Search

Abstract Background Triple-negative breast cancer (TNBC) is highly aggressive with an increased metastatic incidence compared to other breast cancer subtypes. However, due to the absence of clinically reliable biomarkers and targeted therapy in TNBC, outcomes are suboptimal. Hence, there is an urgent need to understand biological mechanisms that lead to identifying novel therapeutic targets for managing metastatic TNBC. Methods The clinical significance of MUC16 and ELAVL1 or Hu antigen R (HuR) was examined using breast cancer TCGA data. Microarray was performed on MUC16 knockdown and scramble TNBC cells and MUC16-associated genes were identified using RNA immunoprecipitation and metastatic cDNA array. Metastatic properties of MUC16 were evaluated using tail vein experiment. MUC16 and HuR downstream pathways were confirmed by ectopic overexpression of MUC16-carboxyl-terminal (MUC16-Cter), HuR and cMyc as well as HuR inhibitors (MS-444 and CMLD-2) in TNBC cells. Results MUC16 was highly expressed in TNBC and correlated with its target HuR. Depletion of MUC16 showed decreased invasion, migration, and colony formation abilities of human and mouse TNBC cells. Mice injected with MUC16 depleted cells were less likely to develop lung metastasis (P = 0.001). Notably, MUC16 and HuR were highly expressed in the lung tropic TNBC cells and lung metastases. Mechanistically, we identified cMyc as a HuR target in TNBC using RNA immunoprecipitation and metastatic cDNA array. Furthermore, MUC16 knockdown and pharmacological inhibition of HuR (MS-444 and CMLD-2) in TNBC cells showed a reduction in cMyc expression. MUC16-Cter or HuR overexpression models indicated MUC16/HuR/cMyc axis in TNBC cell migration. Conclusions Our study identified MUC16 as a TNBC lung metastasis promoter that acts through HuR/cMyc axis. This study will form the basis of future studies to evaluate the targeting of both MUC16 and HuR in TNBC patients.

Background: Triple-negative breast cancer (TNBC) is highly aggressive with an increased metastatic incidence compared to other breast cancer subtypes. However, due to the absence of clinically reliable biomarkers and targeted therapy in TNBC, outcomes are suboptimal. Hence, there is an urgent need to understand biological mechanisms that lead to identifying novel therapeutic targets for managing metastatic TNBC. Methods: The clinical significance of MUC16 and ELAVL1 or Hu antigen R (HuR) was examined using breast cancer TCGA data. Microarray was performed on MUC16 knockdown and scramble TNBC cells and MUC16-associated genes were identified using RNA immunoprecipitation and metastatic cDNA array. Metastatic properties of MUC16 were evaluated using tail vein experiment. MUC16 and HuR downstream pathways were confirmed by ectopic overexpression of MUC16-carboxyl-terminal (MUC16-Cter), HuR and cMyc as well as HuR inhibitors (MS-444 and CMLD-2) in TNBC cells. Results: MUC16 was highly expressed in TNBC and correlated with its target HuR. Depletion of MUC16 showed decreased invasion, migration, and colony formation abilities of human and mouse TNBC cells. Mice injected with MUC16 depleted cells were less likely to develop lung metastasis (P = 0.001). Notably, MUC16 and HuR were highly expressed in the lung tropic TNBC cells and lung metastases. Mechanistically, we identified cMyc as a HuR target in TNBC using RNA immunoprecipitation and metastatic cDNA array. Furthermore, MUC16 knockdown and pharmacological inhibition of HuR (MS-444 and CMLD-2) in TNBC cells showed a reduction in cMyc expression. MUC16-Cter or HuR overexpression models indicated MUC16/HuR/cMyc axis in TNBC cell migration. Conclusions: Our study identified MUC16 as a TNBC lung metastasis promoter that acts through HuR/cMyc axis. This study will form the basis of future studies to evaluate the targeting of both MUC16 and HuR in TNBC patients. [ABSTRACT FROM AUTHOR]

The mechanisms of adaptive resistance to genetic-based targeted therapies of solid malignancies have been the subject of intense research. These studies hold great promise for finding co-targetable hub/pathways which in turn would control the downstream non-genetic mechanisms of adaptive resistance. Many such mechanisms have been described in the paradigmatic BRAF-mutated melanoma model of adaptive response to BRAF inhibition. Currently, a major challenge for these mechanistic studies is to confirm in vivo, at the single-cell proteomic level, the existence of dependencies between the co-targeted hub/pathways and their downstream effectors. Moreover, the drug-induced in vivo modulation of these dependencies needs to be demonstrated. Here, we implement such single-cell-based in vivo expression dependency quantification using immunohistochemistry (IHC)-based analyses of sequential biopsies in two xenograft models. These mimic phase 2 and 3 trials in our own therapeutic strategy to prevent the adaptive response to BRAF inhibition. In this mechanistic model, the dependencies between the targeted Li2CO3-inducible hub HuR and the resistance effectors are more likely time-shifted and transient since the minority of HuRLow cells, which act as a reservoir of adaptive plasticity, switch to a HuRHigh state as they paradoxically proliferate under BRAF inhibition. Nevertheless, we show that a copula/kernel density estimator (KDE)-based quantification of mutual information (MI) efficiently captures, at the individual level, the dependencies between HuR and two relevant resistance markers pERK and EGFR, and outperforms classic expression correlation coefficients. Ultimately, the validation of MI as a predictive IHC-based metric of response to our therapeutic strategy will be carried in clinical trials.

Interferon regulatory factor 2 binding protein 2 (IRF2BP2) suppresses the innate inflammatory response of macrophages. A 9-nucleotide deletion (rs3045215) in the 3′ untranslated region (3′-UTR) of human IRF2BP2 mRNA confers risk of coronary artery disease (CAD) in the Ottawa Heart Genomics Study (OHGS). Here, we sought to identify regulatory mechanisms that may contribute to this risk. We tested how lipopolysaccharides (LPS) affects IRF2BP2 expression in human THP-1 macrophages and primary aortic smooth muscle cells (HAoSMC) genotyped for the deletion allele. Both cell types are implicated in coronary atherosclerosis. We also examined how the deletion affects interaction with RNA binding proteins (RBPs) to regulate IRF2BP2 expression. LPS altered allele-specific binding of RBPs in RNA gel shift assays with the THP-1 macrophage protein extracts. The RBP ELAVL1 suppressed the expression of a luciferase reporter carrying the 3′UTR of IRF2BP2 with the deletion allele. Other RBPs AUF1 or KHSRP did not confer such allele specific regulation. Since it is co-inherited with a risk variant for osteoporosis, a condition tied to arterial calcification, we examined the association of the deletion allele with coronary artery calcification in individuals who had undergone computed tomography angiography in the OHGS. In 323 individuals with a minimal burden of atherosclerosis (50% stenosis), Mendelian randomization revealed that the rs3045215 deletion allele significantly increased coronary artery calcification in men with minimal coronary stenosis. Thus, not only does the rs3045215 deletion allele predict atherosclerosis, but it also predisposes to early-onset calcification in men.

• In vivo dependencies should be quantified in adaptive resistance mechanistic studies • Mutual information (MI) quantifies any type rather than just linear dependencies • MI outperforms classic expression correlation coefficients • Adaptive response to small-molecules inhibitors can be monitored in vivo using MI • Strategies that prevent adaptive resistance can be monitored in vivo using MI The mechanisms of adaptive resistance to genetic-based targeted therapies of solid malignancies have been the subject of intense research. These studies hold great promise for finding co-targetable hub/pathways which in turn would control the downstream non-genetic mechanisms of adaptive resistance. Many such mechanisms have been described in the paradigmatic BRAF-mutated melanoma model of adaptive response to BRAF inhibition. Currently, a major challenge for these mechanistic studies is to confirm in vivo , at the single-cell proteomic level, the existence of dependencies between the co-targeted hub/pathways and their downstream effectors. Moreover, the drug-induced in vivo modulation of these dependencies needs to be demonstrated. Here, we implement such single-cell-based in vivo expression dependency quantification using immunohistochemistry (IHC)-based analyses of sequential biopsies in two xenograft models. These mimic phase 2 and 3 trials in our own therapeutic strategy to prevent the adaptive response to BRAF inhibition. In this mechanistic model, the dependencies between the targeted Li 2 CO 3 -inducible hub HuR and the resistance effectors are more likely time-shifted and transient since the minority of HuRLow cells, which act as a reservoir of adaptive plasticity, switch to a HuRHigh state as they paradoxically proliferate under BRAF inhibition. Nevertheless, we show that a copula/kernel density estimator (KDE)-based quantification of mutual information (MI) efficiently captures, at the individual level, the dependencies between HuR and two relevant resistance markers pERK and EGFR, and outperforms classic expression correlation coefficients. Ultimately, the validation of MI as a predictive IHC-based metric of response to our therapeutic strategy will be carried in clinical trials. [ABSTRACT FROM AUTHOR]

The long non-coding RNA (lncRNA) H19 represents a maternally expressed and epigenetically regulated imprinted gene product and is discussed to have either tumor-promoting or tumor-suppressive actions. Recently, H19 was shown to be regulated under inflammatory conditions. Therefore, aim of this study was to determine the function of H19 in hepatocellular carcinoma (HCC), an inflammation-associated type of tumor. In four different human HCC patient cohorts H19 was distinctly downregulated in tumor tissue compared to normal or non-tumorous adjacent tissue. We therefore determined the action of H19 in three different human hepatoma cell lines (HepG2, Plc/Prf5, and Huh7). Clonogenicity and proliferation assays showed that H19 overexpression could suppress tumor cell survival and proliferation after treatment with either sorafenib or doxorubicin, suggesting chemosensitizing actions of H19. Since HCC displays a highly chemoresistant tumor entity, cell lines resistant to doxorubicin or sorafenib were established. In all six chemoresistant cell lines H19 expression was significantly downregulated. The promoter methylation of the H19 gene was significantly different in chemoresistant cell lines compared to their sensitive counterparts. Chemoresistant cells were sensitized after H19 overexpression by either increasing the cytotoxic action of doxorubicin or decreasing cell proliferation upon sorafenib treatment. An H19 knockout mouse model (H19Δ3) showed increased tumor development and tumor cell proliferation after treatment with the carcinogen diethylnitrosamine (DEN) independent of the reciprocally imprinted insulin-like growth factor 2 (IGF2). In conclusion, H19 suppresses hepatocarcinogenesis, hepatoma cell growth, and HCC chemoresistance. Thus, mimicking H19 action might be a potential target to overcome chemoresistance in future HCC therapy.

The molecular basis of cancer and cancer multiple phenotypes are not yet fully understood. Next Generation Sequencing promises new insight into the role of genetic interactions in shaping the complexity of cancer. Aiming to outline the differences in mutation patterns between familial colorectal cancer cases and controls we analyzed whole exomes of cancer tissues and control samples from an extended colorectal cancer pedigree, providing one of the first data sets of exome sequencing of cancer in an African population against a background of large effective size typically with excess of variants. Tumors showed hMSH2 loss of function SNV consistent with Lynch syndrome. Sets of genes harboring insertions-deletions in tumor tissues revealed, however, significant GO enrichment, a feature that was not seen in control samples, suggesting that ordered insertions-deletions are central to tumorigenesis in this type of cancer. Network analysis identified multiple hub genes of centrality. ELAVL1/HuR showed remarkable centrality, interacting specially with genes harboring non-synonymous SNVs thus reinforcing the proposition of targeted mutagenesis in cancer pathways. A likely explanation to such mutation pattern is DNA/RNA editing, suggested here by nucleotide transition-to-transversion ratio that significantly departed from expected values (p-value 5e-6). NFKB1 also showed significant centrality along with ELAVL1, raising the suspicion of viral etiology given the known interaction between oncogenic viruses and these proteins.

The expression of interferon (IFN) β is highly induced in immune and nonimmune cells in response to virus infection. This upregulation is mediated at both transcriptional and posttranscriptional levels. Whereas the signaling pathways triggered by innate virus receptors leading to transcription factor activation have been extensively studied, the mechanisms by which IFN-β mRNA stability is posttranscriptionally controlled are not fully understood. In this issue of the European Journal of Immunology, Herdy et al. [Eur. J. Immunol. 2015 45:1500-1511] show that a human RNA-binding protein, embryonic lethal, abnormal vision, drosophila-like 1 (ELAVL1)/Hu antigen R (HuR), strongly associates with IFN-β mRNA via AU-rich sequences present in its 3′ untranslated region in various human cell lines. The authors show that ELAVL1/HuR is required for the stabilization of IFN-β mRNA, and suppression of ELAVL1/HuR by its inhibitor MS-444 leads to impaired expression of IFN-β in response to viral dsRNA treatment. Thus, this study uncovers a novel mechanism of posttranscriptional IFN-β mRNA regulation in response to virus infection, through IFN-β stabilization by ELAVL1/HuR. Future studies are expected to identify further regulatory mechanisms of IFN-β stabilization and destabilization in the course of antiviral responses. [ABSTRACT FROM AUTHOR]

The liver-specific microRNA, miR-122, plays an essential role in the propagation of hepatitis C virus (HCV) by binding directly to the 5'-end of its genomic RNA. Despite its significance for HCV proliferation, the host factors responsible for regulating miR-122 remain largely unknown. In this study, we identified the cellular RNA-binding protein, ELAVL1/HuR (embryonic lethal-abnormal vision-like 1/human antigen R), as critically contributing to miR-122 biogenesis by strong binding to the 3'-end of miR-122. The availability of ELAVL1/HuR was highly correlated with HCV proliferation in replicon, infectious, and chronically infected patient conditions. Furthermore, by screening a kinase inhibitor library, we identified rigosertib, an anticancer agent under clinical trials, as having both miR-122-modulating and anti-HCV activities that were mediated by its ability to target polo-like kinase 1 (PLK1) and subsequently modulate ELAVL1/HuR-miR-122 signaling. The expression of PLK1 was also highly correlated with HCV proliferation and the HCV positivity of HCC patients. ELAVL1/HuR-miR-122 signaling and its mediation of PLK1-dependent HCV proliferation were demonstrated by performing various rescue experiments and utilizing an HCV mutant with low dependency on miR-122. In addition, the HCV-inhibitory effectiveness of rigosertib was validated in various HCV-relevant conditions, including replicons, infected cells, and replicon-harboring mice. Rigosertib was highly effective in inhibiting the proliferation of not only wild-type HCVs, but also sofosbuvir resistance-associated substitution-bearing HCVs. Our study identifies PLK1-ELAVL1/HuR-miR-122 signaling as a regulatory axis that is critical for HCV proliferation, and suggests that a therapeutic approach targeting this host cell signaling pathway could be useful for treating HCV and HCV-associated diseases.

Interferon regulatory factor 2 binding protein 2 (IRF2BP2) suppresses the innate inflammatory response of macrophages. A 9-nucleotide deletion (rs3045215) in the 3' untranslated region (3'-UTR) of human IRF2BP2 mRNA confers risk of coronary artery disease (CAD) in the Ottawa Heart Genomics Study (OHGS). Here, we sought to identify regulatory mechanisms that may contribute to this risk. We tested how lipopolysaccharides (LPS) affects IRF2BP2 expression in human THP-1 macrophages and primary aortic smooth muscle cells (HAoSMC) genotyped for the deletion allele. Both cell types are implicated in coronary atherosclerosis. We also examined how the deletion affects interaction with RNA binding proteins (RBPs) to regulate IRF2BP2 expression. LPS altered allele-specific binding of RBPs in RNA gel shift assays with the THP-1 macrophage protein extracts. The RBP ELAVL1 suppressed the expression of a luciferase reporter carrying the 3'UTR of IRF2BP2 with the deletion allele. Other RBPs AUF1 or KHSRP did not confer such allele specific regulation. Since it is co-inherited with a risk variant for osteoporosis, a condition tied to arterial calcification, we examined the association of the deletion allele with coronary artery calcification in individuals who had undergone computed tomography angiography in the OHGS. In 323 individuals with a minimal burden of atherosclerosis (<30% coronary stenosis) and 138 CAD cases (>50% stenosis), Mendelian randomization revealed that the rs3045215 deletion allele significantly increased coronary artery calcification in men with minimal coronary stenosis. Thus, not only does the rs3045215 deletion allele predict atherosclerosis, but it also predisposes to early-onset calcification in men., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Vilmundarson, Duong, Soheili, Chen and Stewart.)

The long non-coding RNA (lncRNA) H19 represents a maternally expressed and epigenetically regulated imprinted gene product and is discussed to have either tumor-promoting or tumor-suppressive actions. Recently, H19 was shown to be regulated under inflammatory conditions. Therefore, aim of this study was to determine the function of H19 in hepatocellular carcinoma (HCC), an inflammation-associated type of tumor. In four different human HCC patient cohorts H19 was distinctly downregulated in tumor tissue compared to normal or non-tumorous adjacent tissue. We therefore determined the action of H19 in three different human hepatoma cell lines (HepG2, Plc/Prf5, and Huh7). Clonogenicity and proliferation assays showed that H19 overexpression could suppress tumor cell survival and proliferation after treatment with either sorafenib or doxorubicin, suggesting chemosensitizing actions of H19 . Since HCC displays a highly chemoresistant tumor entity, cell lines resistant to doxorubicin or sorafenib were established. In all six chemoresistant cell lines H19 expression was significantly downregulated. The promoter methylation of the H19 gene was significantly different in chemoresistant cell lines compared to their sensitive counterparts. Chemoresistant cells were sensitized after H19 overexpression by either increasing the cytotoxic action of doxorubicin or decreasing cell proliferation upon sorafenib treatment. An H19 knockout mouse model ( H19 Δ3) showed increased tumor development and tumor cell proliferation after treatment with the carcinogen diethylnitrosamine (DEN) independent of the reciprocally imprinted insulin-like growth factor 2 (IGF2). In conclusion, H19 suppresses hepatocarcinogenesis, hepatoma cell growth, and HCC chemoresistance. Thus, mimicking H19 action might be a potential target to overcome chemoresistance in future HCC therapy., Competing Interests: Conflict of interest: The authors declare that there is no conflict of interest to disclose.