Platinum-induced upregulation of ITGA6 promotes chemoresistance and spreading in ovarian cancer.

Platinum (PT)-resistant Epithelial Ovarian Cancer (EOC) grows as a metastatic disease, disseminating in the abdomen and pelvis. Very few options are available for PT-resistant EOC patients, and little is known about how the acquisition of PT-resistance mediates the increased spreading capabilities of EOC. Here, using isogenic PT-resistant cells, genetic and pharmacological approaches, and patient-derived models, we report that Integrin α6 (ITGA6) is overexpressed by PT-resistant cells and is necessary to sustain EOC metastatic ability and adhesion-dependent PT-resistance. Using in vitro approaches, we showed that PT induces a positive loop that, by stimulating ITGA6 transcription and secretion, contributes to the formation of a pre-metastatic niche enabling EOC cells to disseminate. At molecular level, ITGA6 engagement regulates the production and availability of insulin-like growth factors (IGFs), over-stimulating the IGF1R pathway and upregulating Snail expression. In vitro data were recapitulated using in vivo models in which the targeting of ITGA6 prevents PT-resistant EOC dissemination and improves PT-activity, supporting ITGA6 as a promising druggable target for EOC patients.

LINC01605 Is a Novel Target of Mutant p53 in Breast and Ovarian Cancer Cell Lines.

TP53 is the most frequently mutated gene in human cancers. Most TP53 genomic alterations are missense mutations, which cause a loss of its tumour suppressor functions while providing mutant p53 (mut_p53) with oncogenic features (gain-of-function). Loss of p53 tumour suppressor functions alters the transcription of both protein-coding and non-protein-coding genes. Gain-of-function of mut_p53 triggers modification in gene expression as well; however, the impact of mut_p53 on the transcription of the non-protein-coding genes and whether these non-protein-coding genes affect oncogenic properties of cancer cell lines are not fully explored. In this study, we suggested that LINC01605 (also known as lincDUSP) is a long non-coding RNA regulated by mut_p53 and proved that mut_p53 directly regulates LINC01605 by binding to an enhancer region downstream of the LINC01605 locus. We also showed that the loss or downregulation of LINC01605 impairs cell migration in a breast cancer cell line. Eventually, by performing a combined analysis of RNA-seq data generated in mut_TP53-silenced and LINC01605 knockout cells, we showed that LINC01605 and mut_p53 share common gene pathways. Overall, our findings underline the importance of ncRNAs in the mut_p53 network in breast and ovarian cancer cell lines and in particular the importance of LINC01605 in mut_p53 pro-migratory pathways.

Integration of Cellular and Humoral Immune Responses as an Immunomonitoring Tool for SARS-CoV-2 Vaccination in Healthy and Fragile Subjects.

Cellular and humoral immunity are both required for SARS-CoV-2 infection recovery and vaccine efficacy. The factors affecting mRNA vaccination-induced immune responses, in healthy and fragile subjects, are still under investigation. Thus, we monitored the vaccine-induced cellular and humoral immunity in healthy subjects and cancer patients after vaccination to define whether a different antibody titer reflected similar rates of cellular immune responses and if cancer has an impact on vaccination efficacy. We found that higher titers of antibodies were associated with a higher probability of positive cellular immunity and that this greater immune response was correlated with an increased number of vaccination side effects. Moreover, active T-cell immunity after vaccination was associated with reduced antibody decay. The vaccine-induced cellular immunity appeared more likely in healthy subjects rather than in cancer patients. Lastly, after boosting, we observed a cellular immune conversion in 20% of subjects, and a strong correlation between pre- and post-boosting IFN-γ levels, while antibody levels did not display a similar association. Finally, our data suggested that integrating humoral and cellular immune responses could allow the identification of SARS-CoV-2 vaccine responders and that T-cell responses seem more stable over time compared to antibodies, especially in cancer patients.

Efficacy and safety of trabectedin for the treatment of advanced uterine or ovarian carcinosarcoma: Results of a phase II multicenter clinical trial (MITO-26).

OBJECTIVE: This open-label phase II clinical trial evaluated the antitumor activity and safety of trabectedin in patients with advanced ovarian (OC) or uterine carcinosarcomas (UC). METHODS: Eligible patients were adults (≥18 years) with histologically proven recurrent OC/UC not amenable to surgery or radiotherapy who received up to two prior chemotherapy lines. Trabectedin 1.3 mg/m2 was administered as a 3-h infusion every three weeks. The primary endpoint was objective response rate (ORR) as per RECIST v.1.1. If at least 8 of 43 patients (18.6%) achieve an objective response, trabectedin would be declared worthy for further investigations. RESULTS: Forty-five patients with either OC (n = 32) or UC (n = 13) from seven MITO centers across Italy were enrolled. The ORR was 11.9% (90% CI: 6-23) and included two patients with a complete response and three with a partial response. Eight patients (19.0%) had disease stabilization for a disease control rate of 31.0% (90% CI: 20-44). Median progression-free survival was 2.01 months (95% CI: 1.78-2.30) and median overall survival was 4.64 months (95% CI: 3.19-8.29). Neutrophil count decreases (n = 8, 18.2%) and transaminase increases (n = 6, 13.6%) were the most common grade 3-5 adverse events related with trabectedin. Two patients died due to trabectedin-related grade 5 hematological toxicity. CONCLUSION: Although trabectedin did not meet the prespecified activity criteria, it confers modest but clinically meaningful benefit to patients with advanced OC/UC as being as effective as any other available treatment for this indication. The toxicity profile appears in line with that previously reported for the drug.

An Exceptional Response to Dostarlimab in Mismatch Repair Deficient, Microsatellite Instability-High and Platinum Refractory Endometrial Cancer.

Until recently, effective therapies for advanced endometrial cancer progressing to a platinum-based combination were lacking. In this setting, immunotherapy with anti PD-1/PDL-1 monoclonal antibodies is rising as a new paradigm in particular for patients with microsatellites instability/mismatch repair deficiency. In this case report, we describe an exceptional and rapid response to dostarlimab in a platinum refractory endometrial cancer patient with high disease burden harboring a mismatch repair deficiency.

CDKN1B mutation and copy number variation are associated with tumor aggressiveness in luminal breast cancer.

The CDKN1B gene, encoding for the CDK inhibitor p27kip1 , is mutated in defined human cancer subtypes, including breast, prostate carcinomas and small intestine neuroendocrine tumors. Lessons learned from small intestine neuroendocrine tumors suggest that CDKN1B mutations could be subclonal, raising the question of whether a deeper sequencing approach could lead to the identification of higher numbers of patients with mutations. Here, we addressed this question and analyzed human cancer biopsies from breast (n = 396), ovarian (n = 110) and head and neck squamous carcinoma (n = 202) patients, using an ultra-deep sequencing approach. Notwithstanding this effort, the mutation rate of CDKN1B remained substantially aligned with values from the literature, showing that essentially only hormone receptor-positive breast cancer displayed CDKN1B mutations in a relevant number of cases (3%). However, the analysis of copy number variation showed that another fraction of luminal breast cancer displayed loss (8%) or gain (6%) of the CDKN1B gene, further reinforcing the idea that the function of p27kip1 is important in this type of tumor. Intriguingly, an enrichment for CDKN1B alterations was found in samples from premenopausal luminal breast cancer patients (n = 227, 4%) and in circulating cell-free DNA from metastatic luminal breast cancer patients (n = 59, 8.5%), suggesting that CDKN1B alterations could correlate with tumor aggressiveness and/or occur later during disease progression. Notably, many of the identified somatic mutations resulted in p27kip1 protein truncation, leading to loss of most of the protein or of its C-terminal domain. Using a gene-editing approach in a luminal breast cancer cell line, MCF-7, we observed that the expression of p27kip1 truncating mutants that lose the C-terminal domains failed to rescue most of the phenotypes induced by CDKN1B gene knockout, indicating that the functions retained by the C-terminal portion are critical for its role as an oncosuppressor, at least in luminal breast cancer. © 2020 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Human epidermal growth factor receptor-2 (HER2) is a potential therapeutic target in extramammary Paget's disease of the vulva.

BACKGROUND: Invasive vulvar Paget's disease with over-expression of the human epidermal growth factor receptor 2 (HER2) protein is potentially suitable for targeted therapy, especially in a metastatic setting where no effective treatments are available. METHODS: Four consecutive patients with HER2 positive advanced vulvar Paget's disease, treated with weekly trastuzumab (loading dose 4 mg/kg, then 2 mg/kg) and paclitaxel (80 mg/m2) followed by 3-weekly trastuzumab maintenance (6 mg/kg), are reported. RESULTS: Median age and follow-up of patients were 62.5 years (45-74) and 16 months (6-54), respectively. Complete or partial responses were observed in all patients. Median time to response was 3 months (range 2-4), while median duration of response was 10 months (range 2-34). Case 1 presented with pulmonary and lymph nodes involvement. She experienced a radiological complete response after 24 treatment administrations, and a progression-free survival of 36 months. At disease progression, treatment re-challenge achieved partial response. She is currently receiving treatment with trastuzumab-emtansine. Case 2 was a 74-year-old woman who developed pulmonary metastasis after first-line cisplatin treatment. She had a partial response and a progression-free survival of 10 months. Case 3 had inguinal and para-aortic lymphadenopathy in complete response after 18 treatment administrations. She developed brain metastasis while receiving trastuzumab maintenance. Case 4 was treated for locally advanced disease and experienced a subjective benefit with relief in perineal pain and itching. No unexpected treatment-related side effects were reported. CONCLUSIONS: Advanced vulvar Paget's disease is a rare disorder and no standard treatment is available. In the sub-group of HER2 positive disease, weekly paclitaxel-trastuzumab appears to be active and safe, and may be considered a therapeutic option in these patients.

Bevacizumab or PARP-Inhibitors Maintenance Therapy for Platinum-Sensitive Recurrent Ovarian Cancer: A Network Meta-Analysis.

INTRODUCTION: Targeted agents such as bevacizumab (BEV) or poly (ADP-ribose) polymerase inhibitors (PARPi) which have been added as concomitant or maintenance therapies have been shown to improve progression-free survival (PFS) in patients with platinum-sensitive recurrent ovarian cancer (PS rOC). In the absence of direct comparison, we performed a network meta-analysis considering BRCA genes status. METHODS: We searched PubMed, EMBASE, and MEDLINE for trials involving patients with PS rOC treated with BEV or PARPi. Different comparisons were performed for patients included in the PARPi trials, according to BRCA genes status as follows: all comers (AC) population, BRCA 1/2 mutated (BRCAm), and BRCA wild type patients (BRCAwt). RESULTS: In the overall population, PARPi prolonged PFS with respect to BEV (hazard ratio (HR) = 0.70, 95% CI 0.54-0.91). In the BRCA mutated carriers, the PFS improvement in favor of PARPi appeared to be higher (HR = 0.46, 95% CI 0.36-0.59) while in BRCAwt patients the superiority of PARPi over BEV failed to reach a statistically significance level (HR = 0.87, 95% CI 0.63-1.20); however, according to the SUCRA analysis, PARPi had the highest probability of being ranked as the most effective therapy (90% and 60%, for PARPi and BEV, respectively). CONCLUSIONS: PARPi performed better as compared with BEV in terms of PFS for the treatment of PS rOC, especially in BRCAm patients who had not previously received PARPi.

TIMP-1 is Overexpressed and Secreted by Platinum Resistant Epithelial Ovarian Cancer Cells.

Epithelial Ovarian Cancer (EOC) is the most lethal gynecological cancer in developed countries, and the development of new strategies to overcome chemoresistance is an awaited clinical need. Angiogenesis, the development of new blood vessels from pre-existing vasculature, has been validated as a therapeutic target in this tumor type. The aim of this study is to verify if EOC cells with acquired resistance to platinum (PT) treatment display an altered angiogenic potential. Using a proteomic approach, we identified the tissue inhibitor of metalloproteinases 1 (TIMP-1) as the only secreted factor whose expression was up-regulated in PT-resistant TOV-112D and OVSAHO EOC cells used as study models. We report that TIMP-1 acts as a double-edged sword in the EOC microenvironment, directly affecting the response to PT treatment on tumor cells and indirectly altering migration and proliferation of endothelial cells. Interestingly, we found that high TIMP-1 levels in stage III-IV EOC patients associate with decreased overall survival, especially if they were treated with PT or bevacizumab. Taken together, these results pinpoint TIMP-1 as a key molecule involved in the regulation of EOC PT-resistance and progression disclosing the possibility that it could be used as a new biomarker of PT-resistance and/or therapeutic target.

Sleeping beauty genetic screen identifies miR-23b::BTBD7 gene interaction as crucial for colorectal cancer metastasis.

BACKGROUND: Metastatic colorectal cancer (CRC) remains a deadly disease. Identifying locally advanced CRC patients with high risk of developing metastasis and improving outcome of metastatic CRC patients require discovering master regulators of metastasis. In this context, the non-coding part of the human genome is still largely unexplored. METHODS: To interrogate the non-coding part of the human genome and disclose regulators of CRC metastasis, we combined a transposon-based forward genetic screen with a novel in vitro assay, which forces cells to grow deprived of cell-substrate and cell-cell contacts (i.e. forced single cell suspension assay - fSCS). FINDINGS: We proved that fSCS selects CRC cells with mesenchymal and pro-metastatic traits. Moreover, we found that the transposon insertions conferred CRC cells resistance to fSCS and thus metastatic advantage. Among the retrieved transposon insertions, we demonstrated that the one located in the 3'UTR of BTBD7 disrupts miR-23b::BTBD7 interaction and contributes to pro-metastatic traits. In addition, miR-23b and BTBD7 correlate with CRC metastasis both in preclinical experiments and in clinical samples. INTERPRETATION: fSCS is a simple and scalable in vitro assay to investigate pro-metastatic traits and transposon-based genetic screens can interrogate the non-coding part of the human genome (e.g. miRNA::target interactions). Finally, both Btbd7 and miR-23b represent promising prognostic biomarkers and therapeutic targets in CRC. FUND: This work was supported by Marie Curie Actions (CIG n. 303877) and Friuli Venezia Giulia region (Grant Agreement n°245574), Italian Association for Cancer Research (AIRC, MFAG n°13589), Italian Ministry of Health (GR-2010-2319387 and PE-2016-02361040) and 5x1000 to CRO Aviano.

Exploring the Role of Fallopian Ciliated Cells in the Pathogenesis of High-Grade Serous Ovarian Cancer.

High-grade serous epithelial ovarian cancer (HGSOC) is the fifth leading cause of cancer death in women and the first among gynecological malignancies. Despite an initial response to standard chemotherapy, most HGSOC patients relapse. To improve treatment options, we must continue investigating tumor biology. Tumor characteristics (e.g., risk factors and epidemiology) are valuable clues to accomplish this task. The two most frequent risk factors for HGSOC are the lifetime number of ovulations, which is associated with increased oxidative stress in the pelvic area caused by ovulation fluid, and a positive family history due to genetic factors. In the attempt to identify novel genetic factors (i.e., genes) associated with HGSOC, we observed that several genes in linkage with HGSOC are expressed in the ciliated cells of the fallopian tube. This finding made us hypothesize that ciliated cells, despite not being the cell of origin for HGSOC, may take part in HGSOC tumor initiation. Specifically, malfunction of the ciliary beat impairs the laminar fluid flow above the fallopian tube epithelia, thus likely reducing the clearance of oxidative stress caused by follicular fluid. Herein, we review the up-to-date findings dealing with HGSOC predisposition with the hypothesis that fallopian ciliated cells take part in HGSOC onset. Finally, we review the up-to-date literature concerning genes that are located in genomic loci associated with epithelial ovarian cancer (EOC) predisposition that are expressed by the fallopian ciliated cells.

Following MicroRNAs Through the Cancer Metastatic Cascade.

Approximately a decade ago the first MicroRNAs (MiRNAs) participating in cancer metastasis were identified and metastmiRs were initially only a handful. Since those first reports, MiRNA research has explosively thrived, mainly due to their revolutionary mechanism of action and the hope of having at hand a novel tool to control cancer aggressiveness. This has ultimately led to delineate an almost impenetrable regulatory network: hundreds of MiRNAs transversally dominating every aspect of normal and cancer biology, each MiRNA having hundreds of targets and context-dependent activity. Providing a comprehensive description of MiRNA roles in cancer metastasis is a daunting task; nevertheless, we still believe that grasping the big picture of MiRNAs in cancer metastasis can give a different perspective on the potential insights and approaches that MiRNAs can offer to understand cancer complexity (e.g., as predictive and prognostic markers) and to tackle cancer metastasis (e.g., as therapeutic targets or tools). This chapter presents a schematic overview of the role of MiRNAs in governing cancer metastasis, describing step by step the cellular and molecular processes whereby cancer cells conquer distant organs and can grow as secondary tumors at different distant sites, and for each step, we will introduce how MiRNAs impinge on each one of them. We deeply apologize with our colleagues for any of their research work that, for clarity, for our effort to streamline and due to space limitations, we did not cite.

N-BLR, a primate-specific non-coding transcript leads to colorectal cancer invasion and migration.

BACKGROUND: Non-coding RNAs have been drawing increasing attention in recent years as functional data suggest that they play important roles in key cellular processes. N-BLR is a primate-specific long non-coding RNA that modulates the epithelial-to-mesenchymal transition, facilitates cell migration, and increases colorectal cancer invasion. RESULTS: We performed multivariate analyses of data from two independent cohorts of colorectal cancer patients and show that the abundance of N-BLR is associated with tumor stage, invasion potential, and overall patient survival. Through in vitro and in vivo experiments we found that N-BLR facilitates migration primarily via crosstalk with E-cadherin and ZEB1. We showed that this crosstalk is mediated by a pyknon, a short ~20 nucleotide-long DNA motif contained in the N-BLR transcript and is targeted by members of the miR-200 family. In light of these findings, we used a microarray to investigate the expression patterns of other pyknon-containing genomic loci. We found multiple such loci that are differentially transcribed between healthy and diseased tissues in colorectal cancer and chronic lymphocytic leukemia. Moreover, we identified several new loci whose expression correlates with the colorectal cancer patients' overall survival. CONCLUSIONS: The primate-specific N-BLR is a novel molecular contributor to the complex mechanisms that underlie metastasis in colorectal cancer and a potential novel biomarker for this disease. The presence of a functional pyknon within N-BLR and the related finding that many more pyknon-containing genomic loci in the human genome exhibit tissue-specific and disease-specific expression suggests the possibility of an alternative class of biomarkers and therapeutic targets that are primate-specific.

Combining Anti-Mir-155 with Chemotherapy for the Treatment of Lung Cancers.

Purpose: The oncogenic miR-155 is upregulated in many human cancers, and its expression is increased in more aggressive and therapy-resistant tumors, but the molecular mechanisms underlying miR-155-induced therapy resistance are not fully understood. The main objectives of this study were to determine the role of miR-155 in resistance to chemotherapy and to evaluate anti-miR-155 treatment to chemosensitize tumors.Experimental Design: We performed in vitro studies on cell lines to investigate the role of miR-155 in therapy resistance. To assess the effects of miR-155 inhibition on chemoresistance, we used an in vivo orthotopic lung cancer model of athymic nude mice, which we treated with anti-miR-155 alone or in combination with chemotherapy. To analyze the association of miR-155 expression and the combination of miR-155 and TP53 expression with cancer survival, we studied 956 patients with lung cancer, chronic lymphocytic leukemia, and acute lymphoblastic leukemia.Results: We demonstrate that miR-155 induces resistance to multiple chemotherapeutic agents in vitro, and that downregulation of miR-155 successfully resensitizes tumors to chemotherapy in vivo We show that anti-miR-155-DOPC can be considered non-toxic in vivo We further demonstrate that miR-155 and TP53 are linked in a negative feedback mechanism and that a combination of high expression of miR-155 and low expression of TP53 is significantly associated with shorter survival in lung cancer.Conclusions: Our findings support the existence of an miR-155/TP53 feedback loop, which is involved in resistance to chemotherapy and which can be specifically targeted to overcome drug resistance, an important cause of cancer-related death. Clin Cancer Res; 23(11); 2891-904. ©2016 AACR.

BNC2 is a putative tumor suppressor gene in high-grade serous ovarian carcinoma and impacts cell survival after oxidative stress.

Rs3814113 is the single-nucleotide polymorphism (SNP) showing the strongest association with high-grade serous ovarian carcinoma (HGSOC) incidence and is located in an intergenic region about 44 kb downstream of basonuclin 2 (BNC2) gene. Lifetime number of ovulations is associated with increased risk to develop HGSOC, probably because of cell damage of extrauterine Müllerian epithelium by ovulation-induced oxidative stress. However, the impact of low-penetrance HGSOC risk alleles (e.g. rs3814113) on the damage induced by oxidative stress remains unclear. Therefore, the purpose of this study was to investigate whether rs3814113 genetic interval regulates BNC2 expression and whether BNC2 expression levels impact on cell survival after oxidative stress. To do this, we analyzed gene expression levels of BNC2 first in HGSOC data sets and then in an isogenic cell line that we engineered to carry a 5 kb deletion around rs3814113. Finally, we silenced BNC2 and measured surviving cells after hydrogen peroxide (H2O2) treatment to simulate oxidative stress after ovulation. In this paper, we describe that BNC2 expression levels are reduced in HGSOC samples compared with control samples, and that BNC2 expression levels decrease following oxidative stress and ovulation in vitro and in vivo, respectively. Moreover, deletion of 5 kb surrounding rs3814113 decreases BNC2 expression levels in an isogenic cell line, and silencing of BNC2 expression levels increases cell survival after H2O2 treatment. Altogether, our findings suggest that the intergenic region located around rs3814113 regulates BNC2 expression, which in turn affects cell survival after oxidative stress response. Indeed, HGSOC samples present lower BNC2 expression levels that probably, in the initial phases of oncogenic transformation, conferred resistance to oxidative stress and ultimately reduced the clearance of cells with oxidative-induced damages.

The clinical and biological significance of MIR-224 expression in colorectal cancer metastasis.

OBJECTIVE: MicroRNA (miRNA) expression profile can be used as prognostic marker for human cancers. We aim to explore the significance of miRNAs in colorectal cancer (CRC) metastasis. DESIGN: We performed miRNA microarrays using primary CRC tissues from patients with and without metastasis, and validated selected candidates in 85 CRC samples by quantitative real-time PCR (qRT-PCR). We tested metastatic activity of selected miRNAs and identified miRNA targets by prediction algorithms, qRT-PCR, western blot and luciferase assays. Clinical outcomes were analysed in six sets of CRC cases (n=449), including The Cancer Genome Atlas (TCGA) consortium and correlated with miR-224 status. We used the Kaplan-Meier method and log-rank test to assess the difference in survival between patients with low or high levels of miR-224 expression. RESULTS: MiR-224 expression increases consistently with tumour burden and microsatellite stable status, and miR-224 enhances CRC metastasis in vitro and in vivo. We identified SMAD4 as a miR-224 target and observed negative correlation (Spearman Rs=-0.44, p<0.0001) between SMAD4 and miR-224 expression in clinical samples. Patients with high miR-224 levels display shorter overall survival in multiple CRC cohorts (p=0.0259, 0.0137, 0.0207, 0.0181, 0.0331 and 0.0037, respectively), and shorter metastasis-free survival (HR 6.51, 95% CI 1.97 to 21.51, p=0.0008). In the TCGA set, combined analysis of miR-224 with SMAD4 expression enhanced correlation with survival (HR 4.12, 95% CI 1.1 to 15.41, p=0.0175). CONCLUSIONS: MiR-224 promotes CRC metastasis, at least in part, through the regulation of SMAD4. MiR-224 expression in primary CRC, alone or combined with its targets, may have prognostic value for survival of patients with CRC.

Epigenetic silencing of microRNA-203 is required for EMT and cancer stem cell properties.

The epithelial-mesenchymal transition (EMT) imparts metastatic competence on otherwise non-metastatic cancer cells through decreased inter-cellular adhesions, increased migratory capacity, stem cell properties and anoikis and chemotherapy resistance. In this study, we profiled changes in microRNA expression during EMT in conjunction with changes in DNA methylation at microRNA promoters to discover essential mediators of EMT-imparted stemness properties. MicroRNA-203 (miR-203) expression is repressed following EMT induced by multiple different stimuli and in established claudin-low cell lines as well as the CD44hi/CD24lo stem cell-enriched fraction. Expression of miR-203 in mesenchymal cells compromises migratory and invasive capacity in vitro, and tumor initiation and metastasis in vivo. Unexpectedly, miR-203 expression affects the sphere-forming capacity of neighboring cells by indirectly enhancing expression of DKK1, a secreted inhibitor of Wnt signaling and stemness resulting in suppression of ß-catenin protein levels. Our data suggest that restoring miR-203 expression levels may inhibit metastasis and combat deregulated Wnt signaling.

Therapeutic synergy between microRNA and siRNA in ovarian cancer treatment.

UNLABELLED: Development of improved RNA interference-based strategies is of utmost clinical importance. Although siRNA-mediated silencing of EphA2, an ovarian cancer oncogene, results in reduction of tumor growth, we present evidence that additional inhibition of EphA2 by a microRNA (miRNA) further "boosts" its antitumor effects. We identified miR-520d-3p as a tumor suppressor upstream of EphA2, whose expression correlated with favorable outcomes in two independent patient cohorts comprising 647 patients. Restoration of miR-520d-3p prominently decreased EphA2 protein levels, and suppressed tumor growth and migration/invasion both in vitro and in vivo. Dual inhibition of EphA2 in vivo using 1,2-dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC) nanoliposomes loaded with miR-520d-3p and EphA2 siRNA showed synergistic antitumor efficiency and greater therapeutic efficacy than either monotherapy alone. This synergy is at least in part due to miR-520d-3p targeting EphB2, another Eph receptor. Our data emphasize the feasibility of combined miRNA-siRNA therapy, and will have broad implications for innovative gene silencing therapies for cancer and other diseases. SIGNIFICANCE: This study addresses a new concept of RNA inhibition therapy by combining miRNA and siRNA in nanoliposomal particles to target oncogenic pathways altered in ovarian cancer. Combined targeting of the Eph pathway using EphA2-targeting siRNA and the tumor suppressor miR-520d-3p exhibits remarkable therapeutic synergy and enhanced tumor suppression in vitro and in vivo compared with either monotherapy alone.

Coordinated targeting of the EGFR signaling axis by microRNA-27a*.

Epidermal growth factor receptor (EGFR) has been characterized as a critical factor in the development and progression of multiple solid tumors, including head and neck squamous cell carcinoma (HNSCC). However, monotherapy with EGFR-specific agents has not been as dramatic as preclinical studies have suggested. Since complex regulation of the EGFR signaling axis might confound current attempts to inhibit EGFR directly, we searched for microRNAs (miRNAs) that may target the EGFR signaling axis. We identified miR-27a (miR-27a-3p) and its complementary or star (*) strand, miR-27a* (miR-27a-5p), as novel miRNAs targeting EGFR, which were significantly downregulated in multiple HNSCC cell lines. Analysis of human specimens demonstrated that miR-27a* is significantly underexpressed in HNSCC as compared to normal mucosa. Increased expression of miR-27a* in HNSCC produced a profound cytotoxic effect not seen with miR-27a. Analysis for potential targets of miR-27a* led to the identification of AKT1 (protein kinase B) and mTOR (mammalian target of rapamycin) within the EGFR signaling axis. Treatment with miR-27a* led to coordinated downregulation of EGFR, AKT1 and mTOR. Overexpression of EGFR signaling pathway components decreased the overall effect of miR-27a* on HNSCC cell viability. Constitutive and inducible expression of miR-27a* in a murine orthotopic xenograft model of oral cavity cancer led to decreased tumor growth. Direct intratumoral injection of miR-27a* inhibited tumor growth in vivo. These findings identify miR-27a* as a functional star sequence that exhibits novel coordinated regulation of the EGFR pathway in solid tumors and potentially represents a novel therapeutic option.