Organoids: An Emerging Precision Medicine Model for Prostate Cancer Research.

Prostate cancer (PCa) has been known as the most prevalent cancer disease and the second leading cause of cancer mortality in men almost all over the globe. There is an urgent need for establishment of PCa models that can recapitulate the progress of genomic landscapes and molecular alterations during development and progression of this disease. Notably, several organoid models have been developed for assessing the complex interaction between PCa and its surrounding microenvironment. In recent years, PCa organoids have been emerged as powerful in vitro 3D model systems that recapitulate the molecular features (such as genomic/epigenomic changes and tumor microenvironment) of PCa metastatic tumors. In addition, application of organoid technology in mechanistic studies (i.e., for understanding cellular/subcellular and molecular alterations) and translational medicine has been recognized as a promising approach for facilitating the development of potential biomarkers and novel therapeutic strategies. In this review, we summarize the application of PCa organoids in the high-throughput screening and establishment of relevant xenografts for developing novel therapeutics for metastatic, castration resistant, and neuroendocrine PCa. These organoid-based studies are expected to expand our knowledge from basic research to clinical applications for PCa diseases. Furthermore, we also highlight the optimization of PCa cultures and establishment of promising 3D organoid models for in vitro and in vivo investigations, ultimately facilitating mechanistic studies and development of novel clinical diagnosis/prognosis and therapies for PCa.

Promising Strategy of mPTP Modulation in Cancer Therapy: An Emerging Progress and Future Insight.

Cancer has been progressively a major global health concern. With this developing global concern, cancer determent is one of the most significant public health challenges of this era. To date, the scientific community undoubtedly highlights mitochondrial dysfunction as a hallmark of cancer cells. Permeabilization of the mitochondrial membranes has been implicated as the most considerable footprint in apoptosis-mediated cancer cell death. Under the condition of mitochondrial calcium overload, exclusively mediated by oxidative stress, an opening of a nonspecific channel with a well-defined diameter in mitochondrial membrane allows free exchange between the mitochondrial matrix and the extra mitochondrial cytosol of solutes and proteins up to 1.5 kDa. Such a channel/nonspecific pore is recognized as the mitochondrial permeability transition pore (mPTP). mPTP has been established for regulating apoptosis-mediated cancer cell death. It has been evident that mPTP is critically linked with the glycolytic enzyme hexokinase II to defend cellular death and reduce cytochrome c release. However, elevated mitochondrial Ca2+ loading, oxidative stress, and mitochondrial depolarization are critical factors leading to mPTP opening/activation. Although the exact mechanism underlying mPTP-mediated cell death remains elusive, mPTP-mediated apoptosis machinery has been considered as an important clamp and plays a critical role in the pathogenesis of several types of cancers. In this review, we focus on structure and regulation of the mPTP complex-mediated apoptosis mechanisms and follow with a comprehensive discussion addressing the development of novel mPTP-targeting drugs/molecules in cancer treatment.

MicroRNA-mRNA Regulatory Network Mediates Activation of mTOR and VEGF Signaling in African American Prostate Cancer.

African American (AA) men exhibit 1.6-fold higher prostate cancer (PCa) incidence and 2.4-fold higher mortality rates compared to European American (EA) men. In addition to socioeconomic factors, emerging evidence suggests that intrinsic biological differences may explain part of PCa disparities. In this study, we applied microRNA (miRNA)-driven bioinformatics to evaluate whether differential miRNA-mRNA regulatory networks play a role in promoting the AA PCa disparities. 10 differentially expressed miRNAs were imported to mirPath V.3 algorithm, leading to identification of 58 signaling pathways differentially regulated in AA PCa versus EA PCa. Among these pathways, we particularly focused on mTOR and VEGF signaling, where we identified 5 reciprocal miRNA-mRNA pairings: miR-34a-5p/HIF1A, miR-34a-5p/PIK3CB, miR-34a-5p/IGFBP2, miR-99b-5p/MTOR and miR-96-5p/MAPKAPK2 in AA PCa versus EA PCa. RT-qPCR validation confirmed that miR-34a-5p, miR-99b-5p and MAPKAPK2 were downregulated, while miR-96-5p, IGFBP2, HIF1A, PIK3CB and MTOR were upregulated in AA PCa versus EA PCa cells. Transfection of miRNA mimics/antagomir followed by RT-qPCR and Western blot analysis further verified that IGFBP2, HIF1A and PIK3CB are negatively regulated by miR-34a-5p, whereas MTOR and MAPKAPK2 are negatively regulated by miR-99b-5p and miR-96-5p, respectively, at mRNA and protein levels. Targeting reciprocal pairings by miR-34a-5p mimic, miR-99b-5p mimic or miR-96-5p antagomir downregulates HIF1α, PI3Kß, mTOR, IGFBP2 but upregulates MAPKAPK2, subsequently reducing cell proliferation and sensitizing docetaxel-induced cytotoxicity in PCa cells. These results suggest that miRNA-mRNA regulatory network plays a critical role in AA PCa disparities, and targeting these core miRNA-mRNA pairings may reduce PCa aggressiveness and overcome the chemoresistance in AA patients.

Downregulation of miR-99b-5p and Upregulation of Nuclear mTOR Cooperatively Promotes the Tumor Aggressiveness and Drug Resistance in African American Prostate Cancer.

Mammalian target of rapamycin (mTOR) regulates various fundamental cellular events including cell proliferation, protein synthesis, metabolism, apoptosis, and autophagy. Tumor suppressive miR-99b-5p has been implicated in regulating PI3K/AKT/mTOR signaling in a variety of types of cancer. Our previous study suggested the reciprocal miR-99b-5p/MTOR (downregulated/upregulated) pairing as a key microRNA-mRNA regulatory component involved in the prostate cancer (PCa) disparities. In this study, we further validated the expression profiles of mTOR and miR-99b-5p in the PCa, colon, breast, and lung cancer specimens and cell lines. The immunohistochemistry (IHC), immunofluorescence, Western blot, and RT-qPCR assays have confirmed that mTOR is upregulated while miR-99b-5p is downregulated in different patient cohorts and a panel of cancer cell lines. Intriguingly, elevated nuclear mTOR expression was observed in African American PCa and other advanced cancers. Transfection of the miR-99b-5p mimic resulted in a significant reduction in nuclear mTOR and androgen receptor (AR), while a slight/moderate to no decrease in cytoplasmic mTOR and AR in PCa and other cancer cells, suggesting that miR-99b-5p inhibits mTOR and AR expression and their nuclear translocation. Moreover, overexpression of miR-99b-5p targets/inhibits AR-mTOR axis, subsequently initiating cell apoptosis and sensitizing docetaxel-induced cytotoxicity in various cancers. In conclusion, our data suggest that reciprocal miR-99b-5p/nuclear mTOR pairing may be a more precise diagnostic/prognostic biomarker for aggressive PCa, than miR-99b-5p/MTOR pairing or mTOR alone. Targeting the AR-mTOR axis using miR-99b-5p has also been suggested as a novel therapeutic strategy to induce apoptosis and overcome chemoresistance in aggressive PCa.

miR-570 interacts with mitochondrial ATPase subunit g (ATP5L) encoding mRNA in stored platelets.

Loss of platelet quality during ex vivo storage is a major concern in the transfusion medicine field and it has been known that platelet mitochondrial dysfunction is associated with storage time. In the last decade, small noncoding RNAs also known as microRNAs (miRNAs) have been reported to regulate key cellular processes through their target sequence interactions with selected mRNAs. In this study, we focused on understanding the mechanisms of platelet mitochondrial dysfunction during storage through miRNA regulation of mRNAs. RNA was isolated from day 0, day 5, and day 9 of stored human leukocyte-depleted platelets and subjected to differential miRNA and mRNA profiling. The miRNA profiling identified several miRNAs at low levels including a set of 12 different miR-548 family members (miR-548a-3p, miR-548aa, miR-548x, miR-548ac, miR-548c-3p, miR-603, miR-548aj, miR-548ae, miR-548z, miR-548u, miR-548al, and miR-570-3p). The mRNA profiling identified, among many, the mitochondrial ATP synthase subunit g (ATP5L) mRNA at high levels during storage. Target Scan algorithm for potential targets of miR-570-3p also identified ATP5L as one of its targets. We further identified two target sites for miR-570-3p in the 3' untranslated region (3'UTR) of ATP5L mRNA. While ATP5L is a subunit of F0ATPase complex, its function is not established yet. Overexpression of miR-570-3p in platelets resulted in reduced levels of ATP5L mRNA and concomitant ATP loss. These experimental results provide first-time insights into the miRNA-mRNA interactions underlying mitochondrial dysfunction in ex vivo stored platelets and warrants further investigation.

Combination of miR-99b-5p and Enzalutamide or Abiraterone Synergizes the Suppression of EMT-Mediated Metastasis in Prostate Cancer.

Prostate cancer (PCa) is the most frequently diagnosed cancer and second leading cause of cancer deaths among American men. Androgen deprivation therapy (ADT) has been systemically applied as a first-line therapy for PCa patients. Despite the initial responses, the majority of patients under ADT eventually experienced tumor progression to castration-resistant prostate cancer (CRPC), further leading to tumor metastasis to distant organs. Therefore, identifying the key molecular mechanisms underlying PCa progression remains crucial for the development of novel therapies for metastatic PCa. Previously, we identified that tumor-suppressive miR-99b-5p is frequently downregulated in aggressive African American (AA) PCa and European American (EA) CRPC, leading to upregulation of mTOR, androgen receptor (AR), and HIF-1α signaling. Given the fact that mTOR and HIF-1α signaling are critical upstream pathways that trigger the activation of epithelial-mesenchymal transition (EMT), we hypothesized that miR-99b-5p may play a critical functional role in regulating EMT-mediated PCa metastasis. To test this hypothesis, a series of cell biology, biochemical, and in vitro functional assays (wound healing, transwell migration, cell/ECM adhesion, and capillary-like tube formation assays) were performed to examine the effects of miR-99b-5p mimic on regulating EMT-mediated PCa metastasis processes. Our results have demonstrated that miR-99b-5p simultaneously targets MTOR and AR signaling, leading to upregulation of E-cadherin, downregulation of Snail/N-cadherin/Vimentin, and suppression of EMT-mediated PCa metastasis. MiR-99b-5p alone and in combination with enzalutamide or abiraterone significantly inhibits the EMT-mediated metastasis of AA PCa and EA CRPC.

Neuroplasticity, axonal guidance and micro-RNA genes are associated with morphine self-administration behavior.

Neuroadaptations in the ventral striatum (VS) and ventral midbrain (VMB) following chronic opioid administration are thought to contribute to the pathogenesis and persistence of opiate addiction. In order to identify candidate genes involved in these neuroadaptations, we utilized a behavior-genetics strategy designed to associate contingent intravenous drug self-administration with specific patterns of gene expression in inbred mice differentially predisposed to the rewarding effects of morphine. In a Yoked-control paradigm, C57BL/6J mice showed clear morphine-reinforced behavior, whereas DBA/2J mice did not. Moreover, the Yoked-control paradigm revealed the powerful consequences of self-administration versus passive administration at the level of gene expression. Morphine self-administration in the C57BL/6J mice uniquely up- or down-regulated 237 genes in the VS and 131 genes in the VMB. Interestingly, only a handful of the C57BL/6J self-administration genes (<3%) exhibited a similar expression pattern in the DBA/2J mice. Hence, specific sets of genes could be confidently assigned to regional effects of morphine in a contingent- and genotype-dependent manner. Bioinformatics analysis revealed that neuroplasticity, axonal guidance and micro-RNAs (miRNAs) were among the key themes associated with drug self-administration. Noteworthy were the primary miRNA genes H19 and micro-RNA containing gene (Mirg), processed, respectively, to mature miRNAs miR-675 and miR-154, because they are prime candidates to mediate network-like changes in responses to chronic drug administration. These miRNAs have postulated roles in dopaminergic neuron differentiation and mu-opioid receptor regulation. The strategic approach designed to focus on reinforcement-associated genes provides new insight into the role of neuroplasticity pathways and miRNAs in drug addiction.

Molecular Insight into Drug Resistance Mechanism Conferred by Aberrant PIK3CD Splice Variant in African American Prostate Cancer.

Targeting PI3Kδ has emerged as a promising therapy for hematologic and non-hematologic malignancies. Previously, we identified an oncogenic splice variant, PIK3CD-S, conferring Idelalisib resistance in African American (AA) prostate cancer (PCa). In the current study, we employed a comprehensive analysis combining molecular biology, biochemistry, histology, in silico simulation, and in vitro functional assays to investigate the PIK3CD-S expression profiles in PCa samples and to elucidate the drug resistance mechanism mediated by PI3Kδ-S (encoded by PIK3CD-S). The immunohistochemistry, RT-PCR, and Western blot assays first confirmed that PI3Kδ-S is highly expressed in AA PCa. Compared with PCa expressing the full-length PI3Kδ-L, PCa expressing PI3Kδ-S exhibits enhanced drug resistance properties, including a higher cell viability, more antiapoptotic and invasive capacities, and constitutively activated PI3K/AKT signaling, in the presence of PI3Kδ/PI3K inhibitors (Idelalisib, Seletalisib, Wortmannin, and Dactolisib). Molecular docking, ATP-competitive assays, and PI3 kinase assays have further indicated a drastically reduced affinity of PI3Kδ inhibitors with PI3Kδ-S vs. PI3Kδ-L, attributed to the lack of core binding residues in the PI3Kδ-S catalytic domain. Additionally, SRSF2 has been identified as a critical splicing factor mediating exon 20 skipping in PIK3CD pre-mRNA. The inhibition of the SRSF2 activity by SRPIN340 successfully sensitizes AA PCa cells to PI3Kδ inhibitors, suggesting a novel therapeutic option for Idelalisib-resistant tumors.

Aberrant PI3Kδ splice isoform as a potential biomarker and novel therapeutic target for endocrine cancers.

Introduction: PI3K/AKT signaling pathway is upregulated in a broad spectrum of cancers. Among the class I PI3Ks (PI3Kδ/ß/δ isoforms), PI3Kδ has been implicated in hematologic cancers and solid tumors. Alternative splicing is a post-transcriptional process for acquiring proteomic diversity in eukaryotic cells. Emerging evidence has highlighted the involvement of aberrant mRNA splicing in cancer development/progression. Methods: Our previous studies revealed that PIK3CD-S is an oncogenic splice variant that promotes tumor aggressiveness and drug resistance in prostate cancer (PCa). To further evaluate the potential of utilizing PI3Kδ-S (encoded from PIK3CD-S) as a cancer biomarker and/or drug target, comprehensive analyses were performed in a series of patient samples and cell lines derived from endocrine/solid tumors. Specifically, IHC, immunofluorescence, western blot and RT-PCR assay results have demonstrated that PI3Kδ isoforms were highly expressed in endocrine/solid tumor patient specimens and cell lines. Results: Differential PIK3CD-S/PIK3CD-L expression profiles were identified in a panel of endocrine/solid tumor cells. SiRNA knockdown of PIK3CD-L or PIK3CD-S differentially inhibits AKT/mTOR signaling in PCa, breast, colon and lung cancer cell lines. Moreover, siRNA knockdown of PTEN increased PI3Kδ levels and activated AKT/mTOR signaling, while overexpression of PTEN reduced PI3Kδ levels and inhibited AKT/mTOR signaling in cancer cells. Intriguingly, PI3Kδ-S levels remained unchanged upon either siRNA knockdown or overexpression of PTEN. Taken together, these results suggested that PTEN negatively regulates PI3Kδ-L and its downstream AKT/mTOR signaling, while PI3Kδ-S promotes AKT/mTOR signaling without regulation by PTEN. Lastly, PI3Kδ inhibitor Idelalisib and SRPK1/2 inhibitor SRPIN340 were employed to assess their efficacies on inhibiting the PI3Kδ-expressing endocrine/solid tumors. Our results have shown that Idelalisib effectively inhibited PI3Kδ-L (but not PI3Kδ-S) mediated AKT/mTOR signaling. In contrast, SRPIN340 reversed the aberrant mRNA splicing, thereby inhibiting AKT/mTOR signaling. In-vitro functional assays have further demonstrated that a combination of Idelalisib and SRPIN340 achieved a synergistic drug effect (with drastically reduced cell viabilities/growths of tumor spheroids) in inhibiting the advanced tumor cells. Conclusion: In summary, our study has suggested a promising potential of utilizing PI3Kδ-S (an oncogenic isoform conferring drug resistance and exempt from PTEN regulation) as a prognostic biomarker and drug target in advanced endocrine cancers.

Deregulated microRNAs Involved in Prostate Cancer Aggressiveness and Treatment Resistance Mechanisms.

Prostate cancer (PCa) is the most frequently diagnosed cancer and the second leading cause of cancer deaths among American men. Complex genetic and epigenetic mechanisms are involved in the development and progression of PCa. MicroRNAs (miRNAs) are short noncoding RNAs that regulate protein expression at the post-transcriptional level by targeting mRNAs for degradation or inhibiting protein translation. In the past two decades, the field of miRNA research has rapidly expanded, and emerging evidence has revealed miRNA dysfunction to be an important epigenetic mechanism underlying a wide range of diseases, including cancers. This review article focuses on understanding the functional roles and molecular mechanisms of deregulated miRNAs in PCa aggressiveness and drug resistance based on the existing literature. Specifically, the miRNAs differentially expressed (upregulated or downregulated) in PCa vs. normal tissues, advanced vs. low-grade PCa, and treatment-responsive vs. non-responsive PCa are discussed. In particular, the oncogenic and tumor-suppressive miRNAs involved in the regulation of (1) the synthesis of the androgen receptor (AR) and its AR-V7 splice variant, (2) PTEN expression and PTEN-mediated signaling, (3) RNA splicing mechanisms, (4) chemo- and hormone-therapy resistance, and (5) racial disparities in PCa are discussed and summarized. We further provide an overview of the current advances and challenges of miRNA-based biomarkers and therapeutics in clinical practice for PCa diagnosis/prognosis and treatment.

Tumor suppressive miR-99b-5p as an epigenomic regulator mediating mTOR/AR/SMARCD1 signaling axis in aggressive prostate cancer.

Introduction: African American (AA) men exhibited 2.3-fold higher PCa incidence and 1.7-fold higher PCa mortality rates when compared to the European American (EA) men. Besides the socioeconomic factors, emerging evidence has highlighted that biological risk factors may play critical roles in the AA PCa disparities. Previously, we have shown that downregulated miR-99b-5p and upregulated mTOR cooperatively promotes the AA PCa aggressiveness and drug resistance. Methods: In this study, we aimed to explore the miR-99b-5p/mTOR/AR/SMARCD1 signaling axis in AA PCa aggressiveness. The analyses used in the study included immunofluorescence, western blot, in-vitro functional assays (TUNEL, colony forming, and MTT), and chromatin immunoprecipitation (ChIP)-qPCR assays in 2D and/or 3D culture model of EA PCa and AA PCa cell lines. Results: Specifically, the immunofluorescence staining, and western blot analysis has revealed that nuclear mTOR, AR, and SMARCD1 were highly expressed in AA PCa (MDA PCa 2b) compared to EA PCa (LNCaP) cell line. Western blot analysis further revealed that miR-99b-5p inhibited protein levels of mTOR, AR/AR-V7 and SMARCD1 in cytoplasm and nuclei of EA and AA PCa. The in-vitro functional (MTT, TUNEL, and clonogenic) assays have demonstrated that miR-99b-5p effectively inhibited cell proliferation/survival and induced cell apoptosis in EA and AA PCa cells. Moreover, combination of miR-99b-5p and enzalutamide (Enz) synergistically enhances the cytotoxicity against aggressive AA PCa and castration-resistant prostate cancer (CRPC). mTOR ChIP-qPCR assays further demonstrated that miR-99b-5p or miR-99b-5p/Enz significantly reduces the recruitment of mTOR to the genes involved in the metabolic reprogramming in CRPC. Discussion: Taken together, miR-99b-5p may function as an epigenomic driver to modulate the mTOR/AR/SMARCD1 signaling axis in AA PCa and resistant CRPC.

Prostate cancer: Alternatively spliced mRNA transcripts in tumor progression and their uses as therapeutic targets.

Prostate cancer is the most frequently diagnosed cancer and second leading cause of cancer deaths among American men. Current therapies show early antitumor responses, but ultimately lead to treatment resistance, relapse and poorer survival in patients. Alternative RNA splicing, a cell mechanism increasing the proteome diversity by producing multiple transcripts from a single gene, has been associated with prostate cancer development/progression. Reports showed that many aberrant mRNA splice variants are upregulated in prostate cancer, promoting malignancy through enhanced proliferation, metastasis, tumor growth, anti-apoptosis, and/or treatment resistance. Here, we discuss the oncogenic properties of aberrant splicing mechanisms underlying prostate cancer pathogenesis, as well as the uses of the splicing variants as potential diagnostics and treatment targets. Finally, we discuss the pharmacologic and molecular approaches for targeting aberrant splicing mechanisms as effective therapies to correct the splicing errors and overcome the drug resistance, ultimately improving the clinical outcome of prostate cancer patients.

Genomics of Black American colon cancer disparities: An RNA sequencing (RNA-Seq) study from an academic, tertiary referral center.

BACKGROUND: Black Americans have a higher incidence and mortality rate from colorectal cancer compared to their non-Hispanic White American counterparts. Even when controlling for sociodemographic differences between these 2 populations, Black Americans remain disproportionately affected by colorectal cancer. The purpose of our study was to determine if differences in gene expression between Black American and non-Hispanic White American colon cancer specimens could help explain differences in the incidence and mortality rate between these 2 populations. METHODS: Black Americans and non-Hispanic White Americans undergoing colon resection for stages I, II, or III colon cancer at a single institution were identified. Black American and non-Hispanic White American patients were matched for age, sex, and colon cancer stage to minimize the risk of confounding variables. Tissue samples were obtained at the time of colon resection and were analyzed using RNA sequencing to determine if there were differences in the expression of genes and biologic processes between the 2 groups. RESULTS: A total of 17 colon cancer specimens were analyzed; 8 (47.1%) patients were Black Americans. A total of 456 genes were identified as being expressed differently (ie, up or downregulated) in Black American compared to non-Hispanic White American colon cancer specimens. Moreover, 500 different genetic pathways were noted to be significantly over-represented with differentially expressed genes in our comparison of Black American and non-Hispanic White American colon cancer specimens, the majority of which plays a role in inflammation and immune cell function. CONCLUSION: Significant differences in gene expression and genetic pathways exist between Black Americans and non-Hispanic White Americans. Additional and multi-institutional and registry-based studies are needed to validate our findings and to further elucidate the contribution that these differences have to the overall incidence and mortality rate from colon cancer in these 2 patient populations.

Prostate apoptosis response protein 4 sensitizes human colon cancer cells to chemotherapeutic 5-FU through mediation of an NF kappaB and microRNA network.

BACKGROUND: Diminished expression or activity of prostate apoptosis response protein 4 (Par-4) has been demonstrated in a number of cancers, although reports on Par-4 expression during colon cancer progression are lacking. An understanding of the molecular events in conjunction with the genetic networks affected by Par-4 is warranted. RESULTS: Colon cancer specimens derived from patients have significantly diminished expression of Par-4 mRNA relative to paired normal colon. Hence, the functional consequences of reintroducing Par-4 into HT29 colon cancer cells were assessed. Overexpression augmented the interaction of Par-4 with NF kappaB in the cytosol but not nucleus, and facilitated apoptosis in the presence of 5-fluorouracil (5-FU). Analogous findings were obtained when AKT1 pro-survival signaling was inhibited. Transcriptome profiling identified approximately 700 genes differentially regulated by Par-4 overexpression in HT29 cells. Nearly all Par-4-regulated genes were shown by promoter analysis to contain cis-binding sequences for NF kappaB, and meta-analysis of patient expression data revealed that one-third of these genes exist as a recurrent co-regulated network in colon cancer specimens. Sets of genes involved in programmed cell death, cell cycle regulation and interestingly the microRNA pathway were found overrepresented in the network. Noteworthy, Par-4 overexpression decreased NF kappaB occupancy at the promoter of one particular network gene DROSHA, encoding a microRNA processing enzyme. The resulting down-regulation of DROSHA was associated with expression changes in a cohort of microRNAs. Many of these microRNAs are predicted to target mRNAs encoding proteins with apoptosis-related functions. Western and functional analyses were employed to validate several predictions. For instance, miR-34a up-regulation corresponded with a down-regulation of BCL2 protein. Treating Par-4-overexpressing HT29 cells with a miR-34a antagomir functionally reversed both BCL2 down-regulation and apoptosis by 5-FU. Conversely, bypassing Par-4 overexpression by direct knockdown of DROSHA expression in native HT29 cells increased miR-34a expression and 5-FU sensitivity. CONCLUSION: Our findings suggest that the initiation of apoptotic sensitivity in colon cancer cells can be mediated by Par-4 binding to NF kappaB in the cytoplasm with consequential changes in the expression of microRNA pathway components.

A Novel FGFR3 Splice Variant Preferentially Expressed in African American Prostate Cancer Drives Aggressive Phenotypes and Docetaxel Resistance.

Alternative splicing (AS) has been shown to participate in prostate cancer development and progression; however, a link between AS and prostate cancer health disparities has been largely unexplored. Here we report on the cloning of a novel splice variant of FGFR3 that is preferentially expressed in African American (AA) prostate cancer. This novel variant (FGFR3-S) omits exon 14, comprising 123 nucleotides that encode the activation loop in the intracellular split kinase domain. Ectopic overexpression of FGFR3-S in European American (EA) prostate cancer cell lines (PC-3 and LNCaP) led to enhanced receptor autophosphorylation and increased activation of the downstream signaling effectors AKT, STAT3, and ribosomal S6 compared with FGFR3-L (retains exon 14). The increased oncogenic signaling imparted by FGFR3-S was associated with a substantial gain in proliferative and antiapoptotic activities, as well as a modest but significant gain in cell motility. Moreover, the FGFR3-S-conferred proliferative and motility gains were highly resistant to the pan-FGFR small-molecule inhibitor dovitinib and the antiapoptotic gain was insensitive to the cytotoxic drug docetaxel, which stands in marked contrast with dovitinib- and docetaxel-sensitive FGFR3-L. In an in vivo xenograft model, mice injected with PC-3 cells overexpressing FGFR3-S exhibited significantly increased tumor growth and resistance to dovitinib treatment compared with cells overexpressing FGFR3-L. In agreement with our in vitro and in vivo findings, a high FGFR3-S/FGFR3-L expression ratio in prostate cancer specimens was associated with poor patient prognosis. IMPLICATIONS: This work identifies a novel FGFR3 splice variant and supports the hypothesis that differential AS participates in prostate cancer health disparities.

Molecular mechanism underlying differential apoptosis between human melanoma cell lines UACC903 and UACC903(+6) revealed by mitochondria-focused cDNA microarrays.

Human malignant melanoma cell line UACC903 is resistant to apoptosis while chromosome 6-mediated suppressed cell line UACC903(+6) is sensitive. Here, we describe identification of differential molecular pathways underlying this difference. Using our recently developed mitochondria-focused cDNA microarrays, we identified 154 differentially expressed genes including proapoptotic (BAK1 [6p21.3], BCAP31, BNIP1, CASP3, CASP6, FAS, FDX1, FDXR, TNFSF10 and VDAC1) and antiapoptotic (BCL2L1, CLN3 and MCL1) genes. Expression of these pro- and anti-apoptotic genes was higher in UACC903(+6) than in UACC903 before UV treatment and was altered after UV treatment. qRT-PCR and Western blots validated microarray results. Our bioinformatic analysis mapped these genes to differential molecular pathways that predict resistance and sensitivity of UACC903 and UACC903(+6) to apoptosis respectively. The pathways were functionally confirmed by the FAS ligand-induced cell death and by siRNA knockdown of BAK1 protein. These results demonstrated the differential molecular pathways underlying survival and apoptosis of UACC903 and UACC903(+6) cell lines.

Condensin binding at distinct and specific chromosomal sites in the Saccharomyces cerevisiae genome.

Mitotic chromosome condensation is chiefly driven by the condensin complex. The specific recognition (targeting) of chromosomal sites by condensin is an important component of its in vivo activity. We previously identified the rRNA gene cluster in Saccharomyces cerevisiae as an important condensin-binding site, but both genetic and cell biology data suggested that condensin also acts elsewhere. In order to characterize the genomic distribution of condensin-binding sites and to assess the specificity of condensin targeting, we analyzed condensin-bound sites using chromatin immunoprecipitation and hybridization to whole-genome microarrays. The genomic condensin-binding map shows preferential binding sites over the length of every chromosome. This analysis and quantitative PCR validation confirmed condensin-occupied sites across the genome and in the specialized chromatin regions: near centromeres and telomeres and in heterochromatic regions. Condensin sites were also enriched in the zones of converging DNA replication. Comparison of condensin binding in cells arrested in G(1) and mitosis revealed a cell cycle dependence of condensin binding at some sites. In mitotic cells, condensin was depleted at some sites while enriched at rRNA gene cluster, subtelomeric, and pericentromeric regions.

Aberrant RNA Splicing in Cancer and Drug Resistance.

More than 95% of the 20,000 to 25,000 transcribed human genes undergo alternative RNA splicing, which increases the diversity of the proteome. Isoforms derived from the same gene can have distinct and, in some cases, opposing functions. Accumulating evidence suggests that aberrant RNA splicing is a common and driving event in cancer development and progression. Moreover, aberrant splicing events conferring drug/therapy resistance in cancer is far more common than previously envisioned. In this review, aberrant splicing events in cancer-associated genes, namely BCL2L1, FAS, HRAS, CD44, Cyclin D1, CASP2, TMPRSS2-ERG, FGFR2, VEGF, AR and KLF6, will be discussed. Also highlighted are the functional consequences of aberrant splice variants (BCR-Abl35INS, BIM-γ, IK6, p61 BRAF V600E, CD19-∆2, AR-V7 and PIK3CD-S) in promoting resistance to cancer targeted therapy or immunotherapy. To overcome drug resistance, we discuss opportunities for developing novel strategies to specifically target the aberrant splice variants or splicing machinery that generates the splice variants. Therapeutic approaches include the development of splice variant-specific siRNAs, splice switching antisense oligonucleotides, and small molecule inhibitors targeting splicing factors, splicing factor kinases or the aberrant oncogenic protein isoforms.

MicroRNA and mRNA expression associated with ectopic germinal centers in thymus of myasthenia gravis.

BACKGROUND: A characteristic pathology of early onset myasthenia gravis is thymic hyperplasia with ectopic germinal centers (GC). However, the mechanisms that trigger and maintain thymic hyperplasia are poorly characterized. Dysregulation of small, non-coding microRNAs (miRNAs) and their target genes has been identified in the pathology of several autoimmune diseases. We assessed the miRNA and mRNA profiles of the MG thymus and have investigated their role in GC formation and maintenance. METHODS: MG thymus samples were assessed by histology and grouped based upon the appearance of GC; GC positive and GC negative. A systems biology approach was used to study the differences between the groups. Our study included miRNA and mRNA profiling, quantitative real-time PCR validation, miRNA target identification, pathway analysis, miRNA-mRNA reciprocal expression pairing and interaction. RESULTS: Thirty-eight mature miRNAs and forty-six annotated mRNA transcripts were differentially expressed between the two groups (>1.5 fold change, ANOVA p<0.05). The miRNAs were found to be involved in immune response pathways and identified in other autoimmune diseases. The cellular and molecular functions of the mRNAs showed involvement in cell death and cell survival, cellular proliferation, cytokine signaling and extra-cellular matrix reorganization. Eleven miRNA and mRNA pairs were reciprocally regulated. The Regulator of G protein Signalling 13 (RGS13), known to be involved in GC regulation, was identified in specimens with GC and was paired with downregulation of miR-452-5p and miR-139-5p. MiRNA target sites were validated by dual luciferase assay. Transfection of miRNA mimics led to down regulation of RGS13 expression in Raji cells. CONCLUSION: Our study indicates a distinct miRNA and mRNA expression pattern in ectopic GC in MG thymus. These miRNAs and mRNAs are involved in regulatory pathways common to inflammation and immune response, cell cycle regulation and anti-apoptotic pathways suggesting their involvement in support of GC formation in the thymus. We demonstrate for the first time that miR-139-5p and miR-452-5p negatively regulate RGS13 expression.

Corrigendum: Alternative splicing promotes tumour aggressiveness and drug resistance in African American prostate cancer.

This corrects the article DOI: 10.1038/ncomms15921.