One of the major limitations associated with platinum use is the resistance that almost invariably develops in different tumor types. In the current study, we sought to identify epigenetically regulated microRNAs as novel biomarkers of platinum resistance in lung and ovarian cancers, the ones with highest ratios of associated chemo-resistance.Methods: We combined transcriptomic data from microRNA and mRNA under the influence of an epigenetic reactivation treatment in a panel of four paired cisplatin -sensitive and -resistant cell lines, followed by real-time expression and epigenetic validations for accurate candidate selection in 19 human cancer cell lines. To identify specific candidate genes under miRNA regulation, we assembled “in silico” miRNAs and mRNAs sequences by using ten different algorithms followed by qRT-PCR validation. Functional assays of site-directed mutagenesis and luciferase activity, miRNAs precursor overexpression, silencing by antago-miR and cell viability were performed to confirm their specificity in gene regulation. Results were further explored in 187 primary samples obtained from ovarian tumors and controls.Results: We identified 4 candidates, miR-7, miR-132, miR-335 and miR-148a, which deregulation seems to be a common event in the development of resistance to cisplatin in both tumor types. miR-7 presented specific methylation in resistant cell lines, and was associated with poorer prognosis in ovarian cancer patients. Our experimental results strongly support the direct regulation of MAFG through miR-7 and their involvement in the development of CDDP resistance in human tumor cells.Conclusion: The basal methylation status of miR-7 before treatment may be a potential clinical epigenetic biomarker, predictor of the chemotherapy outcome to CDDP in ovarian cancer patients. To the best of our knowledge, this is the first report linking the regulation of MAFG by miRNA-7 and its role in chemotherapy response to CDDP. Furthermore, this data highlights the possible role of MAFG as a novel therapeutic target for platinum resistant tumors.

The cumbersome and time-consuming process of generating new mouse strains and multiallelic experimental animals often hinders the use of genetically engineered mouse models (GEMM) in cancer research. Here, we describe the development and validation of an embryonic stem cell (ESC)-GEMM platform for rapid modeling of melanoma in mice. The platform incorporates 12 clinically relevant genotypes composed of combinations of four driver alleles (LSL-Braf V600E , LSL-Nras Q61R , Pten Flox , and Cdkn2a Flox ) and regulatory alleles to spatiotemporally control the perturbation of genes of interest. The ESCs produce high-contribution chimeras, which recapitulate the melanoma phenotypes of conventionally bred mice. Using the ESC-GEMM platform to modulate Pten expression in melanocytes in vivo, we highlighted the utility and advantages of gene depletion by CRISPR-Cas9, RNAi, or conditional knockout for melanoma modeling. Moreover, complemen-tary genetic methods demonstrated the impact of Pten restoration on the prevention and maintenance of Pten-deficient melanomas. Finally, we showed that chimera-derived melanoma cell lines retain regulatory allele competency and are a powerful resource to complement ESC-GEMM chimera experiments in vitro and in syngeneic grafts in vivo. Thus, when combined with sophisticated genetic tools, the ESC-GEMM platform enables rapid, high-throughput, and versatile studies aimed at addressing outstanding questions in melanoma biology.Significance: This study presents a high-throughput and versatile ES cell-based mouse modeling platform that can be combined with state-of-the-art genetic tools to address unanswered questions in melanoma in vivo.See related commentary by Thorkelsson et al., p. 655

Cancer is one of the leading causes of death worldwide and it can affect any part of the organism. It arises as a consequence of the genetic and epigenetic changes that lead to the uncontrolled growth of the cells. The epigenetic machinery can regulate gene expression without altering the DNA sequence, and it comprises methylation of the DNA, histones modifications, and non-coding RNAs. Alterations of these gene-expression regulatory elements can be produced by an imbalance of the intracellular environment, such as the one derived by oxidative stress, to promote cancer development, progression, and resistance to chemotherapeutic treatments. Here we review the current literature on the effect of oxidative stress in the epigenetic machinery, especially over the largely unknown ncRNAs and its consequences toward cancer development and progression.

The cumbersome and time-consuming process of generating new mouse strains and multi-allelic experimental animals often hinders the use of genetically engineered mouse models (GEMM) in cancer research. Here, we describe the development and validation of an embryonic stem cell (ESC)-GEMM platform for rapid modeling of melanoma in mice. Our platform incorporates twelve clinically relevant genotypes composed of combinations of four driver alleles (LSL-BrafV600E, LSL-NrasQ61R, PtenFlox, Cdkn2aFlox) and regulatory alleles to spatiotemporally control the perturbation of genes-of-interest. Our ESCs produce high contribution chimeras, which recapitulate the melanoma phenotypes of conventionally bred mice. Using our ESC-GEMM platform to modulate Pten expression in melanocytes in vivo, we highlight the utility and advantages of gene depletion by CRISPR-Cas9, RNAi, or conditional knockout for melanoma modeling. Moreover, we use complementary genetic methods to demonstrate the impact of Pten restoration on the prevention and maintenance of Pten-deficient melanomas. Finally, we show that chimera-derived melanoma cell lines retain regulatory allele competency and are a powerful resource to complement ESC-GEMM chimera experiments in vitro and in syngeneic grafts in vivo. Thus, when combined with sophisticated genetic tools, our ESC-GEMM platform enables rapid, high-throughput, and versatile studies aimed at addressing outstanding questions in melanoma biology.

Long noncoding RNAs (lncRNAs) are critical regulators of cell biology whose alteration can lead to the development of diseases such as cancer. The potential role of lncRNAs and their epigenetic regulation in response to platinum treatment are largely unknown. We analyzed four paired cisplatin-sensitive/resistant non-small cell lung cancer and ovarian cancer cell lines. The epigenetic landscape of overlapping and cis-acting lncRNAs was determined by combining human microarray data on 30,586 lncRNAs and 20,109 protein coding mRNAs with whole-genome bisulfite sequencing. Selected candidate lncRNAs were further characterized by PCR, gene-ontology analysis, and targeted bisulfite sequencing. Differential expression in response to therapy was observed more frequently in cis-acting than in overlapping lncRNAs (78% vs. 22%, fold change ≥1.5), while significantly altered methylation profiles were more commonly associated with overlapping lncRNAs (29% vs. 8%; P value <0.001). Moreover, overlapping lncRNAs contain more CpG islands (CGIs) (25% vs. 17%) and the majority of CGI-containing overlapping lncRNAs share these CGIs with their associated coding genes (84%). The differences in expression between sensitive and resistant cell lines were replicated in 87% of the selected candidates (P<0.05), while our bioinformatics approach identifying differential methylation was confirmed in all of the selected lncRNAs (100%). Five lncRNAs under epigenetic regulation appear to be involved in cisplatin resistance (AC091814.2, AC141928.1, RP11-65J3.1-002, BX641110, and AF198444). These novel findings provide new insights into epigenetic mechanisms and acquired resistance to cisplatin that highlight specific lncRNAs, some with unknown function, that may signal strategies in epigenetic therapies.

Circular RNAs (circRNAs) are a class of non-coding RNAs featuring a covalently closed ring structure formed through backsplicing. circRNAs are broadly expressed and contribute to biological processes through a variety of functions. Standard gain-of-function and loss-of-function approaches to study gene functions have significant limitations when studying circRNAs. Overexpression studies in particular suffer from the lack of efficient genetic tools. While mammalian expression plasmids enable transient circRNA overexpression in cultured cells, most cell biological studies require long-term ectopic expression. Here we report the development and characterization of genetic tools enabling stable circRNA overexpression in vitro and in vivo . We demonstrated that circRNA expression constructs can be delivered to cultured cells via transposons, whereas lentiviral vectors have limited utility for the delivery of circRNA constructs due to viral RNA splicing in virus-producing cells. We further demonstrated ectopic circRNA expression in a hepatocellular carcinoma mouse model upon circRNA transposon delivery via hydrodynamic tail vein injection. Furthermore, we generated genetically engineered mice harbouring circRNA expression constructs. We demonstrated that this approach enables constitutive, global circRNA overexpression as well as inducible circRNA expression directed specifically to melanocytes in a melanoma mouse model. These tools expand the genetic toolkit available for the functional characterization of circRNAs.

The miR-29 family of microRNAs is encoded by two clusters, miR-29b1~a and miR-29b2~c, and is regulated by several oncogenic and tumor suppressive stimuli. While in vitro evidence suggests a tumor suppressor role for miR-29 in melanoma, the mechanisms underlying its deregulation and contribution to melanomagenesis have remained elusive. Using various in vitro systems, we show that oncogenic MAPK signaling paradoxically stimulates transcription of pri-miR-29b1~a and pri-miR-29b2~c, the latter in a p53-dependent manner. Expression analyses in melanocytes, melanoma cells, nevi, and primary melanoma revealed that pri-miR-29b2~c levels decrease during melanoma progression. Inactivation of miR-29 in vivo with a miRNA sponge in a rapid melanoma mouse model resulted in accelerated tumor development and decreased overall survival, verifying tumor suppressive potential of miR-29 in melanoma. Through integrated RNA sequencing, target prediction, and functional assays, we identified the transcription factors MAFG and MYBL2 as bona fide miR-29 targets in melanoma. Our findings suggest that attenuation of miR-29b2~c expression promotes melanoma development, at least in part, by derepressing MAFG and MYBL2.

The methylation status of the IGFBP-3 gene is strongly associated with cisplatin sensitivity in patients with non-small cell lung cancer (NSCLC). In this study, we found in vitro evidence that linked the presence of an unmethylated promoter with poor response to radiation. Our data also indicate that radiation might sensitize chemotherapy-resistant cells by reactivating IGFBP-3-expression through promoter demethylation, inactivating the PI3K/AKT pathway. We also explored the IGFBP-3 methylation effect on overall survival (OS) in a population of 40 NSCLC patients who received adjuvant therapy after R0 surgery. Our results indicate that patients harboring an unmethylated promoter could benefit more from a chemotherapy schedule alone than from a multimodality therapy involving radiotherapy and platinum-based treatments, increasing their OS by 2.5 y (p = .03). Our findings discard this epi-marker as a prognostic factor in a patient population without adjuvant therapy, indicating that radiotherapy does not improve survival for patients harboring an unmethylated IGFBP-3 promoter.