Targeting NRAS via miR-1304-5p or farnesyltransferase inhibition confers sensitivity to ALK inhibitors in ALK-mutant neuroblastoma.

Targeting Anaplastic lymphoma kinase (ALK) is a promising therapeutic strategy for aberrant ALK-expressing malignancies including neuroblastoma, but resistance to ALK tyrosine kinase inhibitors (ALK TKI) is a distinct possibility necessitating drug combination therapeutic approaches. Using high-throughput, genome-wide CRISPR-Cas9 knockout screens, we identify miR-1304-5p loss as a desensitizer to ALK TKIs in aberrant ALK-expressing neuroblastoma; inhibition of miR-1304-5p decreases, while mimics of this miRNA increase the sensitivity of neuroblastoma cells to ALK TKIs. We show that miR-1304-5p targets NRAS, decreasing cell viability via induction of apoptosis. It follows that the farnesyltransferase inhibitor (FTI) lonafarnib in addition to ALK TKIs act synergistically in neuroblastoma, inducing apoptosis in vitro. In particular, on combined treatment of neuroblastoma patient derived xenografts with an FTI and an ALK TKI complete regression of tumour growth is observed although tumours rapidly regrow on cessation of therapy. Overall, our data suggests that combined use of ALK TKIs and FTIs, constitutes a therapeutic approach to treat high risk neuroblastoma although prolonged therapy is likely required to prevent relapse.

HAR1: an insight into lncRNA genetic evolution.

Long noncoding RNAs (lncRNAs) have a wide range of functions in health and disease, but many remain uncharacterized because of their complex expression patterns and structures. The genetic loci encoding lncRNAs can be subject to accelerated evolutionary changes within the human lineage. HAR1 is a region that has a significantly altered sequence compared to other primates and is a component of two overlapping lncRNA loci, HAR1A and HAR1B. Although the functions of these lncRNAs are unknown, they have been associated with neurological disorders and cancer. Here, we explore the current state of understanding of evolution in human lncRNA genes, using the HAR1 locus as the case study.

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.

The evolutionarily conserved long non-coding RNA LINC00261 drives neuroendocrine prostate cancer proliferation and metastasis via distinct nuclear and cytoplasmic mechanisms.

Metastatic neuroendocrine prostate cancer (NEPC) is a highly aggressive disease, whose incidence is rising. Long noncoding RNAs (lncRNAs) represent a large family of disease- and tissue-specific transcripts, most of which are still functionally uncharacterized. Thus, we set out to identify the highly conserved lncRNAs that play a central role in NEPC pathogenesis. To this end, we performed transcriptomic analyses of donor-matched patient-derived xenograft models (PDXs) with immunohistologic features of prostate adenocarcinoma (AR+ /PSA+ ) or NEPC (AR- /SYN+ /CHGA+ ) and through differential expression analyses identified lncRNAs that were upregulated upon neuroendocrine transdifferentiation. These genes were prioritized for functional assessment based on the level of conservation in vertebrates. Here, LINC00261 emerged as the top gene with over 3229-fold upregulation in NEPC. Consistently, LINC00261 expression was significantly upregulated in NEPC specimens in multiple patient cohorts. Knockdown of LINC00261 in PC-3 cells dramatically attenuated its proliferative and metastatic abilities, which are explained by parallel downregulation of CBX2 and FOXA2 through distinct molecular mechanisms. In the cell cytoplasm, LINC00261 binds to and sequesters miR-8485 from targeting the CBX2 mRNA, while inside the nucleus, LINC00261 functions as a transcriptional scaffold to induce SMAD-driven expression of the FOXA2 gene. For the first time, these results demonstrate hyperactivation of the LINC00261-CBX2-FOXA2 axes in NEPC to drive proliferation and metastasis, and that LINC00261 may be utilized as a therapeutic target and a biomarker for this incurable disease.

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.

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.

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.

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.

T-type calcium channels drive the proliferation of androgen-receptor negative prostate cancer cells.

BACKGROUND: Androgen deprivation therapy (ADT) is the treatment of choice for metastatic prostate cancer (PCa). After an initial response to ADT, PCa cells can generate castration resistant (CRPC) or neuroendocrine (NEPC) malignancies, which are incurable. T-type calcium channels (TTCCs) are emerging as promising therapeutic targets for several cancers, but their role in PCa progression has never been investigated. METHODS: To examine the role of TTCCs in PCa, we analyzed their expression level, copy number variants (CNV) and prognostic significance using clinical datasets (Oncomine and cBioPortal). We then evaluated TTCC expression in a panel of PCa cell lines and measured the effect of their inhibition on cell proliferation and survival using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and caspase assays. RESULTS: TTCCs were upregulated in PCas harboring androgen receptor (AR) mutations; CNV rate was positively associated with PCa progression. Higher expression of one TTCC isoform (CACNA1G) predicted poorer postoperative prognosis in early stage PCa samples. Pharmacological or small interfering RNA (siRNA)-based inhibition of TTCCs caused a decrease in PC-3 cell survival and proliferation. CONCLUSIONS: Our results show that TTCCs are overexpressed in advanced forms of PCa and correlate with a poorer prognosis. TTCC inhibition reduces cell proliferation and survival, suggesting that there may be possible value in the therapeutic targeting of TTCCs in advanced PCa.

The long noncoding RNA HORAS5 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 noncoding RNA (lncRNA) represent an uncharted region of the transcriptome. Several lncRNA have been recently described to mediate oncogenic functions, suggesting that these molecules can be potential therapeutic targets. Here, we identified CRPC-associated lncRNA by analyzing patient-derived xenografts (PDXs) and clinical data. Subsequently, we characterized one of the CRPC-promoting lncRNA, 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 lncRNA function in cancer progression.

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.

Hypoxia and Noncoding RNAs in Taxane Resistance.

Taxanes are chemotherapeutic drugs employed in the clinic to treat a variety of malignancies. Despite their overall efficacy, cancer cells often display resistance to taxanes. Therefore, new strategies to increase the effectiveness of taxane-based chemotherapeutics are urgently needed. Multiple molecular players are linked to taxane resistance; these include efflux pumps, DNA repair mechanisms, and hypoxia-related pathways. In addition, emerging evidence indicates that both non-coding RNAs and epigenetic effectors might also be implicated in taxane resistance. Here we focus on the causes of taxane resistance, with the aim to envisage an integrated model of the 'taxane resistance phenome'. This model could help the development of novel therapeutic strategies to treat taxane-resistant neoplasms.

Mesothelin promoter variants are associated with increased soluble mesothelin-related peptide levels in asbestos-exposed individuals.

BACKGROUND: Soluble mesothelin-related peptide (SMRP) is a promising diagnostic biomarker for malignant pleural mesothelioma (MPM), but various confounders hinder its usefulness in surveillance programmes. We previously showed that a single nucleotide polymorphism (SNP) within the 3'untranslated region (3'UTR) of the mesothelin (MSLN) gene could affect the levels of SMRP. OBJECTIVES: To focus on SNPs located within MSLN promoter as possible critical genetic variables in determining SMRP levels. METHODS: The association between SMRP and SNPs was tested in 689 non-MPM subjects and 70 patients with MPM. Reporter plasmids carrying the four most common haplotypes were compared in a dual luciferase assay, and in silico analyses were performed to investigate the putative biological role of the SNPs. RESULTS: We found a strong association between serum SMRP and variant alleles of rs3764247, rs3764246 (in strong linkage disequilibrium with rs2235504) and rs2235503 in non-MPM subjects. Inclusion of the genotype information led to an increase in SMRP specificity from 79.9% to 85.5%. Although not statistically significant, the group with MPM showed the same trend of association. According to the in vitro luciferase study, rs3764247 itself had a functional role. In silico approaches showed that the binding sites for transcription factors such as Staf and ZNF143 could be affected by this SNP. The other SNPs were shown to interact with each other in a more complex way. CONCLUSIONS: These data support the suggestion that SMRP performance is affected by individual (ie, genetic) variables and that MSLN expression is influenced by SNPs located within the promoter regulatory region.