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About 1 in 8 women will develop invasive breast cancer during their life time and more than 41,000 women die every year in the United States alone. Most of these deaths are attributed to relapse, distant metastasis and therapy resistance. For example, despite initial response to therapy, a significant proportion of ER+ BCs become therapy resistant and progress to incurable metastases. Similarly, TNBCs, which is highly aggressive and are more likely to occur in Hispanic and black women, have higher propensity to relapse and contribute to disproportionate number of deaths. Unfortunately, the patients who do survive, have reduced quality of life due to the chemotherapy-associated toxicity. One mechanism that helps BCs to survive and become therapy-resistant is their unique ability to keep repairing their DNA. The aim of this study was to identify and test whether any FDA-approved non-cancer drug/s can block DNA repair ability and consequently growth of BC cells. Treatment of BC cells with a set of FDA approved drugs showed that antidepressants imipramine can inhibit the BC cell growth. Imipramine treatment significantly reduced the short and long-term viability of TNBC and ER+ breast cancer cells. Further, imipramine treatment inhibited the migration and invasion of breast cancer cells. Systemic delivery of imipramine suppressed the breast cancer cells growth in orthotopic mouse xenograft model. Our results revealed that imipramine treatment induced G1/S cell cycle arrest and apoptosis in breast cancer cells. Importantly, imipramine blocked the DNA repair capacity of BC cells by inhibiting the expression of DNA repair proteins including FOXM1 and RAAD51. Notably, imipramine treatment improved the efficacy of the PARP inhibitor “olapraib” in TNBC and sensitized the tamoxifen response in tamoxifen resistant ER+ breast cancer cells. Our results suggest that imipramine can be used as a single or therapeutic adjuvant for treating therapy sensitive and therapy resistant breast cancers. Based on these results, we are currently conducting a clinical trial to test the therapeutic efficacy of imipramine for treating breast cancer patients (https://www.cancer.gov/about-cancer/treatment/clinical-trials/search/v?id=NCI-2017 01937&r=1). Citation Format: Santosh Timilsina, Subapriya Rajamanickam, Arhan Rao, Panneerdoss Subbarayalu, Ismail Jatoi, Yidong Chen, Ratna Vadlamudi, Virginia G Kaklamani, Manjeet Rao. Repurposing anti-depressant imipramine for treating breast cancers [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS17-25.

Methyltransferase like-3 (METTL3) and METTL14 complex transfers a methyl group from S-adenosyl-L-methionine to N6 amino group of adenosine bases in RNA (m6A) and DNA (m6dA). Emerging evidence highlights a role of METTL3-METTL14 in the chromatin context, especially in processes where DNA and RNA are held in close proximity. However, a mechanistic framework about specificity for substrate RNA/DNA and their interrelationship remain unclear. By systematically studying methylation activity and binding affinity to a number of DNA and RNA oligos with different propensities to form inter- or intra-molecular duplexes or single-stranded molecules in vitro, we uncover an inverse relationship for substrate binding and methylation and show that METTL3-METTL14 preferentially catalyzes the formation of m6dA in single-stranded DNA (ssDNA), despite weaker binding affinity to DNA. In contrast, it binds structured RNAs with high affinity, but methylates the target adenosine in RNA (m6A) much less efficiently than it does in ssDNA. We also show that METTL3-METTL14-mediated methylation of DNA is largely restricted by structured RNA elements prevalent in long noncoding and other cellular RNAs.

Medulloblastoma (MB) is the most common and deadliest brain tumor in children. Proline-, glutamic acid-, and leucine-rich protein 1 (PELP1) is a scaffolding protein and its oncogenic signaling is implicated in the progression of several cancers. However, the role of PELP1 in the progression of MB remains unknown. The objective of this study is to examine the role of PELP1 in the progression of MB. Immunohistochemical analysis of MB tissue microarrays revealed that PELP1 is overexpressed in the MB specimens compared to normal brain. Knockdown of PELP1 reduced cell proliferation, cell survival, and cell invasion of MB cell lines. The RNA-sequencing analysis revealed that PELP1 knockdown significantly downregulated the pathways related to inflammation and extracellular matrix. Gene set enrichment analysis confirmed that the PELP1-regulated genes were negatively correlated with nuclear factor-κB (NF-κB), extracellular matrix, and angiogenesis gene sets. Interestingly, PELP1 knockdown reduced the expression of NF-κB target genes, NF-κB reporter activity, and inhibited the nuclear translocation of p65. Importantly, the knockdown of PELP1 significantly reduced in vivo MB progression in orthotopic models and improved the overall mice survival. Collectively, these results suggest that PELP1 could be a novel target for therapeutic intervention in MB.

Histone deacetylase inhibitors (HDACi) are identified as novel therapeutic agents, however, recent clinical studies suggested that they are marginally effective in treating triple negative breast cancer (TNBC). Here, we show that first-in-class Leukemia Inhibitory Factor Receptor (LIFRα) inhibitor EC359 could enhance the therapeutic efficacy of HDACi against TNBC. We observed that both targeted knockdown of LIFR with CRISPR or treatment with EC359 enhanced the potency of four different HDACi in reducing cell viability, cell survival, and enhanced apoptosis compared to monotherapy in TNBC cells. RNA-seq studies demonstrated oncogenic/survival signaling pathways activated by HDACi were attenuated by the EC359 + HDACi therapy. Importantly, combination therapy potently inhibited the growth of TNBC patient derived explants, cell derived xenografts and patient-derived xenografts in vivo. Collectively, our results suggest that targeted inhibition of LIFR can enhance the therapeutic efficacy of HDACi in TNBC., Li, Viswanadhapalli et al utilized multiple in vitro, ex vivo and in vivo models of TNBC to investigate LIFR inhibition. The authors reported that HDAC inhibition in TNBC cells led to an increase of LIFR expression and over activation of the downstream signaling elements (e.g., STAT3, mTOR, AKT), enhancing the aggressive potential of TNBC cells, and an LIFR inhibitor, EC359, synergistically enhanced the efficacy of HADC inhibitors in suppressing TNBC in vitro and in vivo.

A novel deep learning method links in vitro CRISPR screens to the context of cancer genomics and translates to individual tumors., Genome-wide loss-of-function screens have revealed genes essential for cancer cell proliferation, called cancer dependencies. It remains challenging to link cancer dependencies to the molecular compositions of cancer cells or to unscreened cell lines and further to tumors. Here, we present DeepDEP, a deep learning model that predicts cancer dependencies using integrative genomic profiles. It uses a unique unsupervised pretraining that captures unlabeled tumor genomic representations to improve the learning of cancer dependencies. We demonstrated DeepDEP’s improvement over conventional machine learning methods and validated the performance with three independent datasets. By systematic model interpretations, we extended the current dependency maps with functional characterizations of dependencies and a proof-of-concept in silico assay of synthetic essentiality. We applied DeepDEP to pan-cancer tumor genomics and built the first pan-cancer synthetic dependency map of 8000 tumors with clinical relevance. In summary, DeepDEP is a novel tool for investigating cancer dependency with rapidly growing genomic resources.

Down-regulation of HMBOX1 via METTL3-catalyzed m6A causes genomic instability driven by telomere dysfunction in cancer., The role of RNA methylation on N6-adenosine (m6A) in cancer has been acknowledged, but the underlying mechanisms remain obscure. Here, we identified homeobox containing 1 (HMBOX1) as an authentic target mRNA of m6A machinery, which is highly methylated in malignant cells compared to the normal counterparts and subject to expedited degradation upon the modification. m6A-mediated down-regulation of HMBOX1 causes telomere dysfunction and inactivation of p53 signaling, which leads to chromosome abnormalities and aggressive phenotypes. CRISPR-based, m6A-editing tools further prove that the methyl groups on HMBOX1 per se contribute to the generation of altered cancer genome. In multiple types of human cancers, expression of the RNA methyltransferase METTL3 is negatively correlated with the telomere length but favorably with fractions of altered cancer genome, whereas HMBOX1 mRNA levels show the opposite patterns. Our work suggests that the cancer-driving genomic alterations may potentially be fixed by rectifying particular epitranscriptomic program.

Triple Negative Breast Cancer (TNBC) is an aggressive subtype of breast cancer that lacks effective targeted therapies with the main therapeutic option being cytotoxic chemotherapies, which have toxic side effects on patient’s health. Less toxic alternatives that could target key pathways that are altered in TNBCs could lead to better outcomes for the patients. Important distinguishing traits of TNBCs is the high amounts of genomic instability and high number of alterations in key proteins in DNA repair and cell proliferation that helps the cancer grow, survive and progress. Previous findings from our lab indicate that Imipramine Blue, a derivative of anti-depressant imipramine, is a potent inhibitor of proliferation and DNA repair pathways in TNBC. Therefore, we tested the therapeutic efficacy of Imipramine on TNBCs using various in vitro assays for viability, migration, invasion as well as in vivo xenograft assays for tumor growth. Our results demonstrate that Imipramine inhibits cancer cell viability and progression in vitro. Similarly, in vivo xenograft assays showed reduced tumor growth in pre-clinical models upon imipramine treatment. Importantly, at the molecular level, Imipramine treatment suppressed key players in both cell proliferation, and DNA repair pathways, including FOXM1; a proto-oncogene that is a master regulator of both cell proliferation and DNA Repair pathways. These findings suggest that Imipramine may be a potential therapeutic option for TNBC patients. A clinical trial is ongoing to test the efficacy of Imipramine in Triple Negative Breast Cancer patients. Citation Format: Santosh Timilsina, Subapriya Rajamanickam, Ismail Jatoi, Yidong Chen, Ratna Vadlamudi, Virginia Kaklamani, Manjeet K Rao. Imipramine, an FDA approved anti-depressant, suppresses triple negative breast cancer growth and progression through inhibition of FOXM1 and DNA repair pathways [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-05-05.

Background CDK4/6 inhibitors in combination with endocrine therapy (AE/AI/SERDs) are approved for the treatment of ER+ advanced breast cancer (BCa). However, not all patients benefit from CDK4/6 inhibitors therapy. We previously reported a novel therapeutic agent, ERX-11, that binds to the estrogen receptor (ER) and modulates ER-coregulator interactions. Here, we tested if the combination of ERX-11 with agents approved for ER+ BCa would be more potent. Methods We tested the effect of combination therapy using BCa cell line models, including those that have acquired resistance to tamoxifen, letrozole, or CDK4/6 inhibitors or have been engineered to express mutant forms of the ER. In vitro activity was tested using Cell Titer-Glo, MTT, and apoptosis assays. Mechanistic studies were conducted using western blot, reporter gene assays, RT-qPCR, and mass spectrometry approaches. Xenograft, patient-derived explants (PDEs), and xenograft-derived explants (XDE) were used for preclinical evaluation and toxicity. Results ERX-11 inhibited the proliferation of therapy-resistant BCa cells in a dose-dependent manner, including ribociclib resistance. The combination of ERX-11 and CDK4/6 inhibitor was synergistic in decreasing the proliferation of both endocrine therapy-sensitive and endocrine therapy-resistant BCa cells, in vitro, in xenograft models in vivo, xenograft-derived explants ex vivo, and in primary patient-derived explants ex vivo. Importantly, the combination caused xenograft tumor regression in vivo. Unbiased global mass spectrometry studies demonstrated profound decreases in proliferation markers with combination therapy and indicated global proteomic changes in E2F1, ER, and ER coregulators. Mechanistically, the combination of ERX-11 and CDK4/6 inhibitor decreased the interaction between ER and its coregulators, as evidenced by immunoprecipitation followed by mass spectrometry studies. Biochemical studies confirmed that the combination therapy significantly altered the expression of proteins involved in E2F1 and ER signaling, and this is primarily driven by a transcriptional shift, as noted in gene expression studies. Conclusions Our results suggest that ERX-11 inhibited the proliferation of BCa cells resistant to both endocrine therapy and CDK4/6 inhibitors in a dose-dependent manner and that the combination of ERX-11 with a CDK4/6 inhibitor may represent a viable therapeutic approach.

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.

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.

Ovarian cancer is the deadliest of all gynecologic cancers. Despite success with initial chemotherapy, the majority of patients relapse with an incurable disease. Development of chemotherapy resistance is a major factor for poor long-term survival in ovarian cancer. The biological effects of estrogens are mediated by estrogen receptor alpha (ERα) and estrogen receptor beta (ERβ). Emerging evidence suggests that ovarian cancer cells express ERβ that functions as a tumor suppressor; however, the clinical utility of ERβ agonists in ovarian cancer remains elusive. We tested the utility of two natural ERβ agonists liquiritigenin (Liq), which is isolated from Glycyrrhiza uralensis and S-equol, which is isolated from soy isoflavone daidzein, for treating ovarian cancer. Both natural ERβ ligands had significant growth inhibition in cell viability and survival assays, reduced migration and invasion, and promoted apoptosis. Further, ERβ agonists showed tumor suppressive functions in therapy-resistant ovarian cancer model cells and sensitized ovarian cancer cells to cisplatin and paclitaxel treatment. Global RNA-Seq analysis revealed that ERβ agonists modulate several tumor suppressive pathways, including downregulation of the NF-κB pathway. Immunoprecipitation assays revealed that ERβ interacts with p65 subunit of NF-κB and ERβ overexpression reduced the expression of NF-κB target genes. In xenograft assays, ERβ agonists reduced tumor growth and promoted apoptosis. Collectively, our findings demonstrated that natural ERβ agonists have the potential to significantly inhibit ovarian cancer cell growth by anti-inflammatory and pro-apoptotic actions, and natural ERβ agonists represent novel therapeutic agents for the management of ovarian cancer.

The importance of RNA methylation in biological processes has just begun to emerge. However, our understanding of the role and mechanism by which RNA methylation may play a role in tumorigenesis is unclear. Here, we report that RNA N6-adenosine methyltransferase protein METTL14 supports breast cancer growth and progression. We show that METTL14 mediates its pro-tumorigenic function by activating the expression of RNA binding protein HuR and consequently inducing the transcriptional stability of key cell cycle-associated genes, TGFβ and its signaling partners as well as HMGA2, VEGF and PDGF that are known to play critical roles in cancer growth and metastasis. Importantly, m6 A RNA methylation levels of METTL14 target genes were significantly lower in breast cancer patients compared to normal matched controls. The decreased RNA methylation level resulting in increased activity of these target genes in breast cancer patients is regulated by a feedback loop between RNA demethylase ALKBH5 and METTL14. Our results reveal that ALKBH5 regulates the expression of METTL14 and HuR, which in turn transcriptionally stabilizes ALKBH5. Furthermore, we demonstrate that like METTL14, ALKBH5 also supports breast cancer growth and progression. Taken together, these findings suggest that a collaboration between RNA methylation and demethylation machinery may set up a threshold of RNA methylation that ensures the stability of genes that play a vital role in normal cell proliferation; and any perturbation of that threshold may lead to uncontrolled expression/activity of those genes resulting in tumor growth and progression. Citation Format: Subbarayalu P, Eedunuri VK, Timilsina S, Rajamanickam S, Viswanadhapalli S, Abdelfattah N, Onyeagucha BC, Cui X, Mohammad TA, Huang TH-M, Huang Y, Chen Y, Rao MK. Cross-talk between RNA methyltransferase and demethylase regulates breast cancer growth and progression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P1-04-03.

Despite improvement in overall survival, many patients with breast cancers still succumb to this disease. Identification of new biomarkers and safe therapeutic targets are urgently needed to improve the overall clinical outcome of breast cancer patients. Our studies discovered a RNA binding protein, MATRIN3 (MATR3), as a novel tumor suppressor. MATR3 is expressed at a significantly reduced levels in breast tumors. MATR3 inhibited short and long-term viability as well as migration and invasion of breast cancer cells. Further, MATR3 overexpression suppressed tumor growth, while its depletion induced tumor growth in orthotopic mouse tumor models. RNA seq and RNA immunoprecipitation analyses revealed that MATR3 binds and directly regulates the expression of several microtubule-associated proteins. Mechanistic studies identified MZT2B, a mitotic spindle organizing protein as a down stream effector of MATR3. MZT2B knockdown or knockout using CRISPR-CAS9 resulted in significantly decreased short and long term viability as well as reduced migration and invasion of breast cancer cells. Notably, MZT2B overexpression rescued the inhibitory effect of MATR3 overexpression on breast cancer growth. Furthermore, MATR3 overexpression downregulated expression of key microtubule nucleation protein complex including γ-tubulin and γ-tubulin ring complex protein (TUBGCP). Our data suggest that MATR3 inhibits breast cancer growth and progression by inhibiting MZT2B and consequently microtubule nucleation in breast cancers. Citation Format: Panneerdoss Subbarayalu, Subapriya Rajamanickam, Suryavathi Viswanadhapalli, Fuyang Li, Vijay Eedunuri, Pooja Yadav, Esha Reddy, Santosh Timilsina, Saif SR Nirzhor, Benjamin C Onyeagucha, Li-Ju Wang, Yu-Chiao Chiu, Tabrez Mohammad, Nourhan Abdelfattah, Nicholas Dybdal-Hargreaves, Yidong Chen, Ratna Vadlamudi, Manjeet Rao. Matrin3 inhibits breast cancer growth by suppressing microtubule nucleation protein MZT2B [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS19-14.

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.

Despite improvement in overall survival of breast cancer patients, many women don't survive this disease. Moreover, the quality of life for patients who do survive is often substantially reduced due to the toxicity associated with the chemotherapy. Here, we report that imipramine blue (IB), a novel analogue of anti-depressant imipramine that we recently synthesized, may serve as a safe and potent therapeutic agent for treating breast cancers. We show that IB reduced cell growth, migration and invasion of breast cancer cells. Systemic delivery of IB using nanoparticle-based drug delivery approach suppressed breast cancer growth and metastasis without inducing any toxicity in pre-clinical orthotropic mouse models. Notably, using ex-vivo model of tumor explants from breast cancer patients, we demonstrated that IB inhibited breast cancer growth without affecting normal mammary epithelial cell proliferation. Furthermore, IB improved the sensitivity of breast cancer cells to chemotherapy drugs paclitaxel and doxorubicin. Our results revealed that IB mediated its anti-tumor effect by targeting genes involved in cell cycle progression, microtubule dynamics and DNA damage surveillance pathway including Forkhead Box M1 (FOXM1), stathmin1, S-phase kinase-associated protein 2 (Skp2) and XRCC3, which we show to be highly expressed in breast cancer patients. Importantly, we demonstrated that IB inhibited breast cancer cell's ability to repair DNA strand breaks by impairing homologous recombination events. These findings highlight the potential of IB to be used as a potent therapeutic regimen for treating breast cancer patients. Since IB-1 is derived from a FDA approved drug it has potential to be rapidly translated to the clinic. Citation Format: Rajamanickam S, Subbarayalu P, Timilsina S, Gorthi A, Drake MT, Chen Y, Vadlamudi R, Bishop AJR, Arbiser JL, Rao MK. Imipramine Blue - A safe and potent therapeutic regimen that suppresses breast cancer growth and progression by targeting DNA damage surveillance pathway. [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 P4-07-06.

Background: Triple-negative breast cancer (TNBC) lacks targeted therapies and represents a disproportional share of the breast cancer (BC) mortality rate. TNBC exhibits autocrine stimulation of the LIF/LIFR axis and overexpression of LIF is associated with poorer relapse-free survival in BC patients. Histone deacetylase inhibitors (HDACIs) are emerging as promising multifunctional agents in TNBC to elicit cytotoxic actions. Recent studies have shown that cancer cells elicit feedback activation of leukemia inhibitory factor receptor (LIFR) which in turn curtails response to HDACIs. We developed a first-in-class inhibitor of LIFR, EC359 that directly interacts with LIFR and effectively blocks LIFR downstream signaling. The objective of this study is to examine the therapeutic efficacy of combination therapy using preclinical and patient-derived xenograft (PDX) models. Methods: We tested utility of combination therapy using multiple HDACIs that are currently in clinical trails along with EC359. The effect of combination therapy was evaluated using MTT, invasion, colony formation, and Caspase3/7 assays. Mechanistic studies were performed using Western blotting, qRT-PCR, and STAT3 reporter assays. The efficacy of combination therapy in vivo was examined using xenograft, PDX, and patient-derived explant (PDEx) models. Results: Immunohistochemical analyses of breast tumors using tissue microarrays revealed significant expression of LIFR in TNBC tissues. Treatment of TNBC model cells with four different HDACIs increased the expression of LIFR. LIFR inhibitor EC359 at nM concentration is additive to HDACIs in reducing cell viability. Knockdown of LIFR or treatment with EC359 significantly enhanced the efficacy of HDACIs in reducing the cell viability, colony formation ability, and invasiveness as well as promoted apoptosis compared to monotherapy in TNBC model cells. On the contrary, treatment with STAT3 inhibitor requires µM concentrations to reduce the cell viability of TNBC cells and is not additive to HDACIs. Mechanistic studies utilizing STAT3 reporter gene assays and biochemical studies using multiple TNBC model cells exhibited activation of the LIFR signaling pathway upon HDACIs treatment but was attenuated by EC359 therapy. Treatment of human TNBC utilizing PDEx assays showed that EC359 enhanced the ability of HDACIs to decrease proliferation (Ki-67 positivity) compared to monotherapy. Using TNBC xenografts and PDX models, we demonstrated that EC359 treatment enhanced the ability of HDACIs to reduce in vivo tumor growth compared to monotherapy. Conclusions: Our results suggest that the combination therapy of HDACIs and EC359 provides therapeutic utility in overcoming the limitation of feedback activation of LIFR observed in the treatment of HDACIs in treating TNBC. Supported by DOD BCRP grant W81XWH-18-1-0016 (R.K. Vadlamudi; K.J. Nickisch) Citation Format: Suryavathi Viswanadhapalli, Mengxing Li, Bindu Santhamma, Uday P. Pratap, Yiliao Luo, Junhao Liu, Kristin A. Altwegg, Xiaonan Li, Ahmed Gulzar, Hui Yan, Zhenming Xu, Andrew Brenner, Gangadhara R. Sareddy, Manjeet K. Rao, Rajeshwar R. Tekmal, Hareesh B. Nair, Klaus J. Nickisch, Ratna K. Vadlamudi. Novel combination therapy for treating TNBC using LIFR and HDAC Inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 562.

The development of novel targeted therapies is urgently required for improving the outcome of breast cancer patients. Chemotherapy is the common treatment option for malignant breast cancer. However, resistance and toxicity remain the major obstacles hindering the effectiveness of chemotherapeutic agents in cancer patients. Therefore, identifying genes/factors that sensitize breast cancer cells to chemotherapeutic agents could improve treatment outcome in patients. Using an unbiased high-throughput screen, we identified Signal peptide CUB domain EGF-like 3 (SCUBE3) genes as a novel therapeutic adjuvant that can improve the efficacy of doxorubicin, a chemotherapeutic agent commonly used in treating breast cancer patients. Our findings demonstrated that SCUBE3 promotes breast cancer cells' progression as knockdown of SCUBE3 inhibited the ability of breast cancer cells to form colony, migrate, and invade, while overexpression of SCUBE3 promoted tumor growth in preclinical mouse models. Our results revealed that SCUBE3 mediates its protumor effects by regulating genes involved in growth and survival in the MAPK pathway, DNA damage surveillance pathway including RAD51 and FOXM1, and apoptotic pathway including Mcl-1. Using interaction studies, we demonstrated that EGFR is a true receptor of SCUBE3 as EGFR and SCUBE3 interact and this interaction mediated progrowth signaling of SCUBE3. These findings underline the importance of SCUBE3 as a potent therapeutic target for treating breast cancer patients. Citation Format: Benjamin C. Onyeagucha, Kashish Dhillon, Subapriya Rajamanickam, Subbarayalu Panneerdoss, Vijay K. Eedunuuri, Tabrez A. Mohammad, Santosh Timilsina, Yidong Chen, Manjeet K. Rao. SCUBE3 inhibition improves doxorubicin response in breast cancer [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr B066.

Aberrant activation of Wnt signaling has been implicated in various human cancers including breast cancer. Several intra-cellular signal transduction pathways are induced by Wnt, notably by Wnt/β-catenin dependent pathway. In breast cancer, triple negative breast cancer (TNBC) subtypes have increased Wnt/β-catenin signaling associated with high grade tumors, metastasis, poor prognosis and tumor regression. Importantly, significant upregulation of Wnt/β-catenin pathway and overexpression of Wnt transcriptional targets have emerged in breast cancer stem cells (BCSCs) than normal mammary epithelial stem cells. Blockade of Wnt/β-catenin signaling has been reported to suppress breast cancer growth, invasion, migration and induced apoptosis and “stemness”. WNT pathway inhibition preferentially reduces stem cell like activity in patient-derived metastatic breast cancer compared to normal cells. Collectively, these studies suggest that Wnt/β-catenin pathway could be the potential target in inhibiting TNBC growth, metastatic progression and “stemness”. We recently identified that imipramine, an FDA approved antidepressant used to treat chronic depression, inhibits TNBC growth and progression. Imipramine treatment significantly reduced cell viability, migration and invasion of breast cancer cells in general and TNBC cells in particular. Our preliminary findings demonstrated that systemic delivery of imipramine suppressed TNBC growth in orthotopic mouse xenograft model. Our studies further show that imipramine inhibits several Wnt/β-catenin catenin pathway proteins including WNT1, FZD1, β-catenin, cyclin D1 and c-myc, which are reported to support breast cancer “stemness” and metastasis. Importantly, our preliminary studies revealed that imipramine inhibits TNBC stem cells as evidenced by reduced CD44+ population. Our results suggest the therapeutic efficacy of imipramine as a single/or neo-adjuvant agent for treating TNBC patients. Citation Format: Subapriya Rajamanickam, Santosh Timilsina, Ismoil Jatoi, Virginia Kaklamani, Ratna K Vadlamudi, Manjeet K Rao. Targeting Wnt/β-catenin pathway by antidepressant imipramine for triple negative breast cancer treatment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-10.

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.

The biology of acute myelogenous leukemia (AML) is characterized by a block in differentiation, increase in proliferation and inhibition of apoptosis, all of which when combined lead to an expansion of leukemic blasts.1 AML therapy cures ~20% of those affected,2 highlighting the need for a better understanding of leukemia biology in order to identify novel therapeutic targets. The Wilms’ tumor 1 (WT1) gene has an oncogenic role in leukemogenesis and its overexpression correlates with a poor prognosis.3 WTAP is a highly conserved protein that partners with WT14,5 to function as a switch gene, regulating the balance between quiescence and proliferation.6 WTAP-null mice exhibit embryonic lethality.7 WTAP has been recently described as an oncogenic factor in gliomas.8 We hypothesized that alterations in WTAP, whose role in leukemogenesis is unknown, might provide an alternate means of modulating the WT1 pathway in AML. We performed a series of experiments to test this possibility. First, we examined WTAP expression in several primary AML samples observing increased levels of WTAP in AML compared with normal peripheral blood mononuclear cells (Figure 1a) as well as in AML cell lines (data not shown). Next, the WTAP expression levels were determined in 511 newly diagnosed AML patients using the reverse-phase protein array (RPPA) technique. In comparison with normal bone marrow CD34+ cells, WTAP expression in bulk AML cells was above normal levels in 32% of patients (Figure 1b). Although WTAP levels were not associated with individual cytogenetic abnormalities, some specific molecular mutations such as NPM1 and FLT3-ITD were found to have significant correlation (P ≤ 0.05) with WTAP expression (Supplementary Figure 1A). In addition, RPPA analysis showed that WTAP levels were positively correlated (R > |0.2|) with levels of various cell proliferation-related proteins (cyclins and Hsp90), antiapoptotic proteins (Bcl-2 and Bax), oncoproteins (FLI1) and proteins important for stem cell functions such as Myc and Ash2L (Supplementary Figure 1B). To assess the functional significance of increased WTAP expression, its expression was silenced in K562 and HL-60 cells, leading to a significant reduction (P ≤ 0.05) in proliferation (Figure 1c), and clonogenic survival (P ≤ 0.01) (Figure 1d). Similar effects on proliferation were observed in the AML cell line OCI-AML3 and in primary AML cells (Supplementary Figure 1C), suggesting a pro-proliferative role for WTAP in AML. WTAP knockdown alone did not induce apoptosis but markedly increased (P ≤ 0.01) the extent of apoptosis following etoposide treatment (Figure 1e). These results provide evidence for an association between the increased expression of WTAP and chemoresistance in AML. Figure 1 Expression of WTAP in AML and effect of WTAP silencing on AML cell behavior. (a) Peripheral blood mononuclear cells from normal donors (NL) and AML patients (AML) were obtained by Ficoll–Paque density centrifugation, and protein extracts were ... To examine the role of WTAP in AML progression in vivo, we performed tumor xenograft experiments in nude mice. As shown in Figure 1f, the growth rates and masses of tumors derived from WTAP-knockdown cells were significantly reduced (P ≤ 0.01) compared with control. To complement this analysis, the in vitro transforming activity of WTAP was examined by investigating its effects on growth of the Ba/F3 cell line. This line depends on interleukin 3 (IL-3) for survival and proliferation, but this dependence can be released by the transgenic expression of suitable oncogenes.9 Whereas control Ba/F3 cells were not viable in the absence of IL-3 at 72 h, WTAP-expressing Ba/F3 cells were able to maintain growth factor-independent proliferation, as demonstrated by significantly higher (P ≤ 0.01) number of viable cells (Figure 1g), suggesting that WTAP harbors oncogenic activity. The aberrant cellular proliferation and terminal differentiation block of myeloid cells are two hallmarks of AML.10 Having shown that WTAP regulates growth and survival, we investigated whether WTAP has a role in myeloid cell differentiation. As shown in Figure 1h, knockdown of WTAP promoted phorbol 12-myristate 13-acetate (PMA)-induced myeloid differentiation, as revealed by an increase in the expression of myeloid differentiation markers CD11b and CD14 compared with control cells. These results suggest that increased expression of WTAP in AML not only supports cell proliferation but also induces the differentiation block. Our RPPA analysis suggested a link between WTAP and mammalian target of rapamycin (mTOR) expression; and given that the mTOR pathway is deregulated in a number of cancers including AML,11 we hypothesized a putative regulatory role of WTAP on mTOR activity in AML. As shown in Figure 1i, WTAP knockdown induced a decrease in the phosphorylation levels of mTOR and its downstream effector p70 ribosomal subunit 6 kinase (pS6K) compared with control shRNA. To further understand the participation of WTAP in leukemogenesis, we performed transcriptomic analysis with RNA-Seq on WTAP knockdown in K562 cells. Gene ontology analysis indicated that cell adhesion and regulation of cell proliferation are the most enriched functionalities (Supplementary Figure 1D and Supplementary Table 2). Among the most relevant genes affected by WTAP with recognized roles in leukemia are CD4, CD44, CEBPA, CSF1R, MPO, ABCG2, TCL1A, CYP1A1, CYP3A4, FGFR1, PTPRC (CD45), CD83, CD86, CD9 and CCR4. Consistent with its described role in RNA processing, we determined that WTAP knockdown affected exon usage of 93 genes, including important factors such as MLL and MSI2. Interestingly, we also observed that WTAP affects the processing of its own transcript, very likely via polyA site selection. Complete analysis can be accessed in GEO ({"type":"entrez-geo","attrs":{"text":"GSE46718","term_id":"46718"}}GSE46718—http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jbkjjswaqscegjsa Fisher’s exact test) in their mRNA levels as determined by RNA-Seq. Mutations of WTAP were not observed in the TCGA analysis of AML.12 Therefore, the etiology of increased WTAP expression in AML remains unexplained. We next sought to determine the potential mechanism that may contribute to an increase in WTAP expression in AML. The molecular chaperone Hsp90 maintains the stability of many tumor-promoting oncoproteins,13 including WT1.14 Keeping in mind the connection between WT1 and WTAP, we investigated the potential interaction between Hsp90 and WTAP. First, we determined that WTAP co-immunoprecipitates with Hsp90 (Figure 2a), whereas treatment with the Hsp90 inhibitor ganetespib significantly reduced the binding of Hsp90 to WTAP. Therefore, formation of the WTAP–Hsp90 complex is dependent on the chaperoning activity of Hsp90. Studies have shown that Hsp90 client proteins shift the primary chaperone association from Hsp90 to Hsp70 following inhibition of Hsp90 activity.15 Accordingly, our results showed that ganetespib treatment increased WTAP association with Hsp70 (Figure 2a). Furthermore, the GST pull-down assay showed a direct interaction between WTAP and Hsp90 (Figure 2b). To understand the functional significance of the association of Hsp90 with WTAP, we investigated the effects of Hsp90 inhibitors on WTAP protein stability. Ganetespib treatment promoted the degradation of WTAP in K562 (CML), MV4-11 (AML) and Kasumi-1 (AML) cell lines, previously shown to be highly sensitive to ganetespib (Figure 2c).16 Marked reduction in WTAP expression was similarly observed in blasts exposed to ganetespib (Figure 2d). Furthermore, ganetespib inhibited AML tumor growth in nude mice, as we reported previously,14 and WTAP expression in xenograft tumors (Figure 2e). Inhibition of Hsp90 induces polyubiquitination and proteasomal degradation of Hsp90 client proteins.17 We confirmed that pretreatment with proteasomal inhibitor bortezomib largely prevented ganetespib-induced degradation of WTAP (Figure 2f), and higher levels of ubiquitinated WTAP were observed in the presence of the combination of bortezomib and ganetespib compared with either agent alone (Figure 2g). Together, these data show that ganetespib-mediated degradation of WTAP is dependent on the ubiquitin-proteasome pathway, very similar to other bonafide Hsp90 client proteins. Figure 2 Hsp90 associates with WTAP and is necessary for its stability. (a) The K562 cells were treated with vehicle (−) or Hsp90 inhibitor, ganetespib (1 µm for 6 h) followed by immunoprecipitation (IP) with IgG and WTAP antibody. The immunoprecipitates ... In summary, our study shows that WTAP has an important role in abnormal proliferation and arrested differentiation of leukemia cells, and WTAP is a novel client protein of Hsp90. Therefore, WTAP may be a promising novel therapeutic target in AML. Further investigation aimed to elucidate the mechanism of action of WTAP is warranted.

Osteosarcoma (OS) is the most common primary bone tumor and third most common cancer in children/teens, after lymphomas and brain tumors. OS is treated by surgery and multi-modal chemotherapy. Even though this treatment regimen has improved the 5-year survival rate to 60-70%, once osteosarcoma metastasize to lung and other bones, the survival is significantly reduced. Moreover, the quality of life for patients who do survive is often substantially reduced due to the toxicity associated with the chemotherapy. Since several decades, there hasn’t been any improvement in the osteosarcoma treatment outcome. Therefore, it is important to understand the molecular mechanism of osteosarcoma growth and metastasis and an urgent need to develop effective drug, which can be safe and effective for treating OS patients. We discovered that RNA methylation may regulate OS growth and progression by affecting autophagy. Our studies revealed that RNA demethylase AlkB homolog 5 (ALKBH5) supports OS viability, migration and invasion as well as tumor growth. We discovered several autophagy related genes were significantly altered in ALKBH5-silenced OS cells compared to scrambled control. Importantly, levels of LC3, which is a universal marker for monitoring autophagy, was significantly increased in ALKBH5 depleted OS cells. Furthermore, we found that autophagy initiation complex protein ULK1 level was drastically increased in ALKBH5-silenced cells when compared to scrambled control. To further establish the role of autophagy in osteosarcomogenesis, we treated OS cells with chloroquine, which is a well-known autophagy inhibitor and is currently being investigated for treating adult cancers in clinical trials. Interestingly, silencing of ALKBH5 significantly improved the efficacy of chloroquine as revealed by drastically reduced viability and invasive ability of OS cells. In conclusion, our study is first to shows that RNA methylation plays a critical role osteosarcoma growth and progression by regulating autophagy. Citation Format: Panneerdoss Subbarayalu, Pooja Yadav, Manjeet Rao. RNA methylation regulates osteosarcoma growth and progression via autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4274.

Intrinsic and acquired resistance to antineoplastic agents continues to be a major clinical impediment to successful treatment of cancer in general, and breast cancer in particular. For example, about 40% of ER-positive metastatic breast tumors fail to respond to tamoxifen therapy, and of those that initially respond many will eventually develop resistance. Similarly, taxanes used as a first-line chemotherapy agent will have a response rate of 50%, which comes down to 20–30% in second- or third-line chemotherapy; nearly half of the treated subjects do not respond to it and suffer side effects. These facts underscore the importance of understanding the mechanism of resistance, which is crucial for the optimal systemic therapy of breast cancer. Unfortunately, in spite of extensive inquiry in this field, little is known about the key molecules/signaling pathways that regulate this phenomenon. Recently, we have discovered that small non-coding RNAs, i.e. microRNA (miRNAs), may play a critical role in mediating drug resistance in breast cancers. We have identified miRNAs that are differentially expressed between drug-resistant and sensitive breast cancer cells. Specifically, we have combined a high-throughput screening platform with a library of chemically synthesized inhibitors for all known human miRNAs and identified specific miRNA that selectively sensitize/de-sensitize drug-resistant breast cancer cells to either tamoxifen or trastuzumab (herceptin), drugs that are commonly used as a first line treatment for ER-positive and Her2-positive breast cancers, respectively. We identified 10 and 12 miRNA inhibitors that sensitized resistant cells to tamoxifen and trastuzumab, respectively. Interestingly, expression of cognate miRNA/s that sensitize resistant tumors to drug-induced cell death in our screen was found to be lower in relapsed (drug-resistant) metastatic breast cancer patients compared to normal matched control. In addition to tumor tissues, the levels of sensitizer miRNA/s in the blood sera of relapsed metastatic breast cancer patients was found to be dramatically lower than that of normal healthy individual. These findings are significant as it suggests that miRNAs can be efficiently detected in blood serum and therefore, can be used as a potential biomarker for predicting treatment outcomes. In addition, miRNAs may provide novel therapeutic tools for the treatment of drug-resistant breast cancers. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P4-07-10.

Tumor metastasis is the leading cause of death among breast cancer patients. PELP1 (proline, glutamic acid and leucine rich protein 1) is a nuclear receptor coregulator that is upregulated during breast cancer progression to metastasis and is an independent prognostic predictor of shorter survival of breast cancer patients. Here, we show that PELP1 modulates expression of metastasis-influencing microRNAs (miRs) to promote cancer metastasis. Whole genome miR array analysis using PELP1-overexpressing and PELP1-underexpressing model cells revealed that miR-200 and miR-141 levels inversely correlated with PELP1 expression. Consistent with this, PELP1 knockdown resulted in lower expression of miR-200a target genes ZEB1 and ZEB2. PELP1 knockdown significantly reduced tumor growth and metastasis compared with parental cells in an orthotopic xenograft tumor model. Furthermore, re-introduction of miR-200a and miR-141 mimetics into PELP1-overexpressing cells reversed PELP1 target gene expression, decreased PELP1-driven migration/invasion in vitro and significantly reduced in vivo metastatic potential in a preclinical model of experimental metastasis. Our results demonstrated that PELP1 binds to miR-200a and miR-141 promoters and regulates their expression by recruiting chromatin modifier histone deacetylase 2 (HDAC2) as revealed by chromatin immunoprecipitation, small interfering RNA and HDAC inhibitor assays. Taken together, our results suggest that PELP1 regulates tumor metastasis by controlling the expression and functions of the tumor metastasis suppressors miR-200a and miR-141.

Intrinsic and acquired resistance to antineoplastic agents continues to be a major clinical impediment to successful treatment of cancer in general, and breast cancer in particular. For example, about 40% of ER-positive breast tumors fail to respond to tamoxifen therapy, and of those that initially respond many will eventually develop resistance. Similarly, taxanes used as a first-line chemotherapy agent will have a response rate of 50%, which comes down to 20–30% in second- or third-line chemotherapy; nearly half of the treated subjects do not respond to it and suffer side effects. These facts underscore the importance of understanding the mechanism of resistance, which is crucial for better management of breast cancer. Unfortunately, in spite of extensive inquiry in this field, little is known about the key molecules/signaling pathways that regulate this phenomenon. Recently, we have discovered that small non-coding RNAs, i.e. microRNA (miRNAs), may play a critical role in mediating drug resistance in breast cancers. We have identified miRNAs that are differentially expressed between chemoresistant and sensitive breast cancer cells. Specifically, we have combined a high-throughput screening platform with a library of chemically synthesized inhibitors for all known human miRNAs and identified specific miRNA that selectively sensitize/de-sensitize drug-resistant breast cancer cells to either tamoxifen or trastuzumab (herceptin), drugs that are commonly used as a first line treatment for ER-positive and Her2-positive breast cancers, respectively. Interestingly, we identified 10 and 12 miRNA inhibitors that sensitized resistant cells to tamoxifen and trastuzumab, respectively. Interestingly, expression of cognate miRNA/s that sensitize resistant tumors to drug-induced cell death in our screen was found to be lower in relapsed (drug resistant) metastatic breast cancer patients compared to normal matched control. In addition to tumor tissues, the levels of sensitizer miRNA/s in the blood sera of relapsed metastatic breast cancer patients was found to be dramatically lower than that of normal healthy individual. These findings are significant as it suggests that miRNAs can be efficiently detected in blood serum and therefore, can be used as a potential biomarker for predicting treatment outcomes. In addition, miRNAs may provide novel therapeutic tools for the treatment of drug-resistant breast cancers. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-14.

Approximately 15–20% of all breast cancers account for triple negative breast cancers (TNBCs) that exhibit an aggressive, distinct metastatic pattern and poor prognosis resulting in a disproportionate number of breast cancer deaths. More than 50% of patients with TNBC develop chemoresistance and do not respond to chemotherapeutic drugs, leading to early relapse and shorter survival. Understanding the mechanisms underlying such resistance is therefore crucial for the development of new, efficacious cancer drugs. In spite of extensive inquiry in this field, little is known about the key molecules/signaling pathways that regulate this phenomenon. Recently, we have discovered that small non-coding RNAs-miRNAs play critical roles in mediating drug sensitivity/resistance in TNBCs. We have identified miRNAs that are differentially expressed between chemo-resistant and sensitive breast cancer cells. In particular, through high-throughput miRNA inhibitor library screens, we have identified miRNA inhibitors that sensitize resistant TNBC cells to paclitaxel, a drug commonly used to treat triple negative breast cancers. Interestingly, our studies revealed that specific miRNAs including miR-185 (patent pending) that sensitize resistant TNBCs to paclitaxel are expressed at significantly lower levels in relapsed metastatic TNBC patient sera compared to sera from their healthy siblings. Furthermore, using liposome- or biocompatible PLGA nanoparticle-based approaches, we show that systemic delivery of one of the sensitizer miRNAs suppresses breast cancer lung metastasis without any hepatotoxicity in preclinical mouse tumor models. Importantly, we show that one of the mechanisms by which sensitizer miRNAs regulate paclitaxel sensitivity is by targeting Stathmin, a microtubule destabilizer protein that can bind to tubulin dimers and stimulate microtubule destabilization. These findings suggest that tumor-specific miRNAs render selective cell cytotoxicity in a drug-specific manner, that these miRNAs may serve as detection markers for identifying patients who might benefit most from specific drug treatment, and that these miRNAs may represent novel therapeutic tools for the treatment of the TNBCs. Since miRNAs are endogenously expressed and can be easily manipulated using synthetic oligoribonucleotides, we believe that they represent more attractive targets than the single gene or gene product targeted by conventional cancer treatments that are typically prone to drug resistance. Since paclitaxel is used for the treatment of many cancers, the long-term implications of this study are likely not limited to breast cancer alone but may apply to other tumor types. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P5-10-16.

Tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL) is considered a promising cancer therapeutic agent due to its ability to induce apoptosis in a variety of cancer cells, while sparing normal cells. However, many human tumors including acute myeloid leukemia (AML) are partially or completely resistant to monotherapy with TRAIL, limiting its therapeutic utility. Therefore, identification of factors that contribute to TRAIL resistance may facilitate future development of more effective TRAIL-based cancer therapies. Here, we report a previously unknown role for WT1 in mediating TRAIL resistance in leukemia. Knockdown of WT1 with shRNA rendered TRAIL-resistant myeloid leukemia cells sensitive to TRAIL-induced cell death, and re-expression of shRNA-resistant WT1 restored TRAIL resistance. Notably, TRAIL-mediated apoptosis in WT1-silenced cells was largely due to down-regulation of the antiapoptotic protein Bcl-xL. Moreover, WT1 expression strongly correlated with overexpression of Bcl-xL in AML cell lines and blasts from AML patients. Furthermore, we found that WT1 transactivates Bcl-xL by directly binding to its promoter. We previously showed that WT1 is a novel client protein of heat shock protein 90 (Hsp90). Consistent with this, pharmacological inhibition of Hsp90 resulted in reduced WT1 and Bcl-xL expression leading to increased sensitivity of leukemia cells to TRAIL-mediated apoptosis. Collectively, our results suggest that WT1-dependent Bcl-xL overexpression contributes to TRAIL resistance in myeloid leukemias.

A thorough understanding of the events during mammalian spermatogenesis requires studying specific molecular signatures of individual testicular cell populations as well as their interaction in co-cultures. However, most purification techniques to isolate specific testicular cell populations are timeconsuming, require large numbers of animals, and/or are only able to isolate a few cell types. Here we describe a cost-effective and timesaving approach that uses a single protocol to enrich multiple testicular cell populations (Sertoli, Leydig, and several spermatogenic cell populations) from as few as one mouse. Our protocol combines rigorous enzymatic digestion of seminiferous tubules with counter-current centrifugal elutriation, yielding specific testicular cell populations with >80%–95% purity.

The aberrant overexpression of Wilms tumor 1 (WT1) in myeloid leukemia plays an important role in blast cell survival and resistance to chemotherapy. High expression of WT1 is also associated with relapse and shortened disease-free survival in patients. However, the mechanisms by which WT1 expression is regulated in leukemia remain unclear. Here, we report that heat shock protein 90 (Hsp90), which plays a critical role in the folding and maturation of several oncogenic proteins, associates with WT1 protein and stabilizes its expression. Pharmacologic inhibition of Hsp90 resulted in ubiquitination and subsequent proteasome-dependant degradation of WT1. RNAi-mediated silencing of WT1 reduced the survival of leukemia cells and increased the sensitivity of these cells to chemotherapy and Hsp90 inhibition. Furthermore, Hsp90 inhibitors 17-AAG [17-(allylamino)-17-demethoxygeldanamycin] and STA-9090 significantly reduced the growth of myeloid leukemia xenografts in vivo and effectively down-regulated the expression of WT1 and its downstream target proteins, c-Myc and Bcl-2. Collectively, our studies identify WT1 as a novel Hsp90 client and support the crucial role for the WT1–Hsp90 interaction in maintaining leukemia cell survival. These findings have significant implications for developing effective therapies for myeloid leukemias and offer a strategy to inhibit the oncogenic func-tions of WT1 by clinically available Hsp90 inhibitors.

Despite recent improvements in the overall survival of medulloblastoma (MB), only a modest percentage of patients survive high-risk disease. Furthermore, the toxicity associated with current therapies inflicts debilitating lifelong side effects on surviving MB patients. Using an unbiased microRNA screen, we identified miR-584-5p as a potent tumor suppressor that uniquely sensitizes MB to both ionizing radiation (IR) and vincristine (VCR), which are commonly used in combination to treat MB. Our studies revealed that miR-584-5p inhibited MB stem cell proliferation and consequently growth in vivo. Notably, we discovered that miR-584-5p imparts its tumor-suppressor and therapy-sensitizing effects by inhibiting histone deacetylase 1 (HDAC1), eukaryotic translation initiation factor 4e family member 3 (eIF4E3), and C-MYC. We demonstrated that miR-584-5p overexpression or the knockdown of its target genes including eIF4E3 and HDAC1 resulted in G2-M arrest, DNA damage, spindle defects, and apoptosis in MB cells. Meta-analysis of normal brain and MB tumor samples showed that while miR-584-5p is highly expressed in normal brain and cerebellum, it is highly downregulated across all MB subtypes. miR-584-5p targets, on the other hand, are highly overexpressed in MB tumors compared to normal cerebellum. Taken together, this study highlights a previously unidentified role for miR-584-5p, eIF4E3, and HDAC1 in regulating microtubule dynamics and DNA repair pathways. In addition, our study identifies novel targets that can affect the therapeutic efficacy of VCR and IR in MB and sets the stage for a new paradigm of treating medulloblastoma using microRNA-based therapeutics. Citation Format: Nourhan Abdelfattah, Subapriya Rajamanickam, Santosh Timilsina, Panneerdoss Subbarayalu, Benjamin Onyeagucha, Manjeet Rao. Tumor suppressor miR-584-5p regulates MYC and sensitizes MYC-amplified medulloblastoma to vincristine and ionizing radiation [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr A45.

Osteosarcoma (OS) is the most prevalent primary bone malignancy that affects children and young adults. Despite several years of research, survival outcome of OS patients has not improved in last three decades. OS is treated with multi-modal chemotherapy, which is highly toxic and does not work well for metastatic and chemo-resistant tumors. Currently there is no FDA approved drug that can serve as an alternative to chemotherapy, warranting an urgent need to find more efficacious and targeted therapeutics for OS. Here, we report that RNA demethylase AlkB Homolog 5 (ALKBH5) may serve as a novel therapeutic adjuvant for treating OS. N6 Methyladenosine (m6 A) is the most common internal mRNA modification, which is modulated by the multi-component RNA methyltransferase complex, RNA demethylase (ALKBH5) and m6A readers . We show that ALKBH5 is amplified in sarcomas and its expression is highly elevated in osteosarcoma patients. We demonstrate that silencing of ALKBH5 inhibits the OS growth and migration without affecting the viability of normal human fetal osteoblast cells. Our results reveal that ALKBH5 depletion impairs the cell cycle progression and induces apoptosis in OS cells. Interestingly, we demonstrate that reduction in ALKBH5 levels suppresses the DNA damage repair capacity of osteosarcoma cells rendering them sensitive to DNA damaging agent like Doxorubicin. Using DR-GFP reporter-based homologous recombination (HR) assay, we show that ALKBH5 depletion leads to reduced HR-mediated DNA repair capacity of osteosarcoma cells. Supporting this, we observed significantly reduced expression of several genes that are known to play critical roles in cell cycle progression and DNA damage repair. In summary, this study shows that ALKBH5 is a critical regulator of OS growth and chemosensitivity. Approaches aimed at silencing ALKBH5 can be potentially used to inhibit osteosarcoma growth and progression as well as sensitize osteosarcoma cells to DNA damaging agents. Citation Format: Pooja Yadav, Panneerdoss Subbarayalu, Nourhan Abdelfattah, Vijay K. Eedunuri, Yidong Chen, Manjeet K. Rao. N6Methyladenosine RNA demethylase ALKBH5 as a novel therapeutic target for osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4146.

Homeobox genes encode transcription factors that are essential for normal development and are often dysregulated in cancers. The molecular mechanisms that cause their misregulation in cancers are largely unknown. In this study, we investigate the mechanism by which the Six1 homeobox protein, which has a crucial role during development, is frequently deregulated in several poor outcome, aggressive, metastatic adult human cancers, including breast cancer, ovarian cancer, hepatocellular carcinoma and pediatric malignancies such as rhabdomyosarcoma and Wilms' tumor. Our results reveal that miRNA-185 translationally represses Six1 by binding to its 3'-untranslated region. Analyses of ovarian cancers, pediatric renal tumors and multiple breast cancer cell lines showed decreased miR-185 expression, paralleling an increase in Six1 levels. Further investigation revealed that miR-185 impedes anchorage-independent growth and cell migration, in addition to suppressing tumor growth in vivo, implicating it to be a potent tumor suppressor. Our results indicate that miR-185 mediates its tumor suppressor function by regulating cell-cycle proteins and Six1 transcriptional targets c-myc and cyclin A1. Furthermore, we show that miR-185 sensitizes Six1-overexpressing resistant cancer cells to apoptosis in general and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in particular. Together, our findings suggest that the altered expression of the novel tumor suppressor miR-185 may be one of the central events that leads to dysregulation of oncogenic protein Six1 in human cancers.

Triple-negative breast cancers (TNBCs) represent aggressive heterogeneous subtype of breast cancer with poor clinical outcome. TNBCs have been reported to have high levels of replication stress due to i) various oncogene activations (C-myc or EGFR) ii) germline BRCA mutations iii) “BRCAness” in the absence of BRCA mutations in sporadic TNBCs. Replication stress is known to cause genomic instability, promote tumorigenesis and plays a critical role in therapy resistance in TNBCs. Therefore, targeting replication stress has emerged as an effective approach for better TNBC treatment through further downregulation of the remaining checkpoints to induce catastrophic failure of TNBC cells proliferation. Herein, we evaluated the anticancer efficacy of Carbazole Blue (CB), a synthetic analogue of Carbazole, on TNBC cells growth and progression. Our results demonstrated that CB inhibits short and long term viability of TNBC (MDA-MB-231, MDA-MB-468 and BT549) cells in a dose dependent manner without affecting normal mammary epithelial (MCF-10A) cells. In addition, CB treatment significantly reduced proliferation of TNBC cells, as evidenced by the BrdU proliferation assay. Consistent with this, our results further demonstrated that CB treatment induced G1/S cell cycle arrest and apoptosis in TNBCs. Importantly, systemic delivery of CB using nanoparticle-based delivery approach suppressed breast cancer growth without inducing toxicity, in preclinical orthotopic xenograft and PDX mouse models of TNBC. Furthermore, our gene microarray analysis revealed that CB treatment modulates the expression and activity of several genes known to be involved in DNA replication (CDC6, CDT1, MCMs, Claspin, POLE and PCNA) and associated DNA repair machinery such as (XRCC3, Exo1 and RAD51), which play pivotal roles in replication stress. Our results for the first time highlight the potential use of CB as a novel and potent therapeutic agent for treating TNBCs. As exploiting replication stress to treat cancer is gaining major interest, compound/s that may induce replication stress and inhibit DNA repair ability of cancer cells, has immense translational potential. Citation Format: Rajamanickam S, Park JH, Bates K, Timilsina S, Eedunuri VK, Onyeagucha B, Subbarayalu P, Abdelfattah N, Jung KH, Favours E, Mohammad TA, Chen H-IH, Vadlamudi RK, Chen Y, Kaipparettu BA, Arbiser JL, Rao MK. Targeting replication stress in triple negative breast cancer treatment regimen: An emerging approach [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-06-04.

The reproductive homeobox X-linked, Rhox, genes encode transcription factors that are selectively expressed in reproductive tissues. While there are 33 Rhox genes in mice, only Rhox and Rhox8 are expressed in Sertoli cells, suggesting that they may regulate the expression of somatic-cell gene products crucial for germ cell development. We previously characterized Rhox5-null mice, which are subfertile, exhibiting excessive germ cell apoptosis and compromised sperm motility. To assess the role of Rhox8 in Sertoli cells, we used a tissue-specific RNAi approach to knockdown RHOX8 in vivo, in which the Rhox5 promoter was used to drive Rhox8-siRNA transgene expression in the postnatal Sertoli cells. Western and immunohistochemical analysis confirmed Sertoli-specific knockdown of RHOX8. However, other Sertoli markers, Gata1 and Rhox5, maintained normal expression patterns, suggesting that the knockdown was specific. Interestingly, male RHOX8-knockdown animals showed significantly reduced spermatogenic output, increased germ cell apoptosis, and compromised sperm motility, leading to impaired fertility. Importantly, our results revealed that while some RHOX5-dependent factors were also misregulated in Sertoli cells of RHOX8-knockdown animals, the majority were not, and novel putative RHOX8-regulated genes were identified. This suggests that while reduction in levels of RHOX5 and RHOX8 in Sertoli cells elicits similar phenotypes, these genes are not entirely redundant. Taken together, our study underscores the importance of Rhox genes in male fertility and suggests that Sertoli cell-specific expression of Rhox5 and Rhox8 is critical for complete male fertility.

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-3'-UTR constructs. In contrast, mutant BOK-3'-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.

Although many genes are expressed selectively in Sertoli cells, regulatory sequences sufficient to drive Sertoli cell-specific expression in the postnatal and adult testis in vivo have not been identified. In the present study, we identified promoter sequences from the Pem homeobox gene that direct Sertoli cell-specific expression in an androgen-dependent and stage-specific manner. Immunohistochemical and RNA analysis demonstrated that 0.6-kb 5'-flanking sequence directed transgene expression specifically in the testis and the epididymis but not in any other tissues tested. In the adult testis, this promoter fragment targeted the transgene expression specifically to Sertoli cells during stages IV-VIII of the seminiferous epithelial cycle, thereby mimicking the expression pattern of the endogenous Pem gene. This promoter fragment also recapitulated Pem's normal postnatal expression pattern, as it directed transcript induction between d 6 and d 9 post partum. Deletion of 0.3 kb from the 5'-end of the transgene had no effect on androgen-dependent Sertoli-specific expression but altered stage-specific expression in adult testes and caused premature postnatal expression. Our results suggest that there are at least two regulatory regions in the Pem proximal promoter: one that directs androgen receptor-dependent expression specifically in Sertoli cells within the testis and another that confers stage-specific expression in neonates and adults by acting as a negative regulator. To our knowledge, this is the first identification of regulatory regions that direct faithful developmentally regulated gene expression in postnatal and adult Sertoli cells in vivo.

Background: Triple-negative breast cancers (TNBC) are the most aggressive forms of breast cancer and almost 60% of patients with TNBCs develop chemo-resistance, leading to recurrence, poor prognosis and poor survival. TNBCs have been reported to have high levels of replication stress, which plays pivotal role in genomic instability, and therapy resistance. Targeting replication stress is an emerging approach for better TNBC treatment. Here, we evaluated the anticancer efficacy of carbazole blue (CB), a synthetic analogue of carbazole that we recently synthesized on TNBC cells growth and progression. Experimental Design: The effect of CB on breast cancer growth was assessed in vitro as well as in orthotopic mouse xenograft and PDX-models of breast cancer. In addition, the therapeutic efficacy and safety of CB was determined in long term toxicity studies in mice and also in ex-vivo explants from breast cancer patients. The mechanism of action of CB was evaluated by performing gene expression, cell cycle, apoptosis and DNA repair studies as well as proteins involved in the above mentioned mechanisms. Results: Our results demonstrated that CB inhibits short and long term viability of TNBC cells in a dose dependent manner without affecting normal mammary epithelial cells. We show that the systemic delivery of CB using nanoparticle-based delivery approach suppressed breast cancer growth without inducing toxicity in preclinical and PDX mouse models of triple negative breast cancer. Our long term toxicity studies reveled that CB treatment did not induce any toxicity in Balb/c mice. Using ex-vivo explants from breast cancer patients, we demonstrated that CB modulated breast cancer growth. Consistent with that, our results revealed that CB treatment induced G1/S cell cycle arrest and apoptosis in TNBCs. Interestingly, our gene expression analysis revealed that CB modulates expression and activity of several genes known to be involved in DNA replication and DNA repair machinery. Conclusions: Our results for the first time showed the CB can serve as a novel and potent therapeutic agent for treating breast cancer in general and TNBC in particular. These findings highlight the potential of CB to be applied as a safe regimen for treating breast cancer patients. As exploiting replication stress to treat cancer is gaining major interest, compound/s that may induce replication stress and inhibit DNA repair ability of cancer cells, has immense translational potential. Citation Format: Subapriya Rajamanickam, Kaitlyn Bates, Santosh Timilsina, JunHyoung Park, Benjamin Onyeagucha, Panneerdoss Subbarayalu, Nourhan Abdelfattah, Kwang Hwa Jung, Edward Favours, Tabrez A. Mohammad, Hung-I Harry Chen, Benny A. Kaipparettu, Yidong Chen, Jack L. Arbiser, Manjeet K Rao. Targeting replication stress by carbazole blue- A novel strategy to treat triple negative breast cancers [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 1116. doi:10.1158/1538-7445.AM2017-1116

Medulloblastoma (MB) is the most common malignant pediatric brain tumor. Despite recent improvements in the overall survival, only a modest percentage of patients survive Myc-driven high-risk MB. The quality of life for surviving patients is substantially reduced due to the devastating and often irreversible side effects of radiation and chemotherapy. Recently, in a large unbiased genomic screen, we uncovered a group of microRNAs (miRNAs) capable of meditating drug sensitivity in c-myc amplified high-risk MB. Our functional screen of ~1900 miRNAs identified miR-584-5p as a potent candidate that uniquely sensitizes high-risk MB to radiation as well as vincristine (VCR) (20 to 25-fold dose reduction), an anti-mitotic agent routinely administered alongside radiation and in combination with other chemotherapeutic drugs to treat medulloblastoma patients. Our studies revealed that miR-584 might act as a potent tumor suppressor as it inhibited MB growth in vivo as well as migration and invasion of c-myc amplified MB. We show that miR-584 overexpression results in defective mitosis leading to mitotic catastrophe, apoptosis, DNA damage and G2-M cell-cycle arrest in high-risk MB cells without affecting normal neural stem cell growth. Notably, we discovered that miR-584 directly regulates the expression and activity of genes including histone deacetylase 1 (HDAC1), and eukaryotic translation initiation factor 4e family member 3 (EIF4E3) that are known to play important roles in microtubule dynamics, metaphase-anaphase transition and radio-resistance. Moreover, silencing either of these two target genes resulted in significant inhibition of MB growth and enhanced sensitivity to VCR and ionizing radiation. Overexpressing miR-584 or silencing either HDAC1 or EIF4E3 also inhibited the MB stem cell proliferation and self-renewal. We report that while miR-584-5p is predominantly expressed in normal brain and cerebellum, its expression is significantly reduced in MB patient derived xenografts (PDXs). In contrast to miR-584, EIF4E3 and HDAC1 were found to be overexpressed in medulloblastoma patients. These findings are highly significant, unexpected and innovative as this miRNA and its target genes are the first to be shown to affect the therapeutic efficacy of VCR and radiation in c-myc amplified high-risk medulloblastoma. Citation Format: Nourhan Abdelfattah, Subapriya Rajamanickam, Panneerdoss Subbarayalu, Santosh Timilsina, Benjamin Onyeagucha, Yidong Chen, Manjeet Rao. miRNAs as novel therapeutic adjuvants for improving the efficacy of vincristine and radiation therapy in treating medulloblastoma [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 5440. doi:10.1158/1538-7445.AM2017-5440

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

Microtubules are highly dynamic components of the cytoskeleton that play an important role in a wide range of cellular processes including cell division, cell motility and intracellular transport. Increasing evidence suggests that alterations in microtubule dynamics are critical for cancer growth and metastasis. Microtubule associated proteins (MAPs) regulate microtubule dynamics and consequently can affect sensitivity of cancer cells to microtubule targeting drugs. 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. Immunofluorescence analyses revealed that although MATR3 is predominantly a nuclear protein, it translocates to the cytoplasm and interacts with microtubules when breast cancer cells are treated with paclitaxel. We show that MATR3 associates with stabilized microtubules and it mediates microtubule polymerization in a taxol-dependent manner. Interestingly, our results reveal that MATR3 acts as a effective tumor suppressor as it inhibits breast cancer colony formation, migration and invasion of breast cancer cells in addition to suppressing breast tumor growth in vivo. Analysis of breast cancer samples showed a significantly lower expression of MATR3 when compared to normal adjacent tissues. Our results indicate the possibility that MATR3 mediates its tumor suppressor function by binding and regulating proteins that are known to affect microtubule dynamics. We believe that nuclear-cytoplasmic shuttling of MATR3 is critical for stability of key proteins that might regulate paclitaxel-dependent microtubule dynamics and subsequently cellular effects in cancer cells. Citation Format: Panneerdoss Subbarayalu, Subapriya Rajamanickam, Suryavathi Viswanadhapalli, Nicholas Dybdal-Hargreaves, Santosh Timilsina, Sanjay Bansal, Hima Bansal, Tabrez Mohammad, Yidong Chen, John C Herr, Susan L Mooberry, Manjeet K Rao. Matrin 3: A novel micro-tubule associated RNA binding protein that acts as a potent tumor suppressor [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P4-05-09.

Defects in cellular metabolism have been widely implicated in causing male infertility, but there has been little progress in understanding the underlying mechanism. Here we report that several key metabolism genes are regulated in the testis by Rhox5, the founding member of a large X-linked homeobox gene cluster. Among these Rhox5-regulated genes are insulin 2 (Ins2), resistin (Retn), and adiponectin (Adipoq), all of which encode secreted proteins that have profound and wide-ranging effects on cellular metabolism. The ability of Rhox5 to regulate their levels in the testis has the potential to dictate metabolism locally in this organ, given the existence of the blood-testes barrier. We demonstrate that Ins2 is a direct target of Rhox5 in Sertoli cells, and we show that this regulation is physiologically significant, because Rhox5-null mice fail to up-regulate Ins2 expression during the first wave of spermatogenesis and have insulin-signaling defects. We identify other Rhox family members that induce Ins2 transcription, define protein domains and homeodomain amino acid residues crucial for this property, and demonstrate that this regulation is conserved. Rhox5-null mice also exhibit altered expression of other metabolism genes, including those encoding the master transcriptional regulators of metabolism, PPARG and PPARGC1A, as well as SCD1, the rate-limiting enzyme for fatty acid metabolism. These results, coupled with the known roles of RHOX5 and its target metabolism genes in spermatogenesis in vivo, lead us to propose a model in which RHOX5 is a central transcription factor that promotes the survival of male germ cells via its effects on cellular metabolism.

Prostate cancer cells escape growth inhibition from transforming growth factor β (TGFβ) by downregulating TGFβ receptors. However, the mechanism by which cancer cells downregulate TGFβ receptors in prostate is not clear. Here, we showed that coordinated action of miR-21 and androgen receptor (AR) signaling had a critical role in inhibiting TGFβ receptor II (TGFBR2) expression in prostate cancer cells. Our results revealed that miR-21 suppresses TGFBR2 levels by binding to its 3'-UTR and AR signaling further potentiates this effect in both untransformed and transformed human prostate epithelial cells as well as in human prostate cancers. Analysis of primary prostate cancers showed that increased miR-21/AR expression parallel a significantly reduced expression of TGFBR2. Manipulation of androgen signaling or the expression levels of AR or miR-21 negatively altered TGFBR2 expression in untransformed and transformed human prostate epithelial cells, human prostate cancer xenografts and mouse prostate glands. Importantly, we demonstrated that miR-21 and AR regulated each other's expression resulting in a positive feedback loop. Our results indicated that miR-21/AR mediate its tumor-promoting function by attenuating TGFβ-mediated Smad2/3 activation, cell growth inhibition, cell migration and apoptosis. Together, these results suggest that the AR and miR-21 axis exerts its oncogenic effects in prostate tumors by downregulating TGFBR2, hence inhibiting the tumor-suppressive activity of TGFβ pathway. Targeting miR-21 alone or in combination with AR may restore the tumor inhibitory activity of TGFβ in prostate cancer.

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.

Medulloblastoma is the most common malignant brain tumor in children, accounting for 18% of newly diagnosed brain tumors and 10% of all children cancer-related deaths. Despite improvement in the 5-year survival rate of medulloblastoma in recent years, only a small percentage of patients survive high-risk metastatic disease. The quality of life for those who do survive is often substantially reduced due to the toxicity associated with radiation and chemotherapy. Vincristine is a microtubule-destabilizing antimitotic drug, which is routinely administered in higher dosages to both high and average risk medulloblastoma patients. As a result, these patients suffer from devastating neurotoxic effects that include but not limited to: sensorimotor and autonomic neuropathy, hearing loss, mononeuropathy, and seizures. Using high-throughput microRNA mimic library screens, we identified a group of microRNAs that may improve the efficacy of vincristine against c-MYC amplified medulloblastoma as well as re-sensitize vincristine-resistant medulloblastoma. Our findings revealed that these microRNAs may act as tumor suppressors since their overexpression inhibited colony formation, migration and invasion ability of medulloblastoma cells. Furthermore, these microRNAs suppressed stem cell renewal/proliferation of c-MYC amplified medulloblastoma cells. Expression analysis, gene enrichment analysis and target prediction algorithms revealed that these microRNAs exert their vincristine sensitizing and tumor suppressor effect by targeting genes involved in microtubule organization, cell cycle regulation, DNA damage repair and mRNA translation. One of our most interesting targets is EIF4E3, which is a translation initiation factor. Our preliminary findings indicate that EIF4E3 may regulate medulloblastoma cell growth, progression and vincristine sensitivity by modulating c-MYC translation. Further experiments are underway to test the potential of candidate miRNA and EIF4E3 as vincristine sensitizer in vivo. In conclusion, this study may identify novel factors that have potential not only to decrease the current therapeutic dose of vincristine and therefore eliminate its side effects, but also have potential to multiply the efficacy of lower doses in order to overcome hard to treat high-risk tumors. Citation Format: Nourhan Abdelfattah, Panneerdoss Subbarayalu, Benjamin Onyeagucha, Subapriya Rajamanickam, Hung-I Harry Chen, Manjeet Rao. MicroRNAs as novel therapeutic adjuvants to treat high-risk medulloblastoma.. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr B01.

Decoupling of transcription and translation during postmeiotic germ cell differentiation is critical for successful spermatogenesis. Here we establish that the interaction between microRNAs and actin-associated protein Arpc5 sets the stage for an elaborate translational control mechanism by facilitating the sequestration of germ cell mRNAs into translationally inert ribonucleoprotein particles until they are later translated. Our studies reveal that loss of microRNA-dependent regulation of Arpc5, which controls the distribution of germ cell mRNAs between translationally active and inactive pools, results in abnormal round spermatid differentiation and impaired fertility. Interestingly, Arpc5 functions as a broadly acting translational suppressor, as it inhibits translation initiation by blocking 80S formation and facilitates the transport of mRNAs to chromatoid/P bodies. These findings identify a unique role for actin-associated proteins in translational regulation, and suggest that mRNA-specific and general translational control mechanisms work in tandem to regulate critical germ cell differentiation events and diverse somatic cell functions.

How Sertoli-specific expression is initiated is poorly understood. Here, we address this issue using the proximal promoter (Pp) from the Rhox5 homeobox gene. Its Sertoli cell-specific expression is achieved, in part, through a negative regulatory element that inhibits Pp transcription in non-Sertoli cell lines. Complementing this negative regulation is positive regulation conferred by four androgen-response elements (AREs) that interact with the androgen receptor (AR), a nuclear hormone receptor expressed at high levels in Sertoli cells. A third control mechanism is provided by a consensus GATA-binding site that is crucial for Pp transcription both in vitro and in vivo. Several lines of evidence suggested that GATA factors and AR act cooperatively to activate Pp transcription: (i) the GATA-binding site crucial for Pp transcription is in close proximity to two of the AREs, (ii) GATA and AR form a complex with the Pp in vitro, (iii) overexpression of GATA factors rescued expression from mutant Pp constructs harboring defective AREs, and (iv) incubation of a Sertoli cell line with testosterone triggered corecruitment of AR and GATA4 to the Pp. Collectively, our results suggest that the Rhox5 gene achieves Sertoli cell-specific transcription using a combinatorial strategy involving negative and cooperative positive regulation.

Using a novel tissue-specific RNA interference (RNAi) approach that mimics the principle by which naturally occurring microRNAs (miRNA) are made, we demonstrate that the Wilms' tumor 1 (WT1) transcription factor has an essential role in spermatogenesis. Mice depleted of WT1 in Sertoli nurse cells suffered from increased germ cell apoptosis, loss of adherens junctions, disregulation of adherence junction-associated genes, and impaired fertility. These effects were recapitulated in transgenic mice expressing a dominant-negative form of WT1 in Sertoli cells, demonstrating the validity of our RNAi approach. Our results indicate that the tumor suppressor WT1 promotes Sertoli cell-germ cell signaling events driving spermatogenesis.

Correction to: Leukemia (2014) 28, 1171–1174; doi:10.1038/leu.2014.16 Since the publication of this article, the authors have identified an error concerning one of the author names. D Proia should be listed as DA Proia. The corrected list is shown above. The Authors would like to apologise for any inconvenience this may have caused.

SummaryHomeobox genes encode transcription factors notable for their ability to regulate embryogenesis. Here, we report the discovery of a cluster of 12 related homeobox genes on the X chromosome expressed in male and female reproductive tissues in adult mice. These reproductive homeobox on the X chromosome (Rhox) genes are expressed in a cell type-specific manner; several are hormonally regulated, and their expression pattern during postnatal testis development corresponds to their chromosomal position. Most of the Rhox genes are expressed in Sertoli cells, the nurse cells in direct contact with developing male germ cells, suggesting that they regulate the expression of somatic-cell gene products critical for germ cell development. In support of this, targeted disruption of Rhox5 increased male germ cell apoptosis and reduced sperm production, sperm motility, and fertility. Identification of this family of homeobox genes provides an opportunity to study colinear gene regulation and the transcriptional control of reproduction.

The epididymis is a useful model system to understand the mechanisms that govern region-specific gene expression, as many gene products display spatially restricted expression within this organ. However, surprisingly little is known about how this regulation is achieved. Here, we report regulatory sequences from the Pem homeobox gene that drive expression in different subregions of the mouse epididymis in vivo. We found that the 0.3-kb 5'-flanking sequence (region I) from the Pem proximal promoter (Pem Pp) was sufficient to confer androgen-dependent and developmentally regulated expression in the caput region of the epididymis. Expression was restricted to the normal regions of expression of Pem in the caput (segments 2-4), but there was also aberrant expression in the corpus region. This corpus misexpression was extinguished when 0.6 kb of Pem Pp 5'-flanking sequence was included in the transgene, indicating that one or more negative regulatory elements exist between 0.6 and 0.3 kb upstream of the Pem Pp start site (region II). When heterologous sequences were introduced upstream of the Pem Pp, expression was further restricted, mainly to caput segment 3, implying that the Pem Pp has segment-specific regulatory elements. To our knowledge, the regulatory regions we have identified are the shortest so far defined that dictate regionally localized expression in the epididymis in vivo. They may be useful for identifying the factors that regulate region-specific expression in the epididymis, for expressing and conditionally knocking out genes in different subregions of the epididymis, for treating male infertility, and for generating novel methods of male contraception.

To decipher the mechanism(s) underlying glucocorticoid action on cardiac contractile function, this study investigated the effects of adrenalectomy and dexamethasone treatment on the contents of sarcoplasmic reticulum (SR) Ca2+-cycling proteins, their phosphorylation by endogenous Ca2+/calmodulin-dependent protein kinase II (CaM kinase II), and SR Ca2+sequestration in the rat myocardium. Cardiac SR vesicles from adrenalectomized rats displayed significantly diminished rates of ATP-energized Ca2+uptake in vitro compared with cardiac SR vesicles from control rats; in vivo administration of dexamethasone to adrenalectomized rats prevented the decline in SR function. Western immunoblotting analysis showed that the relative protein amounts of ryanodine receptor/Ca2+-release channel, Ca2+-ATPase, calsequestrin, and phospholamban were neither diminished significantly by adrenalectomy nor elevated by dexamethasone treatment. However, the relative amount of SR-associated CaM kinase II protein was increased 2.5- to 4-fold in dexamethasone-treated rats compared with control and adrenalectomized rats. Endogenous CaM kinase II activity, as judged from phosphorylation of ryanodine receptor, Ca2+-ATPase, and phospholamban protein, was also significantly higher (50–80% increase) in the dexamethasone-treated rats. The stimulatory effect of CaM kinase II activation on Ca2+uptake activity of SR was significantly depressed after adrenalectomy and greatly enhanced after dexamethasone treatment. These findings identify the SR as a major target for glucocorticoid actions in the heart and implicate modification of the SR CaM kinase II system as a component of the mechanisms by which dexamethasone influences SR Ca2+-cycling and myocardial contraction.