Transcriptional epigenetic regulation of Fkbp1/Pax9 genes is associated with impaired sensitivity to platinum treatment in ovarian cancer

Background: In an effort to contribute to overcoming the platinum resistance exhibited by most solid tumors, we performed an array of epigenetic approaches, integrating next-generation methodologies and public clinical data to identify new potential epi-biomarkers in ovarian cancer, which is considered the most devastating of gynecological malignancies. Methods: We cross-analyzed data from methylome assessments and restoration of gene expression through microarray expression in a panel of four paired cisplatin-sensitive/cisplatin-resistant ovarian cancer cell lines, along with publicly available clinical data from selected individuals representing the state of chemoresistance. We validated the methylation state and expression levels of candidate genes in each cellular phenotype through Sanger sequencing and reverse transcription polymerase chain reaction, respectively. We tested the biological role of selected targets using an ectopic expression plasmid assay in the sensitive/resistant tumor cell lines, assessing the cell viability in the transfected groups. Epigenetic features were also assessed in 189 primary samples obtained from ovarian tumors and controls. Results: We identified PAX9 and FKBP1B as potential candidate genes, which exhibited epigenetic patterns of expression regulation in the experimental approach. Re-establishment of FKBP1B expression in the resistant OVCAR3 phenotype in which this gene is hypermethylated and inhibited allowed it to achieve a degree of platinum sensitivity similar to the sensitive phenotype. The evaluation of these genes at a translational level revealed that PAX9 hypermethylation leads to a poorer prognosis in terms of overall survival. We also set a precedent for establishing a common epigenetic signature in which the validation of a single candidate, MEST, proved the accuracy of our computational pipelines. Conclusions: Epigenetic regulation of PAX9 and FKBP1B genes shows that methylation in non-promoter areas has the potential to control gene expression and thus biological consequences, such as the loss of platinum sensitivity. At the translational level, PAX9 behaves as a predictor of chemotherapy response to platinum in patients with ovarian cancer. This study revealed the importance of the transcript-specific study of each gene under potential epigenetic regulation, which would favor the identification of new markers capable of predicting each patient’s progression and therapeutic response.The study was financially supported by FIS (ISCIII) and ERDF/FSE funds (PI15/00186, PI18/0050, and ERDF/FSE, A way to make Europe). The authors gratefully acknowledge the Colombian Ministry for Science, Technology and Innovation (MINCIENCIAS), Code 568-2012, for providing J.S. with partial funding for this study

Dubious effects of methadone as an "anticancer" drug on ovarian cancer cell-lines and patient-derived tumor-spheroids

Background. The opioid agonist D, L-methadone exerts analgesic effects via the mu opioid receptor, encoded by OPRM1 and therefore plays a role in chronic pain management. In preclinical tumor-models D,L-methadone shows apoptotic and chemo-sensitizing effects and was therefore hyped as an off-label "anticancer" drug without substantiation from clinical trials. Its effects in ovarian cancer (OC) are completely unexplored. Methods. We analyzed OPRM1-mRNA expression in six cisplatin-sensitive, two cisplatin-resistant OC cell-lines, 170 OC tissue samples and 12 non-neoplastic control tissues. Pro-angiogenetic, cytotoxic and apoptotic effects of D,L-methadone were evaluated in OC cell-lines and four patient-derived tumor-spheroid models. Results. OPRM1 was transcriptionally expressed in 69% of OC-tissues and in three of eight OC cell-lines. D, L-methadone exposure significantly reduced cell-viability in five OC cell-lines irrespective of OPRM1 expression. D, L-methadone, applied alone or combined with cisplatin, showed no significant effects on apoptosis or VEGF secretion in cell-lines. Notably, in two of the four sphero id models, treatment with D, L-methadone significantly enhanced cell growth (by up to 121%), especially after long-term exposure. This is consistent with the observed attenuation of the inhibitory effects of cisplatin in three spheroid models when adding D, L-methadone. The effect of methadone treatment on VEGF secretion in tumor-spheroids was inconclusive. Conclusions. Our study demonstrates that certain OC samples express OPRM1, which, however, is not a prerequisite for D, L-methadone function. As such, D,L-methadone may exert also detrimental effects by stimulating the growth of certain OC-cells and abrogating cisplatin's therapeutic effect. (C) 2022 The Authors. Published by Elsevier Inc.Peer reviewe

The role of epithelial-to-mesenchymal plasticity in ovarian cancer progression and therapy resistance

Ovarian cancer is the most lethal of all gynecologic malignancies and the eighth leading cause of cancer-related deaths among women worldwide. The main reasons for this poor prognosis are late diagnosis; when the disease is already in an advanced stage, and the frequent development of resistance to current chemotherapeutic regimens. Growing evidence demonstrates that apart from its role in ovarian cancer progression, epithelial-to-mesenchymal transition (EMT) can promote chemotherapy resistance. In this review, we will highlight the contribution of EMT to the distinct steps of ovarian cancer progression. In addition, we will review the different types of ovarian cancer resistance to therapy with particular attention to EMT-mediated mechanisms such as cell fate transitions, enhancement of cancer cell survival, and upregulation of genes related to drug resistance. Preclinical studies of anti-EMT therapies have yielded promising results. However, before anti-EMT therapies can be effectively implemented in clinical trials, more research is needed to elucidate the mechanisms leading to EMT-induced therapy resistance

Liposomal Combination Drug and siRNA Delivery to Combat Drug-Resistant Ovarian Cancer

Ovarian cancer is the deadliest gynecological malignancy and the fifth leading cause of cancer death overall. Due to lack of early symptoms, ovarian cancer is most commonly diagnosed in the distant stages, drastically reducing the 5 year survival rate from 92% in early stage diagnoses to 29% in advanced stage cases. This large difference is thought to be linked to the high rate of recurrence and development of drug resistance to chemotherapeutics in ovarian cancer patients. First-line therapy includes a combination of tumor resection surgery and chemotherapy regimen including cisplatin, a DNA-alkylating agent, and paclitaxel, a microtubule stabilization agent. However, treatment becomes more complex upon recurrence due to the development of drug resistance. Drug resistance has been linked to many mechanisms, including efflux transporters, dysregulation of apoptosis, autophagy, cancer stem cells, epigenetics, and the epithelial-mesenchymal transition. Due to the wide variety of mechanisms involved in resistance, developing and choosing effective therapies is extremely complex. Liposomes demonstrate potential as delivery systems to combat drug-resistance in cancer due to their versatility in loading. Liposomes possess the ability to load multiple therapeutics to re-sensitize resistant cancer cells while simultaneously treating those cells with a chemotherapeutic agent. Here, a liposomal carrier for both paclitaxel and siRNA was designed and synthesized to provide a combinatorial therapy to re-sensitize drug-resistant ovarian cancer cells to paclitaxel and thereby increase the efficacy of paclitaxel. A custom siRNA array was developed, and we identified three possible gene targets, ABCB1, JAK2, and CFLAR, involved in the development of drug resistance in paclitaxel-resistant OVCAR3-TR ovarian cancer cells. Two combinatorial, cationic liposome delivery systems were designed and synthesized via the lipid film hydration method. Liposomes were characterized for size, surface charge, stability, and loading efficiencies. We demonstrated efficient loading of paclitaxel and protection of bound siRNA in both liposome formulations. Cellular uptake of the liposomes was confirmed using fluorescence microscopy. Overall, the liposomes show promise in loading both paclitaxel and siRNA to target genes involved in drug-resistance development in ovarian cancer cells

A novel role for the tumor suppressor gene itf2 in tumorigenesis and chemotherapy response

Despite often leading to platinum resistance, platinum-based chemotherapy continues to be the standard treatment for many epithelial tumors. In this study we analyzed and validated the cytogenetic alterations that arise after treatment in four lung and ovarian paired cisplatin-sensitive/resistant cell lines by 1-million microarray-based comparative genomic hybridization (array-CGH) and qRT-PCR methodologies. RNA-sequencing, functional transfection assays, and gene-pathway activity analysis were used to identify genes with a potential role in the development of this malignancy. The results were further explored in 55 lung and ovarian primary tumors and control samples, and in two extensive in silico databases. Long-term cell exposure to platinum induces the frequent deletion of ITF2 gene. Its expression re-sensitized tumor cells to platinum and recovered the levels of Wnt/β-catenin transcriptional activity. ITF2 expression was also frequently downregulated in epithelial tumors, predicting a worse overall survival. We also identified an inverse correlation between ITF2 and HOXD9 expression, revealing that Non-small cell lung cancer (NSCLC) patients with lower expression of HOXD9 had a better overall survival rate. We defined the implication of ITF2 as a molecular mechanism behind the development of cisplatin resistance probably through the activation of the Wnt-signaling pathway. This data highlights the possible role of ITF2 and HOXD9 as novel therapeutic targets for platinum resistant tumors.This research was funded by the Fondo de Investigación Sanitaria-Instituto de Salud Carlos III, PI15/00186 and PI18/00050, CP19/00063, and CM19/00100 for HR and by MINECO, RTC-2016-5314-1 to I.I.C; by the MINECO, SAF2016-75531-R, by the CAM B2017/BMD-3724 and by the AECC GCB14142311CRES to P.S; and the European Regional Development Fund/European Social Fund FIS (FEDER/FSE, Una Manera de Hacer Europa)

RUNX3 transcript variants have distinct roles in ovarian carcinoma and differently influence platinum sensitivity and angiogenesis

The prognosis of late-stage epithelial ovarian cancer (EOC) patients is affected by chemotherapy response and the malignant potential of the tumor cells. In earlier work, we identified hypermethylation of the runt-related transcription factor 3 gene (RUNX3) as a prognostic biomarker and contrary functions of transcript variants (TV1 and TV2) in A2780 and SKOV3 cells. The aim of the study was to further validate these results and to increase the knowledge about RUNX3 function in EOC. New RUNX3 overexpression models of high-grade serous ovarian cancer (HGSOC) were established and analyzed for phenotypic (IC50 determination, migration, proliferation and angiogenesis assay, DNA damage analysis) and transcriptomic consequences (NGS) of RUNX3 TV1 and TV2 overexpression. Platinum sensitivity was affected by a specific transcript variant depending on BRCA background. RUNX3 TV2 induced an increased sensitivity in BRCA1wt cells (OVCAR3), whereas TV1 increased the sensitivity and induced a G2/M arrest under treatment in BRCA1mut cells (A13-2-12). These different phenotypes relate to differences in DNA repair: homologous recombination deficient A13-2-12 cells show less γH2AX foci despite higher levels of Pt-DNA adducts. RNA-Seq analyses prove transcript variant and cell-line-specific RUNX3 effects. Pathway analyses revealed another clinically important function of RUNX3—regulation of angiogenesis. This was confirmed by thrombospondin1 analyses, HUVEC spheroid sprouting assays and proteomic profiling. Importantly, conditioned media (CM) from RUNX3 TV1 overexpressing A13-2-12 cells induced an increased HUVEC sprouting. Altogether, the presented data support the hypothesis of different functions of RUNX3 transcript variants related to the clinically relevant processes—platinum resistance and angiogenesis

Lapatinib and Poziotinib Overcome ABCB1-Mediated Paclitaxel Resistance in Ovarian Cancer

Conventional frontline treatment for ovarian cancer consists of successive chemotherapy cycles of paclitaxel and platinum. Despite the initial favorable responses for most patients, chemotherapy resistance frequently leads to recurrent or refractory disease. New treatment strategies that circumvent or prevent mechanisms of resistance are needed to improve ovarian cancer therapy. We established in vitro paclitaxel-resistant ovarian cancer cell line and organoid models. Gene expression differences in resistant and sensitive lines were analyzed by RNA sequencing. We manipulated candidate genes associated with paclitaxel resistance using siRNA or small molecule inhibitors, and then screened the cells for paclitaxel sensitivity using cell viability assays. We used the Bliss independence model to evaluate the anti-proliferative synergy for drug combinations. ABCB1 expression was upregulated in paclitaxel-resistant TOV-21G (q \u3c 1x10-300), OVCAR3 (q = 7.4x10-156) and novel ovarian tumor organoid (p = 2.4x10-4) models. Previous reports have shown some tyrosine kinase inhibitors can inhibit ABCB1 function. We tested a panel of tyrosine kinase inhibitors for the ability to sensitize resistant ABCB1-overexpressing ovarian cancer cell lines to paclitaxel. We observed synergy when we combined poziotinib or lapatinib with paclitaxel in resistant TOV-21G and OVCAR3 cells. Silencing ABCB1 expression in paclitaxel-resistant TOV-21G and OVCAR3 cells reduced paclitaxel IC50 by 20.7 and 6.2-fold, respectively. Furthermore, we demonstrated direct inhibition of paclitaxel-induced ABCB1 transporter activity by both lapatinib and poziotinib. In conclusion, lapatinib and poziotinib combined with paclitaxel synergizes to inhibit the proliferation of ABCB1-overexpressing ovarian cancer cells in vitro. The addition of FDA-approved lapatinib to second-line paclitaxel therapy is a promising strategy for patients with recurrent ovarian cancer

Dubious effects of methadone as an “anticancer” drug on ovarian cancer cell-lines and patient-derived tumor-spheroids

BackgroundThe opioid agonist D,L-methadone exerts analgesic effects via the mu opioid receptor, encoded by OPRM1 and therefore plays a role in chronic pain management. In preclinical tumor-models D,L-methadone shows apoptotic and chemo-sensitizing effects and was therefore hyped as an off-label “anticancer” drug without substantiation from clinical trials. Its effects in ovarian cancer (OC) are completely unexplored.MethodsWe analyzed OPRM1-mRNA expression in six cisplatin-sensitive, two cisplatin-resistant OC cell-lines, 170 OC tissue samples and 12 non-neoplastic control tissues. Pro-angiogenetic, cytotoxic and apoptotic effects of D,L-methadone were evaluated in OC cell-lines and four patient-derived tumor-spheroid models.ResultsOPRM1 was transcriptionally expressed in 69% of OC-tissues and in three of eight OC cell-lines. D,L-methadone exposure significantly reduced cell-viability in five OC cell-lines irrespective of OPRM1 expression. D,L-methadone, applied alone or combined with cisplatin, showed no significant effects on apoptosis or VEGF secretion in cell-lines. Notably, in two of the four spheroid models, treatment with D,L-methadone significantly enhanced cell growth (by up to 121%), especially after long-term exposure. This is consistent with the observed attenuation of the inhibitory effects of cisplatin in three spheroid models when adding D,L-methadone. The effect of methadone treatment on VEGF secretion in tumor-spheroids was inconclusive.ConclusionsOur study demonstrates that certain OC samples express OPRM1, which, however, is not a prerequisite for D,L-methadone function. As such, D,L-methadone may exert also detrimental effects by stimulating the growth of certain OC-cells and abrogating cisplatin's therapeutic effect.</p

The RNA-binding protein LARP1 as potential biomarker and therapeutic target in ovarian cancer

Ovarian cancer is the most lethal gynaecological malignancy, responsible for over 4,000 deaths each year in the UK. There is growing evidence that mRNA-binding proteins (RBPs) can be post-transcriptional drivers of cancer progression. Here, I investigated the expression of the RBP LARP1 in ovarian malignancies and role of the protein in ovarian cancer cell biology. LARP1 is highly expressed at both an mRNA and protein level in ovarian cancers compared with benign tumours and normal ovarian tissue. I show that higher levels of LARP1 in tumour tissue are predictive of poor patient survival. Consistent with this clinical finding, in xenograft studies knockdown of LARP1 expression causes a dramatic reduction in tumour growth. In vitro, LARP1 knockdown is associated with increased apoptosis, and is sufficient to restore platinum sensitivity in chemotherapy-resistant cell lines. Furthermore, LARP1 is required to maintain cancer stem cell marker-positive populations, and knockdown decreases tumour-initiating potential, as demonstrated by in vivo limiting dilution assays. Transcriptome deep-sequencing following LARP1 knockdown revealed altered expression of multiple genes linked to survival and evasion of apoptosis, including BCL2 and BIK. Transcripts of both genes are in complex with LARP1 protein, and LARP1 maintains the stability of BCL2 mRNA, whilst actively destabilising BIK transcripts. This effect is mediated at the level of the 3’ untranslated region. I therefore conclude that by differentially regulating mRNA stability, LARP1 is a key post-transcriptional driver of tumourigenicity and cell survival in ovarian cancer.Open Acces

Regulación epigénetica de la expresión de ARNs no codificantes y su implicación en la respuesta terapéutica en cáncer de pulmón no microcítico y cáncer de ovario

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 25-05-2018Esta tesis tiene embargado el acceso al texto completo hasta el 25-11-2019Los ARNs no codificantes, incluyendo microARNs y ARNs no codificantes largos (lncRNAs), son reguladores fundamentales de la biología celular cuya alteración está relacionada con el desarrollo de enfermedades como el cáncer. Sin embargo su papel y regulación en respuesta al tratamiento es aún desconocida. El tratamiento de elección para el cáncer de pulmón no microcítico (CPNM) y el cáncer de ovario es la quimioterapia basada en compuestos de platino. El cisplatino, además de provocar la muerte celular, induce numerosos cambios moleculares que desembocan en el desarrollo de resistencia al fármaco. En el presente trabajo hemos abordado el estudio de la regulación epigenética de microARNs y lncRNAs en CPNM y cáncer de ovario, dos tipos tumorales que desarrollan frecuentemente resistencia al cisplatino. Hemos realizado un análisis a gran escala del transcriptoma, mediante microarrays de expresión y del metiloma, mediante la secuenciación del genoma modificado por bisulfito, en cuatro líneas celulares de CPNM y cáncer de ovario, seguido por la validación de la expresión mediante PCR a tiempo real y la validación epigenética a través de la secuenciación por bisulfito dirigida. De todos los microARNs analizados, el microARN-7 (miR-7) fue el único cuya expresión se encuentra inhibida por metilación de su región reguladora en líneas tumorales resistentes a cisplatino. Nuestros ensayos funcionales mediante mutagénesis dirigida y actividad luciferasa, la sobreexpresión de precursores del miR-7, su silenciamiento por antago-miRs y ensayos de viabilidad celular demuestran la regulación directa del gen MAFG por el miR-7 y su implicación en el desarrollo de resistencia a cisplatino en líneas celulares humanas. Además, hemos determinado el estado de metilación del miR-7 en 291 muestras tumorales quirúrgicas y controles de pacientes con CPNM y cáncer de ovario, lo que se asocia a un peor pronóstico. El análisis de la expresión de MAFG en 99 muestras quirúrgicas y 2505 pacientes con CPNM y cáncer de ovario de bases de datos públicas, ha permitido definir la implicación de MAFG en estas patologías. En este trabajo proponemos que la resistencia al cisplatino mediada por MAFG podría estar asociada con la detoxificación en la situación de estrés oxidativo generado tras el tratamiento con cisplatino. Por otro lado, nuestros resultados en relación con los lncRNAs muestran que la alteración de la expresión es más frecuente en lncRNAs que actúan en cis aunque los patrones de metilación se ven comúnmente más alterados en los solapantes. Además, éstos contienen más islas CpG que, en su mayoría, están compartidas con las de sus genes codificantes asociados. Validamos estos resultados a nivel de expresión y metilación y encontramos cinco lncRNAs con posible implicación en la aparición de resistencia. Globalmente, nuestros resultados ofrecen una nueva visión sobre los mecanismos epigenéticos reguladores de ARN no codificantes y su implicación en la resistencia a cisplatino en cáncer de pulmón y cáncer de ovari