Jey Sabith Ebron

Housekeeping gene selection advisory: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and β-actin are targets of miR-644a.

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.

miR-377-dependent BCL-xL regulation drives chemotherapeutic resistance in B-cell lymphoid malignancies

BackgroundBCL-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.ResultsWe 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.ConclusionsThese findings reveal a novel mechanism by which down-regulation of miR-377 increases BCL-xL expression, promoting chemotherapy resistance in B-cell lymphoid malignancies.

Targeting of Androgen Receptor Expression by Andro-miRs as Novel Adjunctive Therapeutics in Prostate Cancer

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.

Aging and calorie restriction regulate the expression of miR-125a-5p and its target genes Stat3 , Casp2 and Stard13

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.

Molecular characterization of a novel androgen receptor transgene responsive to MicroRNA mediated post‐transcriptional control exerted via 3′‐untranslated region

Androgen Receptor (AR) gene is associated with Prostate cancer (PCa) and hence targeting androgen—and AR—signaling axis remains the most promising primary therapeutic option to treat the disease. The AR mRNA has a 6.8 kb long 3′‐untranslated region (UTR) which harbors several experimentally validated and numerous predicted miRNA binding sites. AR 3′‐UTR is likely to positively or negatively regulate AR expression by interacting with miRNAs and possibly other trans‐acting auxiliary factors including 3′‐UTR RNA binding proteins. In this context, systematic understanding of the regulatory role of AR 3′‐UTR in intrinsic post‐transcriptional control of AR gene expression is of significance to understand AR related diseases including PCa.

Deep sequencing of small RNA libraries from human prostate epithelial and stromal cells reveal distinct pattern of microRNAs primarily predicted to target growth factors

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.

Abstract 2531: Regulation of androgen signaling axis and tumor suppressive function of miR-149-5p in prostate cancer

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

Abstract 1119: Tumor suppressing dual-action miRNA: Targeting Warburg effect and androgen receptor function in CRPC

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.

Additional file 3: Figure S3. of miR-377-dependent BCL-xL regulation drives chemotherapeutic resistance in B-cell lymphoid malignancies

Kaplan-Meier curves for correlation of treatment-free survival with pro-apoptotic BCL-2 expression levels. (A) PUMA, (B) NOXA, and (C) BIM. P values shown are for the log-rank test. (JPEG 309 kb)

Abstract 5212: Regulation of androgen receptor expression by andro-miRs in prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA 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