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Breast Cancer (BC) is a genetically and clinically heterogeneous disease including complex interactions between gene-gene and gene-environment components. This study aimed, to explore whether the Glutathione S- transferase (GSTs) gene polymorphism has role in BC susceptibility. We further evaluated the frequency of four subtypes of BC based on molecular classification followed by microscopic histological analysis to study the grades of invasive ductal carcinoma (IDC).Polymorphism in GST genes in North-Indian BC patients was assessed by multiplex-PCR and PCR-RFLP methods. 105 BC patients and 145 healthy controls were enrolled for this study. Data was analyzed by calculating the odds ratio (OR) and 95% CI from logistic regression analyses.Our findings revealed that GSTM1 null genotype (OR = 2.231; 95% CI = 1.332-3.737; p-value= 0.002) is significantly associated to BC risk in ethnic North- Indian population. However, the risk for BC susceptibility in North-Indians does not appear to be associated with GSTT1 null genotype. The GSTP1 (Val/Val) genotype (OR=1.545; CI=0.663-3.605; p-value= 0.314) was also found to be susceptible for BC risk. Combination of three high risk GST genotypes association exhibiting gene-gene interaction further confirmed the increased risk to BC in this region.The results of present study indicated that polymorphism in GSTM1 and rs1695 of GSTP1 genes may influence BC development among North-Indian women. Thus, the screening of GSTM1 and GSTP1 gene should be recommended for the earlier investigation for BC as a precautionary measure. more...

Androgen receptor (AR) contributes to the progression of glioblastoma (GBM), and antiandrogen agents have the potential to be used for the treatment of GBM. However, AR mutation commonly happens in GBM, which makes the antiandrogen agents less effective. Heat shock 27 kDa protein (HSP27) is a well-documented chaperone protein to stabilize ARs. Inhibition of HSP27 results in AR degradation regardless of the mutation status of ARs, which makes HSP27 a good target to abolish ARs in GBM. Compound I is a HSP27 inhibitor that significantly induces AR degradation in GBM cells via the proteasomal pathway, and it selectively inhibits AR-overexpressed GBM cell growth with IC(50) values around 5 nM. The compound also significantly inhibits in vivo GBM xenograft at 20 mg/kg and does not cause toxicity to mice up to 80 mg/kg. These results suggest that targeting HSP27 to induce AR degradation in GBM is a promising and novel treatment. more...

The novel SARS-CoV-2 is responsible for causing the ongoing outbreak of coronavirus disease 19 (COVID-19), a systemic infection in humans. Ever since it was first detected in December 2019, the number of confirmed cases has continued to increase. Within a short period, this disease has become a global issue, and therefore it is characterized as a pandemic. The current understanding and explanations are based on epidemiological, clinical and physiological observations. Besides, it remains a great challenge, as much remains to be understood about this new disease-causing virus. Therefore, we seek to provide an overview of SARS-CoV-2, including its classification, origin, genomic structure, replication cycle, transmission, pathogenesis, clinical aspects, diagnosis, treatments, prevention and vaccine options. We conducted a literature search for the articles published up to August 2020 using the keywords 'SAR-CoV-2' and 'COVID19' in medical databases; PubMed, google scholar, EMBASE, and web of science. Based on the information collected, the emerging COVID-19, caused by SARS-CoV-2, exhibits strong infectivity but less virulence in terms of severity of disease and mortality rates in certain age groups. It inflicts more damage in terms of peoples' health and well-being, social life, and global economic impacts. Unfortunately, there is no adequate global and standard response to this pandemic to date, and each country is facing a crisis based on its situation, expertise, and hypotheses. While there is no effective therapy and vaccine against the novel SARS-CoV-2 yet, preventive measures are the only tool available to our disposal to control the spread of the COVID-19 pandemic. Ongoing and future research is focused more on developing standard treatment strategies, and efficacious vaccines, which would be useful to tackle this pandemic globally. more...

We have previously reported that stable expression of a dominant negative Death Receptor 5 (dnDR5) in skeletal myoblasts results in decreased basal caspase activity and decreased mRNA and protein expression of the muscle regulatory transcription factor MyoD in growth medium (GM), resulting in inhibited differentation when myoblasts are then cultured in differentiation media (DM). Further, this decreased level of MyoD mRNA was not a consequence of altered message stability, but rather correlated with decreased acetylation of histones in the distal regulatory region (DRR) of the MyoD extended promoter known to control MyoD transcription. As serum response factor (SRF) is the transcription factor known to be responsible for basal MyoD expression in GM, we compared the level of SRF binding to the non-canonical serum response element (SRE) within the DRR in parental and dnDR5 expressing myoblasts. Herein, we report that stable expression of dnDR5 resulted in decreased levels of serum response factor (SRF) binding to the CArG box in the SRE of the DRR. Total SRF expression levels were not affected, but phosphorylation indicative of SRF activation was impaired. This decreased SRF phosphorylation correlated with decreased phosphorylation-induced activation of p38 kinase. Moreover, the aforementioned signaling events affected by expression of dnDR5 could be appropriately recapitulated using either a pharmacological inhibitor of caspase 3 or p38 kinase. Thus, our results have established a signaling pathway from DR5 through caspases to p38 kinase activation, to SRF activation and the basal expression of MyoD. more...

The Androgen Receptor (AR) is a ligand-dependent transcription factor that modulates the expression of androgen-regulated genes involved in the proliferation and differentiation of normal prostate epithelial cells. However, in Prostate Cancer (PCa), these cellular events are dysregulated upon significant changes in AR, such as AR overexpression, leading to uncontrolled cell growth and proliferation. To elucidate the molecular mechanisms by which AR expression and function are controlled, we have explored the post-transcriptional regulatory RNA elements of AR, particularly the 3’ untranslated region (UTR). The AR 3’UTR is 6.8 Kilobases (Kb) long, which indicates that this region is likely a site for multitudinous levels of post-transcriptional regulation. We found that the length of AR 3’UTR negatively correlates with AR expression, with the full length 3’UTR leading to the most downregulation of expression. Currently, we are exploring cis-acting noncoding RNAs and RNA binding proteins (RBPs) predicted to interact with the AR 3’UTR. Citation Format: Eviania Likos, Girish C. Shukla. Post-transcriptional control of androgen receptor expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 869. more...

The androgen receptor (AR) plays a critical role in the development of the normal prostate as well as prostate cancer. Using an integrative transcriptomic analysis of prostate cancer cell lines and tissues, we identified ARLNC1 (AR-regulated long noncoding RNA 1) as an important long noncoding RNA that is strongly associated with AR signaling in prostate cancer progression. Not only was ARLNC1 induced by the AR protein, but ARLNC1 stabilized the AR transcript via RNA-RNA interaction. ARLNC1 knockdown suppressed AR expression, global AR signaling and prostate cancer growth in vitro and in vivo. Taken together, these data support a role for ARLNC1 in maintaining a positive feedback loop that potentiates AR signaling during prostate cancer progression and identify ARLNC1 as a novel therapeutic target. more...

The muscle regulatory transcription factor MyoD is a master regulator of skeletal myoblast differentiation. We have previously reported that MyoD is also necessary for the elevated expression of the pro-apoptotic Bcl2 family member PUMA, and the ensuing apoptosis, that occurs in a subset of myoblasts induced to differentiate. Herein, we report the identification of a functional MyoD binding site within the extended PUMA promoter. In silico analysis of the murine PUMA extended promoter revealed three potential MyoD binding sites within 2 kb of the transcription start site. Expression from a luciferase reporter construct containing this 2 kb fragment was enhanced by activation of MyoD in both myoblasts and fibroblasts and diminished by silencing of MyoD in myoblasts. Experiments utilizing truncated versions of this promoter region revealed that the potential binding site at position - 857 was necessary for expression. Chromatin immunoprecipitation (ChIP) analysis confirmed binding of MyoD to the DNA region encompassing position - 857. The increase in MyoD binding to the PUMA promoter as a consequence of culture in differentiation media (DM) was comparable to the increase in MyoD binding at the myogenin promoter and was diminished in myoblasts silenced for MyoD expression. Finally, ChIP analysis using an antibody specific for the transcription factor p53 demonstrated that, in myoblasts silenced for MyoD expression, p53 binding to the PUMA promoter was diminished in response to culture in DM. These data indicate that MyoD plays a direct role in regulating PUMA expression and reveal functional consequences of MyoD expression on p53 mediated transcription of PUMA. more...

Calorie restriction (CR) is a dietary intervention known to delay aging. In order, to understand molecular mechanisms of CR, we analyzed the expression of 983 MicroRNAs (miRNAs) in the liver of female mice after 2 years of 30% CR using micro-array. 16 miRNAs demonstrated significant changes in their expression upon CR in comparison with age-matched control. mmu-miR-125a-5p (miR-125a-5p) was significantly upregulated upon CR, and in agreement with this, the expression of mRNAs for its three predicted target genes: Stat3, Casp2, and Stard13 was significantly downregulated in the liver of CR animals. The expression of precursor miRNA for miR-125a-5p was also upregulated upon CR, which suggests its regulation at the level of transcription. Upon aging miR-125a-5p expression was downregulated while the expression of its target genes was upregulated. Thus, CR prevented age-associated changes in the expression of miR-125a-5p and its targets. We propose that miR-125a-5p dependent downregulation of Stat3, Casp2, and Stard13 contributes to the calorie restriction-mediated delay of aging. more...

Visual representation of anatomy, structure and physiology of our internal body forms a basis for therapeutic industry and medical research. Despite technological advancement in the medical field still the diagnosis of complex disease like cancer at its onset remains a major challenge. Rapid evolution in the biomedical sciences such as medical physics and nuclear medicine resulted in various non-invasive imaging techniques. This letter to editor discusses the significance, challenges and future of it. Imaging is a powerful non-invasive diagnostic tool which benefits the physicians and other healthcare professionals and it is constantly evolving towards the goal of precision-/personalized-medicine. more...

Dysregulation of miRNAs has been demonstrated in several human malignancies including prostate cancer. Due to tissue limitation and variable disease progression, stage-specific miRNAs changes in prostate cancer is unknown. Using chip-based microarray, we investigated global miRNA expression in human prostate cancer LNCaP, PC3, DU145 and 22Rv1 cells representing early-stage, advanced-stage and castration resistant prostate cancer in comparison with normal prostate epithelial cells. A total of 292 miRNAs were differentially expressed with 125 upregulated and 167 downregulated. These miRNAs were involved in pathways including drug resistance drug-efflux, adipogenesis, epithelial-to-mesenchymal transition, bone metamorphosis, and Th1/Th2 signaling. Regulation of miRNAs were interlinked with upstream regulators such as Argonaut 2 (AGO2), Double-Stranded RNA-Specific Endoribonuclease (DICER1), Sjogren syndrome antigen B (SSB), neurofibromatosis 2 (NF2), and peroxisome proliferator activated receptor alpha (PPARA), activated during stage-specific disease progression. Candidate target genes and pathways dysregulated in stage-specific prostate cancer were identified using CS-miRTar database and confirmed in clinical specimens. Integrative network analysis suggested some genes targeted by miRNAs include miR-17, let7g, miR-146, miR-204, miR-205, miR-221, miR-301 and miR-520 having a major effect on their dysregulation in prostate cancer. MiRNA-microarray analysis further identified miR-130a, miR-181, miR-328, miR146 and miR-200 as a panel of novel miRNAs associated with drug resistance drug-efflux and epithelial-to-mesenchymal transition in prostate cancer. Our findings provide evidence on miRNA dysregulation and its association with key functional components in stage-specific prostate cancer. more...

Diabetes mellitus (DM), a disease of the endocrine pancreas in which either pancreatic islets secrete insufficient insulin, or there is inefficient utilization of insulin; thus, producing hyperglycemic effects and other metabolic complications. more...

Castration-resistant prostate cancer (CRPC) is defined by tumor microenvironment heterogeneity affecting intrinsic cellular mechanisms including dysregulated androgen signaling, aerobic glycolysis (Warburg effect), and aberrant activation of transcription factors including androgen receptor (AR) and c-Myc. Using in vitro, in vivo, and animal models, we find a direct correlation between miR-644a downregulation and dysregulation of essential cellular processes. MiR-644a downregulated expression of diverse tumor microenvironment drivers including c-Myc, AR coregulators, and antiapoptosis factors Bcl-xl and Bcl2. Moreover, miR-644a modulates epithelial–mesenchymal transition (EMT) by directly targeting EMT-promoting factors ZEB1, cdk6, and Snail. Finally, miR-644a expression suppresses the Warburg effect by direct targeting of c-Myc, Akt, IGF1R, and GAPDH expression. RNA sequencing analysis revealed an analogous downregulation of these factors in animal tumor xenografts. These data demonstrate miR-644a mediated fine-tuning of oncogenesis, stimulating pathways and resultant potentiation of enzalutamide therapy in CRPC patients. Significance: This study demonstrates that miR-644a therapeutically influences the CRPC tumor microenvironment by suppressing androgen signaling and additional genes involved in metabolism, proliferation, Warburg effect, and EMT, to potentiate the enzalutamide therapy. more...

Population specific studies in prostate cancer (PCa) reveal a unique heterogeneous etiology. Various factors, such as genetics, environment and dietary regimen seems to determine disease progression, therapeutic resistance and rate of mortality. Enormous disparity documented in disease incidences, aggressiveness and mortality in PCa among AAs (African Americans) and CAs (Caucasian Americans) is attributed to the variations in genetics, epigenetics and their association with metabolism. Scientific and clinical evidences have revealed the influence of variations in Androgen Receptor (AR), RNAse L, macrophage scavenger receptor 1 (MRS1), androgen metabolism by cytochrome P450 3A4, differential regulation of microRNAs, epigenetic alterations and diet in racial disparity in PCa incidences and mortality. Concerted efforts are needed to identify race specific prognostic markers and treatment regimen for a better management of the disease. more...

Prostate cancer (PCa) depends on androgens for growth and maintenance. Despite curative therapeutics of the androgen-sensitive PCa, one-third of patients relapse to a more aggressive form of the disease known as castration-resistant PCa (CRPC). Androgen signaling mediated via Androgen receptor (AR) continues to promote CRPC and therapeutic resistance. The synthesis of testosterone (T) and dihydrotestosterone (DHT) via Intratumoral steroidogenesis appears to play a crucial role in CRPC. It appears that PCa cells synthesize de-novo androgen from cholesterol from acetyl-coA via Terpenoid backbone and Steroid biosynthesis pathways. We found that several enzymes of the intracellular cholesterol synthesis pathways including HMGS1, HMGCR, SREBP1, FASN, SCARB1 are potential targets of hsa-miR-149 5p. Currently, we are characterizing miR-149-5p mediated posttranscriptional regulation of these genes and determining its potential therapeutic application in CRPC. Citation Format: Asmita Bhattarai, Girish C. Shukla. Posttransriptional control of steroid biosynthesis pathway in prostate cancer [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 776. more...

Castration-resistant prostate cancer (CRPC) is defined by affecting intrinsic cellular mechanisms including dysregulated androgen signaling, aerobic glycolysis (Warburg effect), epithelial-mesenchymal transition (EMT) and dysregulation of transcription factors including androgen receptor (AR) and c-Myc. Development of CRPC post Androgen-deprivation therapy is indicated by tumor microenvironment heterogeneity, compensating cellular pathways and recurrence of AR expression leading to the failure of androgen-signaling inhibitors including Enzalutamide and Abiraterone Acetate. We have discovered that miR-644a potentiates Enzalutamide efficacy both, in vitro and in vivo prostate cancer models. In depth analyses revealed that miR-644a downregulated expression of diverse tumor microenvironment regulators including c-Myc, AR, AR co-regulators and the anti-apoptosis factors Bcl-xl and Bcl-2, EMT factors ZEB1, cdk6, and Snail. Enhanced miR-644a expression also suppressed the Warburg effect by direct down-regulating c-Myc, Akt, IGF-1R and GAPDH expression. Taken together, our data demonstrate that the adjunctive miRNA expression down-regulated several oncogenesis promoting cellular pathways and functions cooperatively with Enzalutamide. We propose that a “crowd-control” approach to pacifying aggressive tumorigenesis and metastasis supporting cellular pathways by a miRNA with a wide range of gene targets is likely to potentiate the efficacy of cancer treating drugs and potentially preventing the drug-resistance. Note: This abstract was not presented at the meeting. Citation Format: Girish C. Shukla. A crowd-control approach for castration-resistant prostate cancer therapeutics [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 3559. more...

Formation of catalytic core of the U12-dependent spliceosome involves U6atac and U12 interaction with the 5′ splice site and branch site regions of a U12-dependent intron, respectively. Beyond the formation of intermolecular helix I region between U6atac and U12 snRNAs, several other regions within these RNA molecules are predicted to form stem-loop structures. Our previous work demonstrated that the 3′ stem-loop region of U6atac snRNA contains a U12-dependent spliceosome-specific targeting activity. Here, we show a detailed structure-function analysis and requirement of a substructure of U6atac 3′ stem-loop in U12-dependent in vivo splicing. We show that the C-terminal RNA recognition motif of p65, a U12 snRNA binding protein, also binds to the distal 3′ stem-loop of U6atac. By using a binary splice site mutation suppressor assay we demonstrate that p65 protein-binding apical stem-loop of U12 snRNA can be replaced by this U6atac distal 3′ stem-loop. Furthermore, we tested the compatibility of the U6atac 3′ end from phylogenetically distant species in a human U6atac background, to establish the evolutionary relatedness of these structures and in vivo function. In summary, we demonstrate that RNA-RNA and RNA-protein interactions in the minor spliceosome are highly plastic as compared to the major spliceosome. more...

U12 snRNA is analogous to U2 snRNA of the U2-dependent spliceosome and is essential for the splicing of U12-dependent introns in metazoan cells. The essential region of U12 snRNA, which base pairs to the branch site of minor class introns is well characterized. However, other regions which are outside of the branch site base pairing region are not yet characterized and the requirement of these structures in U12-dependent splicing is not clear. U12 snRNA is predicted to form an intricate secondary structure containing several stem–loops and single-stranded regions. Using a previously characterized branch site genetic suppression assay, we generated second-site mutations in the suppressor U12 snRNA to investigate the in vivo requirement of structural elements in U12-dependent splicing. Our results show that stem–loop IIa is essential and required for in vivo splicing. Interestingly, an evolutionarily conserved stem–loop IIb is dispensable for splicing. We also show that stem–loop III, which binds to a p65 RNA binding protein of the U11-U12 di.snRNP complex, is essential for in vivo splicing. The data validate the existence of proposed stem–loops of U12 snRNA and provide experimental support for individual secondary structures. more...

Androgen receptor (AR) is a ligand-dependent transcription factor, which plays a significant role in prostate carcinogenesis. Blockade of AR and its ligand, androgen is the basis for the treatment of prostate cancer (PCa). Nevertheless, a modest increase in the critical levels of AR mRNA and corresponding protein is sufficient for the development of resistance to antiandrogen therapy. A strategy to further downregulate AR mRNA and protein expression in combination with antiandrogen therapy may prevent or delay the development of androgen-independent PCa. Recent studies show that microRNAs (miRNAs) perform tumor suppressor functions in various cancers. In this study, we demonstrate that the overexpression of miR 488* downregulates the transcriptional activity of AR and inhibits the endogenous AR protein production in both androgen-dependent and androgen-independent PCa cells. In addition, miR 488* blocks the proliferation and enhances the apoptosis of PCa cells. Our data indicate that miR 488* targets AR and is a potential modulator of AR mediated signaling. Our findings provide insight for utilizing miRNAs as novel therapeutics to target AR in PCa. more...

This Nano Focus article highlights recent advances in RNA nanotechnology as presented at the First International Conference of RNA Nanotechnology and Therapeutics, which took place in Cleveland, OH, USA (October 23-25, 2010) (http;//www.eng.uc.edu/nanomedidne/RNA2010/), chaired by Peixuan Guo and co-chaired by David Rueda and Scott Tenenbaum. The conference was the first of its kind to bring together more than 30 invited speakers in the frontier of RNA nanotechnology from France, Sweden, South Korea, China, and throughout the United States to discuss RNA nanotechnology and Its applications. It provided a platform for researchers from academia, government, and the pharmaceutical industry to share existing knowledge, vision, technology, and challenges in the field and promoted collaborations among researchers interested in advancing this emerging scientific discipline. The meeting covered a range of topics, including biophysical and single-molecule approaches for characterization of RNA nanostructures; structure studies on RNA nanoparticles by chemical or biochemical approaches, computation, prediction, and modeling of RNA nanoparticle structures; methods for the assembly of RNA nanoparticles; chemistry for RNA synthesis, conjugation, and labeling; and application of RNA nanoparticles in therapeutics. A special invited talk on the well-established principles of DNA nanotechnology was arranged to provide models for RNA nanotechnology. An Administrator from National Institutes of Health (NIH) National Cancer Institute (NCI) Alliance for Nanotechnology in Cancer discussed the current nanocancer research directions and future funding opportunities at NCl. As indicated by the feedback received from the invited speakers and the meeting participants, this meeting was extremely successful, exciting, and informative, covering many groundbreaking findings, pioneering ideas, and novel discoveries. more...

Prostate Cancer (PCa) is the most commonly diagnosed cancer in males with 161,360 people diagnosed in the United States and is predicted to cause 26,730 deaths in 2017. The current treatment of androgen deprivation therapy (ADT) initially depletes circulating androgens; however, intratumoral androgens rescue androgen receptor (AR) signaling and promotes the development of castration-resistant prostate cancer (CRPC). The dysregulation of the Wnt/B-catenin signaling pathway has been implicated in the development of many cancers including PCa. The disruption of this signaling leads to the stabilization of B-catenin which upregulates many genes involved in tumorigenesis. Additionally, B-catenin acts as AR cofactor. Our previous studies show that AR is a direct target of miR-644a. We hypothesized that targeting the expression of both the B-catenin pathway and AR by conventional drugs and/or tumor suppressor miR-644a would have synergistic therapeutic benefits. In this study, we are investigating miR-644a mediated posttranscriptional downregulation of GSK3-B and B-catenin in the wnt/B-catenin signaling pathway. The effect of miR-644a in combination with inhibitors (B-catenin or GSK3-B) was also assessed using viability assays and a significant downregulation was observed. We will further study the posttranscriptional effects of tumor suppressor miR-644a on the wnt/B-catenin pathway as well as assess if miR-644a can inhibit the cross-talk of these pathways. Citation Format: Alexis R. Plaga, Girish C. Shukla. Regulation of crosstalk between AR and Wnt/Beta-catenin pathways by miR-644a in prostate cancer [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 480. more...

U6 and U6atac snRNAs play analogous critical roles in the major U2-dependent and minor U12-dependent spliceosomes, respectively. Previous results have shown that most of the functional cores of these two snRNAs are either highly similar in sequence or functionally interchangeable. Thus, a mechanism must exist to restrict each snRNA to its own spliceosome. Here we show that a chimeric U6 snRNA containing the unique and highly conserved 3′ end domain of U6atac snRNA is able to function in vivo in U12-dependent spliceosomal splicing. Function of this chimera required the coexpression of a modified U4atac snRNA; U4 snRNA could not substitute. Partial deletions of this element in vivo, as well as in vitro antisense experiments, showed that the 3′ end domain of U6atac snRNA is necessary to direct the U4atac/U6atac.U5 tri-snRNP to the forming U12-dependent spliceosome. In vitro experiments also uncovered a role for U4atac snRNA in this targeting. more...

Prostate cancer begins as an androgen-responsive disease. However, subsequent accumulation of multiple sequential genetic and epigenetic alterations transforms the disease into an aggressive, castration-resistant prostate cancer (CRPC). The monoallelic Androgen Receptor (AR) is associated with the onset, growth and development of Prostate cancer. The AR is a ligand-dependent transcription factor, and the targeting of androgen- and AR-signaling axis remains the primary therapeutic option for Prostate cancer (PCa) treatment. A durable and functional disruption of AR signaling pathways combining both traditional and novel therapeutics is likely to provide better treatment options for CRPC. Recent work has indicated that expression of AR is modulated at the posttranscriptional level by regulatory miRNAs. Due to a relatively long 3’ untranslated region (UTR) of AR mRNA, the posttranscription expression is likely to be regulated by hundreds of miRNAs in normal as well as in disease state. The main objective of the article is to offer a thought-provoking concept of “andro-miRs” and their potential application in AR gene expression targeting. This new paradigm for targeting constitutively active AR and its tumor specific splicing isoforms using andro-miRs may pave the way for a novel adjunctive therapy and improved treatment of CRPC. more...

Background BCL-xL is an anti-apoptotic BCL-2 family protein that inhibits apoptosis and is overexpressed in many cancers. We have reported that acquired resistance to the BCL-2 inhibitor ABT-199 (venetoclax) is associated with increased BCL-xL expression. Yet, how BCL-xL mediates chemoresistance in hematopoietic malignancies is not clear. This finding may help in design of new strategies for therapeutic intervention to overcome acquired chemoresistance mediated by BCL-xL. Results We now show that the increased BCL-xL expression was inversely correlated with that of miR-377 in ABT-199-resistant cells. This finding was also extended to a panel of B-cell lymphoid lines and primary chronic lymphocytic leukemia (CLL) cells. miR-377 suppressed BCL-xL expression by recognizing two binding sites in the BCL-xL 3’-UTR. Mutation of these two miR-377 consensus-binding sites completely abolished its regulatory effect. Expression of a miR-377 mimic downregulated BCL-xL protein expression and significantly increased apoptotic cell death. Expression of a miR-377 inhibitor restored BCL-xL protein expression and limited cell death caused by the hypomethylating agent 5-azacytidine. Thus, miR-377-dependent BCL-xL regulation drives acquired therapeutic resistance to ABT-199. We further show that CLL patients who received a diverse array of chemotherapy regimens also had significantly higher BCL-xL and lower miR377 expression, indicating that exposure to chemotherapy might trigger transcriptional silencing of miR-377, which results in high levels of BCL-xL. Importantly, CLL patients with high BCL-xL/low miR-377 expression had an advanced tumor stage. Moreover, the high BCL-xL expression correlated with short treatment-free survival in 76 CLL patients. miR-377 is located at 14q32 in the DLK1-DIO3 region, which encodes the largest tumor suppressor miRNA cluster in humans. Examination of five additional 14q32 miRNAs revealed that the majority were significantly down-regulated in most CLL patients as well as in ABT-199-resistant cell lines. Remarkably, four of these miRNAs had significantly decreased expression in chemotherapy-treated CLL patients as compared to those untreated. These findings indicate a reduced expression of multiple miRNAs that may reflect a global silencing of this miRNA cluster in therapy-resistant lymphoid cells. Conclusions These findings reveal a novel mechanism by which down-regulation of miR-377 increases BCL-xL expression, promoting chemotherapy resistance in B-cell lymphoid malignancies. Electronic supplementary material The online version of this article (doi:10.1186/s12943-015-0460-8) contains supplementary material, which is available to authorized users. more...

Complex epithelial and stromal cell interactions are required during the development and progression of prostate cancer. Regulatory small non-coding microRNAs (miRNAs) participate in the spatiotemporal regulation of messenger RNA (mRNA) and regulation of translation affecting a large number of genes involved in prostate carcinogenesis. In this study, through deep-sequencing of size fractionated small RNA libraries we profiled the miRNAs of prostate epithelial (PrEC) and stromal (PrSC) cells. Over 50 million reads were obtained for PrEC in which 860,468 were unique sequences. Similarly, nearly 76 million reads for PrSC were obtained in which over 1 million were unique reads. Expression of many miRNAs of broadly conserved and poorly conserved miRNA families were identified. Sixteen highly expressed miRNAs with significant change in expression in PrSC than PrEC were further analyzed in silico. ConsensusPathDB showed the target genes of these miRNAs were significantly involved in adherence junction, cell adhesion, EGRF, TGF-β and androgen signaling. Let-7 family of tumor-suppressor miRNAs expression was highly pervasive in both, PrEC and PrSC cells. In addition, we have also identified several miRNAs that are unique to PrEC or PrSC cells and their predicted putative targets are a group of transcription factors. This study provides perspective on the miRNA expression in PrEC and PrSC, and reveals a global trend in miRNA interactome. We conclude that the most abundant miRNAs are potential regulators of development and differentiation of the prostate gland by targeting a set of growth factors. Additionally, high level expression of the most members of let-7 family miRNAs suggests their role in the fine tuning of the growth and proliferation of prostate epithelial and stromal cells. more...

U4 small nuclear RNA (snRNA) and U6 snRNA form a base-paired di-snRNP complex that is essential for pre-mRNA splicing of the major class of metazoan nuclear introns. The functionally analogous but highly diverged U4atac and U6atac snRNAs form a similar complex that is involved in splicing of the minor class of introns. Previous results with mutants of U6atac in which a substructure was replaced by the analogous structure from U6 snRNA suggested that wild-type U4 snRNA might be able to interact productively with the mutant U6atac snRNA. Here we show that a mutant U4 snRNA designed to base pair with a mutant U6atac snRNA can activate U12-dependent splicing when coexpressed in an in vivo genetic suppression assay. This genetic interaction could also be demonstrated in an in vitro crosslinking assay. These results show that a U4/U6atac di-snRNP can correctly splice a U12-dependent intron and suggest that the specificity for spliceosome type resides in the U6 and U6atac small nuclear ribonucleoproteins. Further experiments suggest that expression of a mutant U4 snRNA that can bind to wild-type U6atac snRNA alters the specificity of some splice sites, providing an evolutionary rationale for maintaining two U4-like snRNAs. more...

Glutathione S-transferase P1 (GSTP1), an enzyme involved in detoxification process, is frequently inactivated in prostate cancer due to epigenetic modifications. Through in silico analysis, we identified a subset of microRNAs (miRNAs) that are putative targets in regulating GSTP1. miRNAs are small endogenous non-coding RNA that are critical regulators of various physiologic and pathologic processes, and their level of expression may play a precise role in the early diagnosis and prognosis of cancer. These small molecules have been detected in a wide variety of human biological specimens including blood, serum, urine, ejaculate, and tissues, which could be utilized as clinically useful biomarker in early detection and prognosis of prostate cancer. The chapter summarizes the current knowledge about miRNA involved in GSTP1 regulation in prostate cancer and their potential as useful biomarkers of the disease for early detection and prognosis, along with challenges and limitations in this development. more...

Both spliceosomal and self-splicing group II introns require the function of similar small, metal binding RNA stem-loop elements located in U6 or U6atac snRNAs of the spliceosome or domain 5 (D5) of group II introns. Here we report that two different D5 elements can functionally replace the U6atac snRNA stem-loop in an in vivo splicing assay. For efficient function in vivo, a single base pair from the upper helical section of the D5 sequence had to be removed. Introducing the equivalent base pair deletion into the D5 element of a group II intron reduced but did not eliminate self-splicing activity. Our results strengthen the case that these RNA elements play similar roles in the catalytic centers of both the spliceosome and a self-splicing ribozyme. more...

Intratumoral Prostate cancer (PCa) heterogeneity is characterized by multiple intrinsic mechanisms including Androgen signaling, aerobic glycolysis, aberrant transcriptional activation of c-Myc and dysregulation of miRNAs. The ability of prostate cancer cells to alter growth and metabolism in a manner distinct from benign cells is a major concern in the treatment of castration-resistant prostate cancer. Therefore, targeting the diverse oncogenesis promoting pathways involved in PCa progression and resistance at the posttranscriptional level using micro RNAs (miRNAs) offers a novel therapeutic option. Using a miRNA mimic screen, we identified a human-specific miRNA miR-644a, which modulates expression of a diverse set of oncogenic pathways in PCa including androgen signaling, AKT/IGF-1R signaling, c-myc mediated signaling and EMT pathways. We have also demonstrated a potent tumor suppressive function of miR-644a in mouse prostate cancer xenograft model. miR-644a also sensitizes the PCa tumors to current therapeutic options including AR antagonist Enzalutamide by regulating the key modulators in development of drug resistance. In conclusion, our study demonstrates that miR-644a is a novel tumor suppressor miRNA regulating key prostate carcinogenesis promoting pathways. Citation Format: Jey Sabith Ebron, Jagjit Singh, Eswar Shankar, Crystal Weyman, Sanjay Gupta, Daniel J. Lindner, Girish C. Shukla. Fine-tuning the expression of heterogeneous network of genes involved in androgen signaling, aerobic glycolysis, apoptosis and epithelial-mesenchymal transition by microRNA-644a in prostate cancer potentiation of AR signaling therapy by miR-644a: Selective manipulation of the prostate cancer transcriptome by miR-644a [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 1470. doi:10.1158/1538-7445.AM2017-1470 more...

Prostate Cancer (PCa) is the second most commonly diagnosed cancer in the United States with 180,890 males diagnosed and is predicted to cause approximately 26,000 deaths in 2016. The current treatment of androgen deprivation therapy (ADT) initially depletes circulating androgens; however, intratumoral androgens rescue androgen receptor (AR) signaling and promotes the development of castration resistant prostate cancer (CRPC). The dysregulation of the Wnt/β-catenin signaling pathway has been implicated in the development of many cancers including PCa. The disruption of this signaling leads to the stabilization of β-catenin which upregulates many genes involved in tumorigenesis. Additionally, β-catenin acts as AR cofactor. Our previous studies show that AR is a direct target of miR-644a. We hypothesized that targeting the expression of both the β-catenin pathway and AR by conventional drugs and/or tumor suppressor miR-644a would have synergistic therapeutic benefits. In this study, we are investigating miR-644a mediated posttranscriptional downregulation of GSK3-β and β-catenin in the wnt/β-catenin signaling pathway. The effect of miR-644a in combination with inhibitors (β-catenin or GSK3-β) was also assessed using proliferation assays and a significant downregulation was observed. We will further study the posttranscriptional effects of tumor suppressor miR-644a on the wnt/β-catenin pathway and also the processing and regulation of miR-644a in PCa. Citation Format: Alexis Plaga, Girish C. Shukla. Regulation of cross talk between AR and wnt/beta-catenin pathways in prostate 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 478. doi:10.1158/1538-7445.AM2017-478 more...

Prostate cancer growth and proliferation depends on androgen signaling mediated by transactivation of Androgen Receptor (AR). Androgen ablation remains the mainstay therapy for treatment of the disease. However, despite androgen ablation, the disease relapses to more aggressive form known as castration-resistant prostate cancer (CRPC). Androgen Signaling Inhibitor such as Abiraterone Acetate and Enzalutamide are the most effective treatment methods currently being used to treat CRPC. However, more than one-third of CRPC patients develop resistance to these treatments, mostly due to the gain of function in the AR protein and increase in intratumoral dihydrotestosterone (DHT) synthesis. Intratumoral DHT synthesis from steroid precursors in tumors is augmented by up-regulation of enzymes of cholestrogenesis and steroidogenesis, such as HMG-CoA reductase, SCARB1, and 17βHSD respectively. Tumor-specific downregulation of microRNAs which regulate the AR and steroid biosynthesis has been implicated in tumor growth and resistance to therapeutics in CRPC. We are focusing on tumor suppressive role of miR-149-5p in PCa. We discovered that miR-149-5p expression was significantly lower in prostate cancer tissues compared to normal tissues. The mechanistic investigation revealed that miR-149-5p downregulates wild type and alternatively spliced variant of AR. It also down regulates the expression of SCARB1, HMGCS and HMG-CoA reductase, the proteins known to facilitate intratumoral DHT synthesis. Ectopic expression of miR-149-5p negatively regulated the expression of prostate-specific antigen, a downstream target of androgen signaling and it also inhibited invasion and proliferation of CRPC cells. Our findings indicate a significant role of miR-149-5p in regulating androgen signaling and possibly DHT synthesis in CRPC. This provides a strong rationale for further investigating the significance of miR-149-5p for generation of new therapeutic for CRPC. Citation Format: Savita Singh, Jey Sabith Ebron, Eswar Shankar, Sanjay Gupta, Daniel Lindner, Girish Shukla. Regulation of androgen signaling axis and tumor suppressive function of miR-149-5p in prostate 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 2531. doi:10.1158/1538-7445.AM2017-2531 more...

The ability of prostate cancer cells to alter their metabolism in a manner distinct from benign cells and energy reprogramming is a major concern in treatment of castration resistant prostate cancer (CRPC). On the other hand, development of resistance to next-generation drugs directed against the AR signaling axis (Abiraterone acetate and Enzalutamide) confers a grim prognosis. Hence, targeting aerobic glycolysis of tumor cells as well as AR signaling axis in CRPC is highly desirable. Here, we report the role of a dual-action miRNA which targets the Androgen Receptor function as well as action of key factors involved in tumor energy metabolism. In addition, the miRNA also targets the key anti-apoptotic factors and enhance the apoptosis in cultured cells. Furthermore, together with Enzalutamide, the miRNA shows a potent synergistic anti-tumor activity in 22RV1 mouse xenografts. We will present the detailed results showing the therapeutic potential of dual-action miRNA in the treatment of CRPC. Citation Format: Jey Sabith Ebron, Jagjit Singh, Kavleen Sikand, Eswar Shankar, Sanjay Gupta, Girish C. Shukla. Tumor suppressing dual-action miRNA: Targeting Warburg effect and androgen receptor function in CRPC. [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 1119. more...

Results of overexpression or downregulation of a microRNA (miRNA) on its target mRNA expression are often validated by reverse-transcription and quantitative PCR analysis using an appropriate housekeeping gene as an internal control. The possible direct or indirect effects of a miRNA on the expression of housekeeping genes are often overlooked. Among many housekeeping genes, expressions of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin have been used extensively for normalization of gene expression data. Here, we show that GAPDH and β-actin are direct targets of miR-644a. Our data demonstrate the unsuitability of GAPDH and β-actin as internal controls in miR-644a functional studies and emphasize the need to carefully consider the choice of a reference gene in miRNA experiments. more...

The remarkable discovery of small noncoding microRNAs (miRNAs) and their role in posttranscriptional gene regulation have revealed another fine-tuning step in the expression of genetic information. A large number of cellular pathways, which act in organismal development and are important in health and disease, appear to be modulated by miRNAs. At the molecular level, miRNAs restrain the production of proteins by affecting the stability of their target mRNA and/or by down-regulating their translation. This review attempts to offer a snapshot of aspects of miRNA coding, processing, target recognition and function in animals. Our goal here is to provide the readers with a thought-provoking and mechanistic introduction to the miRNA world rather than with a detailed encyclopedia. more...

Background Recent data show aberrant and altered expression of regulatory noncoding micro (mi) RNAs in prostate cancer (PCa). A large number of miRNAs are encoded in organized intronic clusters within many protein coding genes. While expression profiling studies of miRNAs are common place, little is known about the host gene and their resident miRNAs coordinated expression in PCa cells. Furthermore, whether expression of a subset of miRNAs is distinct in androgen-responsive and androgen-independent cells is not clear. Here we have examined the expression of mature miRNAs of miR 17–92, miR 106b-25 and miR 23b-24 clusters along with their host genes C13orf25, MCM7 and AMPO respectively in PCa cell lines. Results The expression profiling of miRNAs and host genes was performed in androgen-sensitive MDA PCa 2b and LNCaP as well as in androgen-refractory PC-3 and DU 145 cell culture models of PCa. No significant correlation between the miRNA expression and the intrinsic hormone-responsive property of PCa cells was observed. Androgen-sensitive MDA PCa 2b cells exhibited the highest level of expression of most miRNAs studied in this report. We found significant expression variations between host genes and their resident miRNAs. The expressions of C13orf25 and miR 17–92 cluster as well as MCM7 and miR 106b-25 cluster did not reveal statistically significant correlation, thus suggesting that host genes and resident miRNAs may be expressed independent of each other. Conclusion Our results suggest that miRNA expression profiles may not predict intrinsic hormone-sensitive environment of PCa cells. More importantly, our data indicate the possibility of additional novel mechanisms for intronic miRNA processing in PCa cells. more...

U4atac snRNA forms a base-paired complex with U6atac snRNA. Both snRNAs are required for the splicing of the minor U12-dependent class of eukaryotic nuclear introns. We have developed a new genetic suppression assay to investigate the in vivo roles of several regions of U4atac snRNA in U12-dependent splicing. We show that both the stem I and stem II regions, which have been proposed to pair with U6atac snRNA, are required for in vivo splicing. Splicing activity also requires U4atac sequences in the 5′ stem–loop element that bind a 15.5 kDa protein that also binds to a similar region of U4 snRNA. In contrast, mutations in the region immediately following the stem I interaction region, as well as a deletion of the distal portion of the 3′ stem–loop element, were active for splicing. Complete deletion of the 3′ stem–loop element abolished in vivo splicing function as did a mutation of the Sm protein binding site. These results show that the in vivo sequence requirements of U4atac snRNA are similar to those described previously for U4 snRNA using in vitro assays and provide experimental support for models of the U4atac/U6atac snRNA interaction. more...

The discovery of a second type of spliceosomal intron in the genomes of many multicellular eukaryotes raised the exciting possibility of comparing the structural and functional features of the two splicing systems with the hope of shedding light on important features of each (Tarn & Steitz, 1997)+ This hope was amply fulfilled with the discovery of the parallel roles played in the two systems by the respective sets of spliceosomal snRNAs+ The initial identification of functions for the previously known U11 and U12 snRNAs (Hall & Padgett, 1994; Tarn & Steitz, 1996b) was magnified by the discovery of the novel U4atac and U6atac snRNAs (Tarn & Steitz, 1996a)+ The similarities of these latter two snRNAs to the U4 and U6 snRNAs of the major splicing system were apparent and led to detailed comparisons of the features of these RNAs and their interactions (Tarn & Steitz, 1996a)+ For both the U4/U6 pair and the U4atac/ more...

Androgen receptor (AR) is a member of the steroid receptor family which plays a significant role in the regulation of both prostate cell proliferation and growth suppression. The targeting of androgen- and AR- signaling axis remains the primary therapeutic option for prostate cancer (PCa) treatment. AR mRNA has a long 3′UTR of 6.8 kb length and is predicted to be a target of many miRNAs. The existence of long 3′UTR proposes a complex mechanism of regulation of AR gene expression through miRNAs and other factors including RNA binding proteins and polyadenylation signals. Differential expression of various miRNAs has been reported to be associated with the progression of the disease to harmone-independence and castration-resistant prostact cancer (CRPC). However, the direct regulation of AR transcriptional activity through miRNAs is not clearly understood. Our lab has discovered that AR is a target of andro-miRs (miR-488*, miR-644, miR-149, miR-512-5p). These andro-miRs bind to target sequences in the 3′UTR of AR and downregulates luciferase expression in the target-validation validation experiments as well as endogeneous AR expression. Our ongoing study of andro-miRs also indicates that, in addition to downregulation of AR gene expression by the action of andro-miRs alone and synergistically, these miRNAs can regulate AR signaling leading to androgen-independence and resistance. This post transcriptional regulation of AR gene expression through andro-miRs makes them an interesting candidate for miR-based adjunctive treatment in CRPC treatment. Note: This abstract was not presented at the meeting. Citation Format: Jey Sabith Ebron, Kavleen Sikand, Jagjit Singh, Girish C. Shukla. Regulation of androgen receptor expression by andro-miRs in prostate cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5212. doi:10.1158/1538-7445.AM2014-5212 more...

The U6 spliceosomal snRNA forms an intramolecular stem-loop structure during spliceosome assembly that is required for splicing and is proposed to be at or near the catalytic center of the spliceosome. U6atac snRNA, the analog of U6 snRNA used in the U12-dependent splicing of the minor class of spliceosomal introns, contains a similar stem-loop whose structure but not sequence is conserved between humans and plants. To determine if the U6 and U6atac stem-loops are functionally analogous, the stem-loops from human and budding yeast U6 snRNAs were substituted for the U6atac snRNA structure and tested in an in vivo genetic suppression assay. Both chimeric U6/U6atac snRNA constructs were active for splicing in vivo. In contrast, several mutations of the native U6atac stem-loop that either delete putatively unpaired residues or disrupt the putative stem regions were inactive for splicing. Compensatory mutations that are expected to restore base pairing within the stem regions restored splicing activity. However, other mutants that retained base pairing potential were inactive, suggesting that functional groups within the stem regions may contribute to function. These results show that the U6atac snRNA stem-loop structure is required for in vivo splicing within the U12-dependent spliceosome and that its role is likely to be similar to that of the U6 snRNA intramolecular stem-loop. more...

Splicing of U12-dependent introns requires the function of U11, U12, U6atac, U4atac, and U5 snRNAs. Recent studies have suggested that U6atac and U12 snRNAs interact extensively with each other, as well as with the pre-mRNA by Watson-Crick base pairing. The overall structure and many of the sequences are very similar to the highly conserved analogous regions of U6 and U2 snRNAs. We have identified the homologs of U6atac and U12 snRNAs in the plant Arabidopsis thaliana. These snRNAs are significantly diverged from human, showing overall identities of 65% for U6atac and 55% for U12 snRNA. However, there is almost complete conservation of the sequences and structures that are implicated in splicing. The sequence of plant U6atac snRNA shows complete conservation of the nucleotides that base pair to the 5' splice site sequences of U12-dependent introns in human. The immediately adjacent AGAGA sequence, which is found in human U6atac and all U6 snRNAs, is also conserved. High conservation is also observed in the sequences of U6atac and U12 that are believed to base pair with each other. The intramolecular U6atac stem-loop structure immediately adjacent to the U12 interaction region differs from the human sequence in 9 out of 21 positions. Most of these differences are in base pairing regions with compensatory changes occurring across the stem. To show that this stem-loop was functional, it was transplanted into a human suppressor U6atac snRNA expression construct. This chimeric snRNA was inactive in vivo but could be rescued by coexpression of a U4atac snRNA expression construct containing compensatory mutations that restored base pairing to the chimeric U6atac snRNA. These data show that base pairing of U4atac snRNA to U6atac snRNA has a required role in vivo and that the plant U6atac intramolecular stem-loop is the functional analog of the human sequence. more...

Master transcriptional regulator Androgen Receptor (AR) remained a fascinating and an enigmatic molecule, particularly in Prostate cancer biology. Major impetus in PCa research is currently focused on the development of new generation of therapeutics to target AR expression, especially in castration-resistant stage of the disease. 920 amino acid coding AR gene encompasses over 180 kilobase (kb) of X-chromosome containing eight exons that produces a nearly 10.7 kb AR mRNA. The fully processed mature AR mRNA account for only 26% of total coding capacity and most of the transcript codes for noncoding regions that includes 5’ and 3’ UTRs. However, functional roles of UTRs in AR gene expression, principally its regulatory involvement in development of CRPC remained indefinable. Our research is focusing on understanding the functional roles of 6.8 kb long AR 3’ UTR in AR gene expression and especially the molecular mechanism that revitalizes AR expression in CRPC. We have shown that a number of regulatory noncoding miRNAs play a critical 3’ UTR targeting role in AR gene expression and has potential therapeutic implications. Here we show a synergistic approach of targeting AR expression mediated via its 3’ UTR, using a miRNA cocktail combinatorial approach. In addition, we are investigating the potential functional association of 3’ UTR in AR gene expression and implication in PCa. Citation Format: Girish C. Shukla, Kavleen Sikand, Jey S. Ebron. Androgen receptor 3’ untranslated region (UTR): a conundrum and a significant therapeutic opportunity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3196. doi:10.1158/1538-7445.AM2013-3196 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend. more...