistically, mutational selection, epigenetic alteration, activation of the Wnt pathway or impaired p53 function have been implicated. The present study describes that the surviving fraction of cisplatin resistant cells co- upregulate MDR1, BMI1 and acetyl transferase activity of TIP60. Using complementary gain and loss of function approaches, we demonstrate that the expression of MDR1 is positively regulated by BMI1, a stem-cell factor classically known as a transcriptional repressor. Our study establishes a functional interaction between TIP60 and BMI-1 resulting in upregulation of MDR1 expression. Chromatin immunoprecipitation (ChIP) assays further establish that the proximal MDR1 promoter responds to cisplatin in a BMI1 dependent manner. BMI1 interacts with a cluster of E-box elements on the MDR1 promoter and recruits TIP60 resulting in acetylation of histone H2A and H3. Collectively, our data establish a hitherto unknown liaison among MDR1, BMI1 and TIP60 and provide mechanistic insights into cisplatin-induced MDR1 expression resulting in acquired cross-resistance against paclitaxel, doxorubicin and likely other drugs. In conclusion, our results advocate utilizing anti-BMI1 strategies to alleviate acquired resistance to chemotherapy.

BMI1, a new target of CK2α.

BACKGROUND: The polycomb group protein, BMI1 plays important roles in chromatin modification, stem cell function, DNA damage repair and mitochondrial bioenergetics. Such diverse cellular functions of BMI1 could be, in part, due to post-translational modifications, especially phosphorylation. To date, AKT has been reported as a kinase that by site specific phosphorylation of BMI1 modulates its oncogenic functions. METHODS: Immunoprecipitation in conjunction with kinase assay and mass spectrometry was used to determine association with and site specific phosphorylation of BMI1 by CK2α. Functional implications of the BMI1/CK2α axis was examined in cancer cells utilizing siRNA and exogenous gene expression followed by biochemical and phenotypic studies. Correlations between expression of CK2α and BMI1 were determined from cell lines and formalin fixed paraffin embedded tissues representing the normal fallopian tube epithelium and high grade serous ovarian cancer samples. RESULTS: Here we report that CK2α, a nuclear serine threonine kinase, phosphorylates BMI1 at Serine 110 as determined by in-vitro/ex-vivo kinase assay and mass spectrometry. In ovarian cancer cell lines, expression of CK2α correlated with the phospho-species, as well as basal BMI1 levels. Preventing phosphorylation of BMI1 at Serine 110 significantly decreased half-life and stability of the protein. Additionally, re-expression of the phosphorylatable but not non-phosphorylatable BMI1 rescued clonal growth in endogenous BMI1 silenced cancer cells leading us to speculate that CK2α-mediated phosphorylation stabilizes BMI1 and promotes its oncogenic function. Clinically, compared to normal fallopian tube epithelial tissues, the expression of both BMI1 and CK2α were significantly higher in tumor tissues obtained from high-grade serous ovarian cancer patients. Among tumor samples, the expression of BMI1 and CK2α positively correlated (Spearman coefficient = 0.62, P = 0.0021) with each other. CONCLUSION: Taken together, our findings establish an important regulatory role of CK2α on BMI1 phosphorylation and stability and implicate the CK2α/BMI1 axis in ovarian cancer.

Mitochondrial BMI1 maintains bioenergetic homeostasis in cells.

The polycomb complex proto-oncogene BMI1 [B lymphoma Mo-MLV insertion region 1 homolog (mouse)] is essential for self-renewal of normal and cancer stem cells. BMI1-null mice show severe defects in growth, development, and survival. Although BMI1 is known to exert its effect in the nucleus via repression of 2 potent cell-cycle regulators that are encoded by the Ink4a/Arf locus, deletion of this locus only partially rescues BMI1-null phenotypes, which is indicative of alternate mechanisms of action of BMI1. Here, we show that an extranuclear pool of BMI1 localizes to inner mitochondrial membrane and directly regulates mitochondrial RNA (mtRNA) homeostasis and bioenergetics. These mitochondrial functions of BMI1 are independent of its previously described nuclear functions because a nuclear localization-defective mutant BMI1 rescued several bioenergetic defects that we observed in BMI1-depleted cells, for example, mitochondrial respiration, cytochrome c oxidase activity, and ATP production. Mechanistically, BMI1 coprecipitated with polynucleotide phosphorylase, a ribonuclease that is responsible for decay of mtRNA transcripts. Loss of BMI1 enhanced ribonuclease activity of polynucleotide phosphorylase and reduced mtRNA stability. These findings not only establish a novel extranuclear role of BMI1 in the regulation of mitochondrial bioenergetics, but also provide new mechanistic insights into the role of this proto-oncogene in stem cell differentiation, neuronal aging, and cancer.-Banerjee Mustafi, S., Aznar, N., Dwivedi, S. K. D., Chakraborty, P. K., Basak, R., Mukherjee, P., Ghosh, P., Bhattacharya, R. Mitochondrial BMI1 maintains bioenergetic homeostasis in cells.

Cystathionine ß-synthase regulates endothelial function via protein S-sulfhydration.

Deficiencies of the human cystathionine ß-synthase (CBS) enzyme are characterized by a plethora of vascular disorders and hyperhomocysteinemia. However, several clinical trials demonstrated that despite reduction in homocysteine levels, disease outcome remained unaffected, thus the mechanism of endothelial dysfunction is poorly defined. Here, we show that the loss of CBS function in endothelial cells (ECs) leads to a significant down-regulation of cellular hydrogen sulfide (H2S) by 50% and of glutathione (GSH) by 40%. Silencing CBS in ECs compromised phenotypic and signaling responses to the VEGF that were potentiated by decreased transcription of VEGF receptor (VEGFR)-2 and neuropilin (NRP)-1, the primary receptors regulating endothelial function. Transcriptional down-regulation of VEGFR-2 and NRP-1 was mediated by a lack in stability of the transcription factor specificity protein 1 (Sp1), which is a sulfhydration target of H2S at residues Cys68 and Cys755. Reinstating H2S but not GSH in CBS-silenced ECs restored Sp1 levels and its binding to the VEGFR-2 promoter and VEGFR-2, NRP-1 expression, VEGF-dependent proliferation, and migration phenotypes. Thus, our study emphasizes the importance of CBS-mediated protein S-sulfhydration in maintaining vascular health and function.-Saha, S., Chakraborty, P. K., Xiong, X., Dwivedi, S. K. D., Mustafi, S. B., Leigh, N. R., Ramchandran, R., Mukherjee, P., Bhattacharya, R. Cystathionine ß-synthase regulates endothelial function via protein S-sulfhydration.

VEGF signaling: Role in angiogenesis and beyond.

Angiogenesis is a crucial process for tissue development, repair, and tumor survival. Vascular endothelial growth factor (VEGF) is a key driver secreted by cancer cells, promoting neovascularization. While VEGF's role in angiogenesis is well-documented, its influence on the other aspects in tumor microenvironemt is less discussed. This review elaborates on VEGF's impact on intercellular interactions within the tumor microenvironment, including how VEGF affects pericyte proliferation and migration and mediates interactions between tumor-associated macrophages and cancer cells, resulting in PDL-1-mediated immunosuppression and Nrf2-mediated epithelial-mesenchymal transition. The review discusses VEGF's involvement in intra-organelle crosstalk, tumor metabolism, stemness, and epithelial-mesenchymal transition. It also provides insights into current anti-VEGF therapies and their limitations in cancer treatment. Overall, this review aims to provide a thorough overview of the current state of knowledge concerning VEGF signaling and its impact, not only on angiogenesis but also on various other oncogenic processes.

Secrets of DNA-PKcs beyond DNA repair.

The canonical role of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) in repairing DNA double-strand breaks combined with its reported dysregulation in several malignancies has driven the development of DNA-PKcs inhibitors as therapeutics. However, until recently the relationship between DNA-PKcs and tumorigenesis has been primarily investigated with regard to its role in non-homologous end joining (NHEJ) repair. Emerging research has uncovered non-canonical DNA-PKcs functions involved with transcriptional regulation, telomere maintenance, metabolic regulation, and immune signaling all of which may also impinge on tumorigenesis. This review mainly discusses these non-canonical roles of DNA-PKcs in cellular biology and their potential contribution to tumorigenesis, as well as evaluating the implications of targeting DNA-PKcs for cancer therapy.

Development of a novel PCR restriction analysis of the hsp65 gene as a rapid method to screen for the Mycobacterium tuberculosis complex and nontuberculous mycobacteria in high-burden countries.

The limitations of conventional methods of identification of Mycobacterium tuberculosis have led to the development of several nucleic acid amplification techniques which have the advantage of being rapid, sensitive, and specific. However, their expense or the need for technical expertise makes it difficult to use them in regions in which tuberculosis is endemic. A novel PCR restriction analysis (PRA) of the hsp65 gene was therefore developed for rapid screening of clinical isolates to identify Mycobacterium spp. The restriction enzymes NruI and BamHI were selected to obtain a limited number of restriction patterns to further differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). Three hundred ten isolates from clinical specimens and 24 reference strains were tested. The assay correctly identified 295 of the 310 culture isolates as MTBC, while the remaining 15 isolates were identified as NTM. Of the isolates tested, 135 MTBC strains and all 15 NTM were also confirmed by PRA using Sau96I and CfoI. Thirty-eight randomly selected MTBC strains and all 15 NTM were further confirmed by sequencing. The NruI/BamHI PRA was simple, as it did not require any elaborate analyses. It was cost-effective, rapid, highly sensitive, and specific and did not require technical expertise. The assay can, therefore, be used as a simple screening test not only to detect Mycobacterium spp. but also to differentiate MTBC from NTM in peripheral laboratories with minimal availability of funds.

Amino acids derived benzoxazepines: design, synthesis and antitumor activity.

Two series of new benzoxazepines substituted with different alkyl amino ethyl chains were synthesized comprising synthetic steps of inter and intramolecular Mitsunobu reaction, lithium aluminium hydride (LAH) reduction, debenzylation, bimolecular nucleophilic substitution (SN2) reaction. The present study investigates the effect of a tyrosine-based benzoxazepine derivative in human breast cancer cells MCF-7 and MDA-MB-231 and in breast cancer animal model. The anti-proliferative effect of 15a on MCF-7 cells was associated with G1 cell-cycle arrest. This G1 growth arrest was followed by apoptosis as 15a dose dependently increased phosphatidylserine exposure, PARP cleavage and DNA fragmentation that are hallmarks of apoptotic cell death. Interestingly, 15a activated components of both intrinsic and extrinsic pathways of apoptosis characterized by activation of caspase-8 and -9, mitochondrial membrane depolarization and increase in Bax/Bcl2 ratio. However, use of selective caspase inhibitors revealed that the caspase-8-dependent pathway is the major contributor to 15a-induced apoptosis. Compound 15a also significantly reduced the growth of MCF-7 xenograft tumors in athymic nude mice. Together, 15a could serve as a base for the development of a new group of effective breast cancer therapeutics.

A role for the cystathionine-ß-synthase /H2S axis in astrocyte dysfunction in the aging brain.

Astrocytic dysfunction is central to age-related neurodegenerative diseases. However, the mechanisms leading to astrocytic dysfunction are not well understood. We identify that among the diverse cellular constituents of the brain, murine and human astrocytes are enriched in the expression of CBS. Depleting CBS in astrocytes causes mitochondrial dysfunction, increases the production of reactive oxygen species (ROS) and decreases cellular bioenergetics that can be partially rescued by exogenous H2S supplementation or by re-expressing CBS. Conversely, the CBS/H2S axis, associated protein persulfidation and proliferation are decreased in astrocytes upon oxidative stress which can be rescued by exogenous H2S supplementation. Here we reveal that in the aging brain, the CBS/H2S axis is downregulated leading to decreased protein persulfidation, together augmenting oxidative stress. Our findings uncover an important protective role of the CBS/H2S axis in astrocytes that may be disrupted in the aged brain.

The deubiquitinase USP10 protects pancreatic cancer cells from endoplasmic reticulum stress.

The ubiquitin-specific peptidase 10 (USP10) plays a context-specific, pro or anti-tumorigenic role in different malignancies. However, the role of USP10 in pancreatic cancer remains unclear. Our protein and RNA level analysis from archived specimens and public databases show that USP10 is overexpressed in pancreatic ductal adenocarcinoma (PDAC) and expression correlates with poor overall patient survival. Phenotypically, silencing USP10 decreased viability, clonal growth and invasive properties of pancreatic cancer cells. Mechanistically, silencing USP10 upregulated BiP and induced endoplasmic reticulum (ER) stress that led to an unfolded protein response (UPR) and upregulation of PERK, IRE1α. Decreased cell viability of USP10 silenced cells could be rescued by a chemical chaperone that promotes protein folding. Our studies suggest that USP10 by protecting pancreatic cancer cells from ER stress may support tumor progression.

Gold Nanoparticles Disrupt the IGFBP2/mTOR/PTEN Axis to Inhibit Ovarian Cancer Growth.

By exploiting the self-therapeutic properties of gold nanoparticles (GNPs) a molecular axis that promotes the growth of high-grade serous ovarian cancer (HGSOC), one of the deadliest gynecologic malignancies with poorly understood underlying molecular mechanisms, has been identified. The biodistribution and toxicity of GNPs administered by intravenous or intraperitoneal injection, both as a single dose or by repeated dosing over two weeks are first assessed; no biochemical or histological toxicity to vital organs is found. Using an orthotopic patient-derived xenograft (PDX) model of HGSOC, the authors then show that GNP treatment robustly inhibits tumor growth. Investigating the molecular mechanisms underlying the GNP efficacy reveals that GNPs downregulate insulin growth factor binding protein 2 (IGFBP2) by disrupting its autoregulation via the IGFBP2/mTOR/PTEN axis. This mechanism is validated by treating a cell line-based human xenograft tumor with GNPs and an mTOR dual-kinase inhibitor (PI-103), either individually or in combination with GNPs; GNP and PI-103 combination therapy inhibit ovarian tumor growth similarly to GNPs alone. This report illustrates how the self-therapeutic properties of GNPs can be exploited as a discovery tool to identify a critical signaling axis responsible for poor prognosis in ovarian cancer and provides an opportunity to interrogate the axis to improve patient outcomes.

Small Non-Coding-RNA in Gynecological Malignancies.

Gynecologic malignancies, which include cancers of the cervix, ovary, uterus, vulva, vagina, and fallopian tube, are among the leading causes of female mortality worldwide, with the most prevalent being endometrial, ovarian, and cervical cancer. Gynecologic malignancies are complex, heterogeneous diseases, and despite extensive research efforts, the molecular mechanisms underlying their development and pathology remain largely unclear. Currently, mechanistic and therapeutic research in cancer is largely focused on protein targets that are encoded by about 1% of the human genome. Our current understanding of 99% of the genome, which includes noncoding RNA, is limited. The discovery of tens of thousands of noncoding RNAs (ncRNAs), possessing either structural or regulatory functions, has fundamentally altered our understanding of genetics, physiology, pathophysiology, and disease treatment as they relate to gynecologic malignancies. In recent years, it has become clear that ncRNAs are relatively stable, and can serve as biomarkers for cancer diagnosis and prognosis, as well as guide therapy choices. Here we discuss the role of small non-coding RNAs, i.e., microRNAs (miRs), P-Element induced wimpy testis interacting (PIWI) RNAs (piRNAs), and tRNA-derived small RNAs in gynecological malignancies, specifically focusing on ovarian, endometrial, and cervical cancer.

Anti-tumor activity of a new series of benzoxazepine derivatives in breast cancer.

A series of new benzoxazepine derivatives substituted with different alkoxy and aryloxy group were synthesized comprising synthetic steps of Mitsunobu reaction, lithium aluminum hydride (LAH) reduction, followed by debenzylation and finally intramolecular Mitsunobu cyclization. The new benzoxazepines specifically inhibited growth of breast cancer cell lines, MCF-7 and MDA-MB-231, but lack cytotoxicity to normal HEK-293 cells. The cell growth inhibition induced by the active compounds was due to cell cycle arrest at G(0)/G(1) phase. The active compound could cause significant reduction in tumor volume of MCF-7 xenograft tumor in nude mice model and their activity was comparable to that of tamoxifen citrate at 16mgkg(-1) dose at 30days of treatment. The identified most active compounds of the series have specific advantages as anti-cancer agent in breast cancer than tamoxifen.

Targeting the TGFß pathway in uterine carcinosarcoma.

Uterine carcinosarcoma (UCS) is a relatively infrequent, but extremely aggressive endometrial malignancy. Although surgery and chemotherapy have improved outcomes, overall survival (OS) remains dismal due to the lack of targeted therapy and biphasic (epithelial and mesenchymal) nature that renders the tumor aggressive and difficult to manage. Here we report a role of transforming growth factor-ß (TGFß) in maintaining epithelial to mesenchymal transition (EMT) phenotype and aggressiveness in UCS. Using a 3D-culture system, we evaluated the efficacy of the transforming growth factor-ß receptor-I (TGFßR1) kinase inhibitor Galunisertib (GLT), alone and in combination with standard chemotherapeutic drugs used for the management of UCS. We demonstrate that GLT by inhibiting canonical and non-canonical signaling emanating from transforming growth factor-ß1 (TGFß1) reduces cellular viability, invasion, clonal growth and differentiation. Interestingly, GLT sensitizes UCS cells to chemotherapy both in vitro and in in vivo preclinical tumor model. Hence, targeting TGFß signaling, in combination with standard chemotherapy, may be exploited as an important strategy to manage the clinically challenging UCS.

Cystathione ß-synthase regulates HIF-1α stability through persulfidation of PHD2.

The stringent expression of the hypoxia inducible factor-1α (HIF-1α) is critical to a variety of pathophysiological conditions. We reveal that, in normoxia, enzymatic action of cystathionine ß-synthase (CBS) produces H2S, which persulfidates prolyl hydroxylase 2 (PHD2) at residues Cys21 and Cys33 (zinc finger motif), augmenting prolyl hydroxylase activity. Depleting endogenous H2S either by hypoxia or by inhibiting CBS via chemical or genetic means reduces persulfidation of PHD2 and inhibits activity, preventing hydroxylation of HIF-1α, resulting in stabilization. Our in vitro findings are further supported by the depletion of CBS in the zebrafish model that exhibits axis defects and abnormal intersegmental vessels. Exogenous H2S supplementation rescues both in vitro and in vivo phenotypes. We have identified the persulfidated residues and defined their functional significance in regulating the activity of PHD2 via point mutations. Thus, the CBS/H2S/PHD2 axis may provide therapeutic opportunities for pathologies associated with HIF-1α dysregulation in chronic diseases.

Synthesis and biological evaluation of 3,4,6-triaryl-2-pyranones as a potential new class of anti-breast cancer agents.

A series of 3,4,6-triaryl-2-pyranones, new class of anti-breast cancer agents, have been synthesized as a structural variants of cyclic triphenylethylenes by replacing the fused benzene ring with pendant phenyl ring to mimic the phenolic A ring of estradiol. Nine of these newly synthesized pyranones exhibited significant anti-proliferative activity in both ER+ve and ER-ve breast cancer cell lines. Four active non-cytotoxic compounds 5c, 5d, 5g and 5h showed specific and selective cytotoxicity and two compounds 5d and 5h induced significant DNA fragmentation in both MCF-7 and MDA-MB-231 cell lines. Based on RBA studies, the molecules probably act in an ER-independent mechanism. The involved pathway was observed as caspase-dependant apoptosis in MCF-7 cells. However, the particular caspases involved and the possible cellular target through which this series of compounds mediate cell death are not known.