Santosh Timilsina

Inhibition of FoxM1-Mediated DNA Repair by Imipramine Blue Suppresses Breast Cancer Growth and Metastasis

Purpose: The approaches aimed at inhibiting the ability of cancer cells to repair DNA strand breaks have emerged as promising targets for treating cancers. Here, we assessed the potential of imipramine blue (IB), a novel analogue of antidepressant imipramine, to suppress breast cancer growth and metastasis by inhibiting the ability of breast cancer cells to repair DNA strand breaks by homologous recombination (HR). Experimental Design: The effect of IB on breast cancer growth and metastasis was assessed in vitro as well as in preclinical mouse models. Besides, the therapeutic efficacy and safety of IB was determined in ex vivo explants from breast cancer patients. The mechanism of action of IB was evaluated by performing gene-expression, drug–protein interaction, cell-cycle, and DNA repair studies. Results: We show that the systemic delivery of IB using nanoparticle-based delivery approach suppressed breast cancer growth and metastasis without inducing toxicity in preclinical mouse models. Using ex vivo explants from breast cancer patients, we demonstrated that IB inhibited breast cancer growth without affecting normal mammary epithelial cells. Furthermore, our mechanistic studies revealed that IB may interact and inhibit the activity of proto-oncogene FoxM1 and associated signaling that play critical roles in HR-mediated DNA repair. Conclusions: These findings highlight the potential of IB to be applied as a safe regimen for treating breast cancer patients. Given that FoxM1 is an established therapeutic target for several cancers, the identification of a compound that inhibits FoxM1- and FoxM1-mediated DNA repair has immense translational potential for treating many aggressive cancers. Clin Cancer Res; 22(14); 3524–36. ©2016 AACR.

Cross-talk between miR-471-5p and autophagy component proteins regulates LC3-associated phagocytosis (LAP) of apoptotic germ cells

Phagocytic clearance of apoptotic germ cells by Sertoli cells is vital for germ cell development and differentiation. Here, using a tissue-specific miRNA transgenic mouse model, we show that interaction between miR-471-5p and autophagy member proteins regulates clearance of apoptotic germ cells via LC3-associated phagocytosis (LAP). Transgenic mice expressing miR-471-5p in Sertoli cells show increased germ cell apoptosis and compromised male fertility. Those effects are due to defective engulfment and impaired LAP-mediated clearance of apoptotic germ cells as miR-471-5p transgenic mice show lower levels of Dock180, LC3, Atg12, Becn1, Rab5 and Rubicon in Sertoli cells. Our results reveal that Dock180 interacts with autophagy member proteins to constitute a functional LC3-dependent phagocytic complex. We find that androgen regulates Sertoli cell phagocytosis by controlling expression of miR-471-5p and its target proteins. These findings suggest that recruitment of autophagy machinery is essential for efficient clearance of apoptotic germ cells by Sertoli cells using LAP.Although phagocytic clearance of apoptotic germ cells by Sertoli cells is essential for spermatogenesis, little of the mechanism is known. Here the authors show that Sertoli cells employ LC3-associated phagocytosis (LAP) by recruiting autophagy member proteins to clear apoptotic germ cells.

Downregulation of KCNMB4 expression and changes in BK channel subtype in hippocampal granule neurons following seizure activity

A major challenge is to understand maladaptive changes in ion channels that sets neurons on a course towards epilepsy development. Voltage- and calcium-activated K+ (BK) channels contribute to early spike timing in neurons, and studies indicate that the BK channel plays a pathological role in increasing excitability early after a seizure. Here, we have investigated changes in BK channels and their accessory β4 subunit (KCNMB4) in dentate gyrus (DG) granule neurons of the hippocampus, key neurons that regulate excitability of the hippocampus circuit. Two days after pilocarpine-induced seizures, we found that the predominant effect is a downregulation of the β4 accessory subunit mRNA. Consistent with reduced expression, single channel recording and pharmacology indicate a switch in the subtype of channels expressed; from iberiotoxin-resistant, type II BK channels (BK α/β4) that have higher channel open probability and slow gating, to iberiotoxin-sensitive type I channels (BK α alone) with low open probability and faster gating. The switch to a majority of type I channel expression following seizure activity is correlated with a loss of BK channel function on spike threshold while maintaining the channel’s contribution to increased early spike frequency. Using heterozygous β4 knockout mice, we find reduced expression is sufficient to increase seizure sensitivity. We conclude that seizure-induced downregulation of KCNMB4 is an activity dependent mechanism that increases the excitability of DG neurons. These novel findings indicate that BK channel subtypes are not only defined by cell-specific expression, but can also be plastic depending on the recent history of neuronal excitability.

Topological Characterization of Human and Mouse m5C Epitranscriptome Revealed by Bisulfite Sequencing

Background Compared with the well-studied 5-methylcytosine (m5C) in DNA, the role and topology of epitranscriptome m5C remain insufficiently characterized. Results Through analyzing transcriptome-wide m5C distribution in human and mouse, we show that the m5C modification is significantly enriched at 5′ untranslated regions (5′UTRs) of mRNA in human and mouse. With a comparative analysis of the mRNA and DNA methylome, we demonstrate that, like DNA methylation, transcriptome m5C methylation exhibits a strong clustering effect. Surprisingly, an inverse correlation between mRNA and DNA m5C methylation is observed at CpG sites. Further analysis reveals that RNA m5C methylation level is positively correlated with both RNA expression and RNA half-life. We also observed that the methylation level of mitochondrial RNAs is significantly higher than RNAs transcribed from the nuclear genome. Conclusions This study provides an in-depth topological characterization of transcriptome-wide m5C modification by associating RNA m5C methylation patterns with transcriptional expression, DNA methylations, RNA stabilities, and mitochondrial genome.

Cross-talk among writers, readers, and erasers of m6A regulates cancer growth and progression

Collaboration among writers-readers-erasers of m6A regulates the stability of tumor-specific genes. The importance of RNA methylation in biological processes is an emerging focus of investigation. We report that altering m6A levels by silencing either N6-adenosine methyltransferase METTL14 (methyltransferase-like 14) or demethylase ALKBH5 (ALKB homolog 5) inhibits cancer growth and invasion. METTL14/ALKBH5 mediate their protumorigenic function by regulating m6A levels of key epithelial-mesenchymal transition and angiogenesis-associated transcripts, including transforming growth factor–β signaling pathway genes. Using MeRIP-seq (methylated RNA immunoprecipitation sequencing) analysis and functional studies, we find that these target genes are particularly sensitive to changes in m6A modifications, as altered m6A status leads to aberrant expression of these genes, resulting in inappropriate cell cycle progression and evasion of apoptosis. Our results reveal that METTL14 and ALKBH5 determine the m6A status of target genes by controlling each other’s expression and by inhibiting m6A reader YTHDF3 (YTH N6-methyladenosine RNA binding protein 3), which blocks RNA demethylase activity. Furthermore, we show that ALKBH5/METTL14 constitute a positive feedback loop with RNA stability factor HuR to regulate the stability of target transcripts. We discover that hypoxia alters the level/activity of writers, erasers, and readers, leading to decreased m6A and consequently increased expression of target transcripts in cancer cells. This study unveils a previously undefined role for m6A in cancer and shows that the collaboration among writers-erasers-readers sets up the m6A threshold to ensure the stability of progrowth/proliferation-specific genes, and protumorigenic stimulus, such as hypoxia, perturbs that m6A threshold, leading to uncontrolled expression/activity of those genes, resulting in tumor growth, angiogenesis, and progression.

MiR-584-5p potentiates vincristine and radiation response by inducing spindle defects and DNA damage in medulloblastoma

Despite improvements in overall survival, only a modest percentage of patients survives high-risk medulloblastoma. The devastating side effects of radiation and chemotherapy substantially reduce quality of life for surviving patients. Here, using genomic screens, we identified miR-584-5p as a potent therapeutic adjuvant that potentiates medulloblastoma to radiation and vincristine. MiR-584-5p inhibited medulloblastoma growth and prolonged survival of mice in pre-clinical tumor models. MiR-584-5p overexpression caused cell cycle arrest, DNA damage, and spindle defects in medulloblastoma cells. MiR-584-5p mediated its tumor suppressor and therapy-sensitizing effects by targeting HDAC1 and eIF4E3. MiR-584-5p overexpression or HDAC1/eIF4E3 silencing inhibited medulloblastoma stem cell self-renewal without affecting neural stem cell growth. In medulloblastoma patients, reduced expression of miR-584-5p correlated with increased levels of HDAC1/eIF4E3. These findings identify a previously undefined role for miR-584-5p/HDAC1/eIF4E3 in regulating DNA repair, microtubule dynamics, and stemness in medulloblastoma and set the stage for a new way to treat medulloblastoma using miR-584-5p.The radiation and chemotherapy used for treating medulloblastoma patients cause debilitating side effects. Here, the authors show that miR-584 acts as a therapeutic adjuvant as it sensitizes medulloblastoma to radiation and chemotherapy by targeting HDAC1 or eIF4E3 to enhance spindle defects and DNA damage.

Novel post-transcriptional and post-translational regulation of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 determine the fate of breast cancer cells to survive or die

Deregulation of apoptosis is central to cancer progression and a major obstacle to effective treatment. The Bcl-2 gene family members play important roles in the regulation of apoptosis and are frequently altered in cancers. One such member is pro-apoptotic protein Bcl-2-related Ovarian Killer (BOK). Despite its critical role in apoptosis, the regulation of BOK expression is poorly understood in cancers. Here, we discovered that miR-296-5p regulates BOK expression by binding to its 3’-UTR in breast cancers. Interestingly, miR-296-5p also regulates the expression of anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1), which is highly expressed in breast cancers. Our results reveal that Mcl-1 and BOK constitute a regulatory feedback loop as ectopic BOK expression induces Mcl-1, whereas silencing of Mcl-1 results in reduced BOK levels in breast cancer cells. In addition, we show that silencing of Mcl-1 but not BOK reduced the long-term growth of breast cancer cells. Silencing of both Mcl-1 and BOK rescued the effect of Mcl-1 silencing on breast cancer cell growth, suggesting that BOK is important for attenuating cell growth in the absence of Mcl-1. Depletion of BOK suppressed caspase-3 activation in the presence of paclitaxel and in turn protected cells from paclitaxel-induced apoptosis. Furthermore, we demonstrate that glycogen synthase kinase (GSK3) α/β interacts with BOK and regulates its level post-translationally in breast cancer cells. Taken together, our results suggest that fine tuning of the levels of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 may decide the fate of cancer cells to either undergo apoptosis or proliferation.

Abstract 2336: Novel regulatory mechanisms for Bcl2-related Ovarian Killer (BOK) expression in breast cancer

Deregulation of apoptosis is central to cancer progression and a major obstacle to effective treatment. The Bcl-2 gene family members play important roles in the regulation of apoptosis and are frequently altered in cancers. One such member is Bcl-2-related Ovarian Killer (BOK), which is a pro-apoptotic protein. Despite its critical role in apoptosis, the regulation of BOK expression is poorly understood in cancers. Here, we discovered that miR-296-5p, regulates BOK expression by binding to its 3’UTR in breast cancers. Furthermore, we show that depletion of BOK by either miR-296-5p or siRNA against BOK protected breast cancer cells from undergoing paclitaxel-induced apoptosis. Interestingly, miR-296-5p also regulates the expression of Mcl-1, which is an anti-apoptotic protein and is highly expressed in breast cancers. Our results reveal that Mcl-1 is important for suppression of BOK function as ectopic BOK expression induced Mcl-1, while silencing of BOK resulted in reduced Mcl-1 levels in breast cancer cells. In addition, we show that specific silencing of Mcl-1 reduced the long-term growth of breast cancer cells, whereas BOK inhibition didn’t have any effect on the growth of breast cancer cells. Surprisingly, silencing of both Mcl-1 and BOK rescued the effect of Mcl-1 silencing on breast cancer cell growth, suggesting that BOK is important for attenuating cell growth in the absence of Mcl-1, and also showing a tight feedback regulatory loop between BOK and Mcl-1 in breast cancer cells. Furthermore, we demonstrated that BOK protein level is regulated post-translationally by GSK3α and to some extent GSK3β as GSK3 inhibitor (CHIR99021) or silencing of GSK3 significantly increased BOK protein levels in breast cancer cells. Notably, we found that Mcl-1 interacts with GSK3α/β and silencing of Mcl-1 using siRNA significantly attenuated endogenous GSK3α/β levels in breast cancer cells. Taken together, our results suggest that fine tuning (either post-transcriptionally by miR-296-5p or post-translationally by GSK3) of the levels of pro-apoptotic protein BOK and anti-apoptotic protein Mcl-1 decide the fate of cancer cells to either undergo Apoptosis or proliferation. Citation Format: Benjamin Chidi Onyeagucha, Panneerdoss Subbarayalu, Subapriya Rajamanickam, Nourhan Abdelfattah, Santosh Timilsina, Rosa M. Guzman, Carla Zeballos, Vijay Eedunuri, Sanjay Bansal, Hima Bansal, Tabrez A. Mohammad, Yidong Chen, Manjeet K. Rao. Novel regulatory mechanisms for Bcl2-related Ovarian Killer (BOK) expression in breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2336. doi:10.1158/1538-7445.AM2017-2336

Abstract 3658: A novel microtubule associated RNA binding protein matrin 3 act as a tumor suppressor by regulating mitotic spindle organizing proteins in triple negative breast cancers

Microtubule-targeting drugs are widely used as cancer chemotherapeutic agents and they have been shown to inhibit mitotic progression and interphase signaling. Despite their use as a first line treatment for cancer, many patients develop resistance to microtubule-targeting drugs leading to early relapse and shorter survival. Moreover, the quality of life for patients who do survive is often substantially reduced due to the toxicity associated with these drugs. Therefore, identification of new factors that determine the effectiveness of microtubule-targeting agents will not only facilitate a better understanding of the mechanisms of acquired drug resistance but will also be amenable to therapeutic interventions. We discovered a novel microtubule associated protein “Matrin 3 (MATR3)” that is known to bind to RNA and play a critical role in RNA transport and RNA stabilization. Our results revealed that MATR3 acts as a potent tumor suppressor as it inhibits long-term growth, migration, invasion as well as tumor growth of triple negative breast cancer (TNBC) cells in vivo. We demonstrated that MATR3 overexpression induced cell cycle arrest and apoptosis in TNBC cells. Furthermore, analysis of breast cancer samples showed a significantly lower expression of MATR3 when compared to normal adjacent tissues. Importantly, our RNA immunoprecipitation (RIP)-seq analysis showed that MATR3 controls expression of several mitotic spindle organizing proteins by binding to their RNA. Interestingly, we found that these MATR3 regulated proteins were highly altered in breast cancer patients. In conclusion, we identified a novel RNA-binding protein that inhibits breast cancer growth and progression suppressor by regulating microtubule dynamics. Citation Format: Panneerdoss Subbarayalu, Subapriya Rajamanickam, Suryavathi Viswanadhapalli, Benjamin C. Onyeagucha, Vijay K. Eedunuri, Nicholas Dybdal-Hargreaves, Santosh Timilsina, Hima Bansal, Sanjay Bansal, Tabrez Mohammad, Yidong Chen, John C. Herr, Susan L. Mooberry, Manjeet K. Rao. A novel microtubule associated RNA binding protein matrin 3 act as a tumor suppressor by regulating mitotic spindle organizing proteins in triple negative breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3658.

Abstract P2-03-04: Down-regulation of Bcl2-related ovarian killer (BOK) by miR-296-5p protects breast cancer cells from paclitaxel-induced apoptosis

Accumulating evidence shows that miRNAs play a role in drug resistance. Despite these observations, little is known about the identities of the miRNAs involved in drug resistance and their downstream targets. In the present study, we identified miR-296-5p for which a tumor suppressive role has been previously described, as a miRNA that is involved in paclitaxel drug resistance in triple negative breast cancer (TNBC) cells. Enforced expression of miR-296-5p suppressed cell growth, migration, and invasion in MDA-MB-231 breast cancer cells. Using a microarray approach, we identified BCL2-related Ovarian Killer (BOK), a pro-apoptotic gene as a target of miR-296-5p. BOK levels were validated BOK levels in miR-296-5p transfected MDA-MB-231 and MDA-MB-468 cells using real-time PCR and Western blot. Our results demonstrated that over-expression of miR-296-5p down-regulated BOK expression in TNBC cells. Transfection of miR-296-5p significantly suppressed luciferase reporters containing wild-type BOK-39-UTR constructs. In contrast, mutant BOK-39-UTR constructs were unaffected by ectopic miR-296-5p. Furthermore, BOK expression was induced in the presence of paclitaxel, but ectopic miR-296-5p significantly suppressed BOK induction by paclitaxel treatment compared to the control cells. These data provide new insights on the role of miRNAs in drug resistance and suggests that therapeutic strategies against miR-296-5p may be warranted. Citation Format: Onyeagucha BC, Rajamanickam S, Subbarayalu P, Bansal B, Bansal H, Chang Y-F, Timilsina S, Abdelfaltah N, Eedunuri VK, Rao MK. Down-regulation of Bcl2-related ovarian killer (BOK) by miR-296-5p protects breast cancer cells from paclitaxel-induced apoptosis. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P2-03-04.