The Mechanistic Role of Long Non-coding RNAs in Advanced Prostate Cancer Response to Therapy

Prostate cancer is one of the most common malignancies and the fifth leading cause of cancer related deaths in males, worldwide. It is commonly treated using androgen deprivation therapy (ADT), but around 25% develop ADT resistance and are called Castration-Resistant Prostate Cancers (CRPCs). Therapies currently used for CRPC treatment include: the new generation anti-androgen enzalutamide, the taxane cabazitaxel and carboplatin. Despite the prolonged survival resulting from these treatments, CRPC is still incurable. Recent evidence suggests that long non-coding RNAs (lncRNAs) promote drug resistance. The lncRNA HORAS5 (i.e. linc00161) regulates drug response in different cancers and is upregulated in CRPC versus hormone-sensitive patient-derived xenografts (PDXs), thereby stimulating pro-survival mechanisms. This project has investigated whether HORAS5 has a role in CRPC response to therapy. CRPC cells have been treated with different concentrations of cabazitaxel, carboplatin and enzalutamide. Cabazitaxel exposure increases HORAS5 expression, in androgen receptor-positive (AR+) and -negative (AR–) prostate cancer cells and HORAS5 overexpression decreases cabazitaxel sensitivity and cell apoptosis. HORAS5 RNA interference (RNAi) increases cabazitaxel sensitivity and cell apoptosis. Next-generation RNA sequencing and real time qPCR have shown that the anti-apoptotic factor BCL2A1 is significantly upregulated upon HORAS5 overexpression in AR- prostate cancer cells exposed to cabazitaxel. BCL2A1 silencing decreases cell count and increases apoptosis of prostate cancer cells exposed to cabazitaxel. HORAS5 and BCL2A1 upregulation is associated with decreased survival in prostate cancer patients and HORAS5 is upregulated in clinical samples from prostate cancer patients exposed to taxanes. Transfection of CRPC cells with HORAS5-targeting antisense oligonucleotides (ASOs) efficiently reduces HORAS5 expression, thereby decreasing cabazitaxel IC50 when tested in combination with this drug. Overall, this project shows that HORAS5 stimulates BCL2A1 expression in prostate cancer cells, thereby reducing caspase-mediated apoptosis. This leads to increased cabazitaxel resistance. The clinical relevance of HORAS5 expression and the effect of HORAS5-targeting ASOs shown in this thesis highlight the translational potential of HORAS5 modulation. This work sheds light on the relevance of lncRNAs in cancer drug resistance and proposes the use of HORAS5 as a future therapeutic target to increase therapy efficacy in CRPC

The role of histone lysine demethylases in cancer cells' resistance to tyrosine kinase inhibitors.

Current cancer therapies are often associated with treatment failure and reduced patients' survival due to drug resistance. There are various mechanisms involved in the acquisition of cancer drug resistance, including the selection of advantageous mutations, overexpression of transporter proteins and epigenetic alterations. In this context, epigenetic alterations refer to chromatin-mediated regulation of gene expression that results in heritable changes in the cellular phenotype. There is an ever-growing body of evidence suggesting that epigenetic mechanisms play an important role in bringing about drug resistance in cancer cells. While the relationship between chemotherapy and epigenetics has been widely discussed, emerging evidence indicates that specific epigenetic effectors are also crucial for the development of resistance to tyrosine kinase inhibitors (TKIs). One particular gene that encodes the histone lysine demethylase KDM5A is overexpressed in several cancers. In breast cancer tissues, cells with KDM5A gene amplification were found to be more resistant to erlotinib, an inhibitor of the tyrosine kinase epidermal growth factor receptor (EGFR), when compared to cells without the same amplification. KDM5A was also shown to mediate resistance to a second EGFR inhibitor called gefitinib, in EGFR-mutant lung cancer cell lines. This evidence indicates that KDM5A could activate alternative survival pathways involved in overcoming EGFR inhibition. In line with these results, another histone demethylase (i.e., KDM1A) promotes liver cancer cells' resistance to the TKI sorafenib. Current evidence provides a suitable rationale to consider the use of specific KDMs inhibitors to sensitize cells to tyrosine kinase targeted therapies and thus, presents an opportunity to prevent the further development of drug resistance. This review discusses the involvement of histone lysine demethylases in the development of resistance to TKI and highlights the importance to develop new cancer treatment regimens to counteract this phenomenon

Deep analysis of neuroblastoma core regulatory circuitries using online databases and integrated bioinformatics shows their pan-cancer roles as prognostic predictors.

AIM: Neuroblastoma is a heterogeneous childhood cancer derived from the neural crest. The dual cell identities of neuroblastoma include Mesenchymal (MES) and Adrenergic (ADRN). These identities are conferred by a small set of tightly-regulated transcription factors (TFs) binding super enhancers, collectively forming core regulatory circuitries (CRCs). The purpose of this study was to gain a deep understanding of the role of MES and ADRN TFs in neuroblastoma and other cancers as potential indicators of disease prognosis, progression, and relapse. METHODS: To that end, we first investigated the expression and mutational profile of MES and ADRN TFs in neuroblastoma. Moreover, we established their correlation with neuroblastoma risk groups and overall survival while establishing their extended networks with long non-coding RNAs (lncRNAs). Furthermore, we analysed the pan-cancer expression and mutational profile of these TFs and their correlation with patient survival and finally their network connectivity, using a panel of bioinformatic tools including GEPIA2, human pathology atlas, TIMER2, Omicsnet, and Cytoscape. RESULTS: We show the association of multiple MES and ADRN TFs with neuroblastoma risk groups and overall survival and find significantly higher expression of various MES and ADRN TFs compared to normal tissues and their association with overall survival and disease-free survival in multiple cancers. Moreover, we report the strong correlation of the expression of these TFs with the infiltration of stromal and immune cells in the tumour microenvironment and with stemness and metastasis-related genes. Furthermore, we reveal extended pan-cancer networks comprising these TFs that influence the tumour microenvironment and metastasis and may be useful indicators of cancer prognosis and patient survival. CONCLUSION: Our meta-analysis shows the significance of MES and ADRN TFs as indicators of patient prognosis and the putative utility of these TFs as potential novel biomarkers

The long non-coding RNA <i>HORAS5</i> mediates castration-resistant prostate cancer survival by activating the androgen receptor transcriptional program

Prostate Cancer (PCa) is driven by the androgen receptor (AR)-signaling axis. Hormonal therapy often mitigates PCa progression, but a notable number of cases progress to castration-resistant PCa (CRPC). CRPC retains AR-activity and is incurable. Long non-coding RNAs (lncRNAs) represent an uncharted region of the transcriptome. Several lncRNAs have been recently described to mediate oncogenic functions, suggesting that these molecules can be potential therapeutic targets. Here, we identified CRPC-associated lncRNAs by analyzing patient-derived xenografts (PDXs) and clinical data. Subsequently, we characterized one of the CRPC-promoting lncRNAs, HORAS5, in vitro and in vivo. We demonstrated that HORAS5 is a stable, cytoplasmic lncRNA that promotes CRPC proliferation and survival by maintaining AR activity under androgen-depleted conditions. Most strikingly, knockdown of HORAS5 causes a significant reduction in the expression of AR itself and oncogenic AR targets such as KIAA0101. Elevated expression of HORAS5 is also associated with worse clinical outcomes in patients. Our results from HORAS5 inhibition in in vivo models further confirm that HORAS5 is a viable therapeutic target for CRPC. Thus, we posit that HORAS5 is a novel, targetable mediator of CRPC through its essential role in the maintenance of oncogenic AR activity. Overall, this study adds to our mechanistic understanding of how lncRNAs function in cancer progression

Long non-coding RNAs are key players in Prostate cancer tumorigenesis and drug resistance

"Long non-coding RNAs (lncRNAs) have been characterized as key players in several cancer-associated processes such as tumorigenesis and drug resistance. Emerging evidence indicates that lncRNAs affect initiation and progression of several cancers, including prostate cancer (PCa) and its advanced forms, such as metastatic castration resistant prostate cancer (mCRPC). Among others, H19 is one of the most studied oncogenic lncRNAs in cancer and has been associated with cancer tumorigenesis and progression, via mediating several pathways an acting in different mechanisms of action, including epigenetic and miRNA regulation. We have investigated lncRNA roles in PCa tumorigenesis by analysing the E006AA cell model. Emerging evidence has shown that parental E006AA are not tumorigenic in nude mice, while its derived E006AA-ht, is highly tumorigenic. Via high-throughput RNA sequencing we have shown that E006AA-ht overexpress different lncRNAs compared to E006AA. Interestingly, we found a high upregulation of H19 in E006AA-ht vs the parental cell line. Our in vitro validation has confirmed the sequencing data and further research could unravel a crucial role of H19 in PCa tumorigenesis, opening a new challenging chapter of lncRNAs research. We hypothesize that H19 could be involved in in vivo immunity suppression and our further studies aim at investigating H19 in this molecular and clinical context. Furthermore, we have studied lncRNAs as key players in cancer aggressiveness by using cellular models of mCRPC. From our studies, HORAS5 (i.e. linc00161) emerged as the most consistently upregulated lncRNA in CRPC patient-derived xenografts. This lncRNAs was previously associated with cancer drug-response in osteosarcoma, ovarian cancer and hepatocellular carcinoma. Our study has shown for the first time that HORAS5 promotes drug resistance in CRPC. After a preliminary drug screen, we have selected the chemotherapeutics cabazitaxel for further investigation. Via lentiviral-mediated overexpression and siRNA-based silencing we have regulated HORAS5 expression and analysed cell count and apoptosis of CRPC cells exposed to clinically achievable concentrations of cabazitaxel. The overexpression of HORAS5 increases cabazitaxel resistance, while HORAS5 silencing has an opposite effect, via inhibition of apoptosis. RNA sequencing and RT-qPCR revealed that BCL2A1 is the most upregulated transcript in HORAS5 overexpressing cells exposed to cabazitaxel, and that BCL2A1 silencing decreases cell count and increases caspase activity. Our data suggest that BCL2A1 expression is induced by HORAS5, thereby enhancing CRPC cells resistance to cabazitaxel. Transfection of CRPC cells with HORAS5-targeting ASOs can effectively silence this lncRNA and determine a decrease of cabazitaxel resistance. We have also shown that both HORAS5 and BCL2A1 upregulation results in decreased survival in PCa patients and samples from mCRPC patients treated with taxanes have upregulation of HORAS5. Overall, our studies bring novel insights into crucial roles of lncRNAs in PCa progression and aggressiveness making them emerging targets for cancer treatment at different stages.

Variation rs2235503 C > A Within the Promoter of MSLN Affects Transcriptional Rate of Mesothelin and Plasmatic Levels of the Soluble Mesothelin-Related Peptide.

Soluble mesothelin-related peptide (SMRP) is a promising biomarker for malignant pleural mesothelioma (MPM), but several confounding factors can reduce SMRP-based test's accuracy. The identification of these confounders could improve the diagnostic performance of SMRP. In this study, we evaluated the sequence of 1,000 base pairs encompassing the minimal promoter region of the MSLN gene to identify expression quantitative trait loci (eQTL) that can affect SMRP. We assessed the association between four MSLN promoter variants and SMRP levels in a cohort of 72 MPM and 677 non-MPM subjects, and we carried out in vitro assays to investigate their functional role. Our results show that rs2235503 is an eQTL for MSLN associated with increased levels of SMRP in non-MPM subjects. Furthermore, we show that this polymorphic site affects the accuracy of SMRP, highlighting the importance of evaluating the individual's genetic background and giving novel insights to refine SMRP specificity as a diagnostic biomarker

LncRNA HORAS5 promotes taxane resistance in castration-resistant prostate cancer via a BCL2A1-dependent mechanism.

Background: Castration-resistant prostate cancer (CRPC) is an incurable malignancy. Long noncoding RNAs (lncRNAs) play key roles in drug resistance. Materials & methods: LncRNA HORAS5 role in cabazitaxel resistance (i.e., cell-count, IC50 and caspase activity) was studied via lentiviral-mediated overexpression and siRNA-based knockdown. Genes expression was analyzed with RNA-sequencing, reverse transcription quantitative PCR (RT-qPCR) and western blot. HORAS5 expression was queried in clinical database. Results: Cabazitaxel increased HORAS5 expression that upregulated BCL2A1, thereby protecting CRPC cells from cabazitaxel-induced apoptosis. BCL2A1 knockdown decreased cell-count and increased apoptosis in CRPC cells. HORAS5-targeting antisense oligonucleotide decreased cabazitaxel IC50. In CRPC clinical samples, HORAS5 expression increased upon taxane treatment. Conclusion:HORAS5 stimulates the expression of BCL2A1 thereby decreasing apoptosis and enhancing cabazitaxel resistance in CRPC cells

The Role of Autophagy and lncRNAs in the Maintenance of Cancer Stem Cells

Cancer stem cells (CSCs) possess properties such as self-renewal, resistance to apoptotic cues, quiescence, and DNA-damage repair capacity. Moreover, CSCs strongly influence the tumour microenvironment (TME) and may account for cancer progression, recurrence, and relapse. CSCs represent a distinct subpopulation in tumours and the detection, characterisation, and understanding of the regulatory landscape and cellular processes that govern their maintenance may pave the way to improving prognosis, selective targeted therapy, and therapy outcomes. In this review, we have discussed the characteristics of CSCs identified in various cancer types and the role of autophagy and long noncoding RNAs (lncRNAs) in maintaining the homeostasis of CSCs. Further, we have discussed methods to detect CSCs and strategies for treatment and relapse, taking into account the requirement to inhibit CSC growth and survival within the complex backdrop of cellular processes, microenvironmental interactions, and regulatory networks associated with cancer. Finally, we critique the computationally reinforced triangle of factors inclusive of CSC properties, the process of autophagy, and lncRNA and their associated networks with respect to hypoxia, epithelial-to-mesenchymal transition (EMT), and signalling pathways

The Contribution of Autophagy and LncRNAs to MYC-Driven Gene Regulatory Networks in Cancers

MYC is a target of the Wnt signalling pathway and governs numerous cellular and developmental programmes hijacked in cancers. The amplification of MYC is a frequently occurring genetic alteration in cancer genomes, and this transcription factor is implicated in metabolic reprogramming, cell death, and angiogenesis in cancers. In this review, we analyse MYC gene networks in solid cancers. We investigate the interaction of MYC with long non-coding RNAs (lncRNAs). Furthermore, we investigate the role of MYC regulatory networks in inducing changes to cellular processes, including autophagy and mitophagy. Finally, we review the interaction and mutual regulation between MYC and lncRNAs, and autophagic processes and analyse these networks as unexplored areas of targeting and manipulation for therapeutic gain in MYC-driven malignancies

BRG1 and NPM-ALK Are Co-Regulated in Anaplastic Large-Cell Lymphoma; BRG1 Is a Potential Therapeutic Target in ALCL.

Anaplastic large-cell lymphoma (ALCL) is a T-cell malignancy driven in many cases by the product of a chromosomal translocation, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK). NPM-ALK activates a plethora of pathways that drive the hallmarks of cancer, largely signalling pathways normally associated with cytokine and/or T-cell receptor-induced signalling. However, NPM-ALK is also located in the nucleus and its functions in this cellular compartment for the most part remain to be determined. We show that ALCL cell lines and primary patient tumours express the transcriptional activator BRG1 in a NPM-ALK-dependent manner. NPM-ALK regulates expression of BRG1 by post-translational mechanisms dependent on its kinase activity, protecting it from proteasomal degradation. Furthermore, we show that BRG1 drives a transcriptional programme associated with cell cycle progression. In turn, inhibition of BRG1 expression with specific shRNA decreases cell viability, suggesting that it may represent a key therapeutic target for the treatment of ALCL