Francesca Orso

microRNA-214 contributes to melanoma tumour progression through suppression of TFAP2C

Malignant melanoma is fatal in its metastatic stage. It is therefore essential to unravel the molecular mechanisms that govern disease progression to metastasis. MicroRNAs (miRs) are endogenous non‐coding RNAs involved in tumourigenesis. Using a melanoma progression model, we identified a novel pathway controlled by miR‐214 that coordinates metastatic capability. Pathway components include TFAP2C, homologue of a well‐established melanoma tumour suppressor, the adhesion receptor ITGA3 and multiple surface molecules. Modulation of miR‐214 influences in vitro tumour cell movement and survival to anoikis as well as extravasation from blood vessels and lung metastasis formation in vivo. Considering that miR‐214 is known to be highly expressed in human melanomas, our data suggest a critical role for this miRNA in disease progression and the establishment of distant metastases.

microRNA-222 Controls Neovascularization by Regulating Signal Transducer and Activator of Transcription 5A Expression

Objective—Inflammatory stimuli released into atherosclerotic plaque microenvironment regulate vessel formation by modulating gene expression and translation. microRNAs are a class of short noncoding RNAs, acting as posttranscriptional regulators of protein-coding genes involved in various biological processes, including vascular cell biology. Among them, microRNA-221/222 (miR-221/222) seem to negatively modulate vascular remodeling by targeting different target genes. Here, we investigated their potential contribution to inflammation-mediated neovessel formation. Methods and Results—We used quantitative real-time RT-PCR amplification to analyze expression of 7 microRNAs previously linked to vascular biology, such as miR-17-5p, miR-21, miR-126, miR-210, miR-221, miR-222, and miR-296 and found high levels of expression for all of them in quiescent endothelial cells. However, miR-126, miR-221, miR-222, and miR-296 turned out to be down-modulated in endothelial cells exposed to inflammatory stimuli. Applying a gain-of-function approach, we demonstrated that, among them, only miR-222 was involved in inflammation-mediated vascular remodeling. In addition, we identified signal transducer and activator of transcription 5A (STAT5A) as a bona fide target of miR-222 and observed that miR-222 negatively correlated with STAT5A expression in human endothelial cells from advanced neovascularized atherosclerotic lesions. Conclusion—We identified STAT5A as a novel miR-222 target, and this finding opens up new perspectives for treatment of vascular diseases.

miR148b is a major coordinator of breast cancer progression in a relapse-associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1

Breast cancer is often fatal during its metastatic dissemination. To unravel the role of microRNAs (miRs) during malignancy, we analyzed miR expression in 77 primary breast carcinomas and identified 16 relapse‐associated miRs that correlate with survival and/or distinguish tumor subtypes in different datasets. Among them, miR‐148b, down‐regulated in aggressive breast tumors, was found to be a major coordinator of malignancy. In fact, it is able to oppose various steps of tumor progression when overexpressed in cell lines by influencing invasion, survival to anoikis, extravasation, lung metastasis formation, and chemotherapy response. miR‐148b controls malignancy by coordinating a novel pathway involving over 130 genes and, in particular, it directly targets players of the integrin signaling, such as ITGA5, ROCK1, PIK3CA/p110α, and NRAS, as well as CSF1, a growth factor for stroma cells. Our findings reveal the importance of the identified 16 miRs for disease outcome predictions and suggest a critical role for miR‐148b in the control of breast cancer progression.—Cimino, D., De Pittà, C., Orso, F., Zampini, M., Casara, C., Penna, E., Quaglino, E., Forni, M., Damasco, C., Pinatel, E., Ponzone, R., Romualdi, C., Brisken, C., De Bortoli, M., Biglia, N., Provero, P., Lanfranchi, G., Taverna, D. miR148b is a major coordinator of breast cancer progression in a relapse‐associated microRNA signature by targeting ITGA5, ROCK1, PIK3CA, NRAS, and CSF1. FASEB J. 27, 1223–1235 (2013). www.fasebj.org

AP-2α and AP-2γ regulate tumor progression via specific genetic programs

The events occurring during tumor formation and progression display similarities to some of the steps in embryonic morphogenesis. The family of AP‐2 proteins consists of five different transcription factors (α, β, γ, δ, and ε) that play relevant roles in embryonic development, as demonstrated by the phenotypes of the corresponding knockout mice. Here, we show that AP‐2α and AP‐2γ proteins play an essential role in tumorigenesis. Down‐modulation of AP‐2 expression in tumor cells by RNA interference (RNAi) led to enhanced tumor growth and reduced chemotherapy‐induced cell death, as well as migration and invasion. Most of these biological modulations were rescued by AP‐2 overexpression. We observed that increased xeno‐transplant growth was mostly due to highly enhanced proliferation of the tumor cells together with reduced innate immune cell recruitment. Moreover, we showed that migration impairment was mediated, at least in part, by secreted factors. To identify the genetic programs involved in tumorigenesis, we performed whole genome microarray analysis of AP‐2α knockdown cells and observed that AP‐2α regulates specific genes involved in cell cycle, cell death, adhesion, and migration. In particular, we showed that ESDN, EREG, and CXCL2 play a major role in AP‐2 controlled migration, as ablation of any of these genes severely altered migration.—Orso, F., Penna, E., Cimino, D., Astanina, E., Maione, F., Valdembri, D., Giraudo, E., Serini, G., Sismondi, P., De Bortoli, M., Taverna, D. AP‐2α and AP‐2γ regulate tumor progression via specific genetic programs. FASEB J. 22, 2702‐2714 (2008)

miR-214 Coordinates Melanoma Progression by Upregulating ALCAM through TFAP2 and miR-148b Downmodulation

Malignant melanoma is one of the most aggressive human cancers, but the mechanisms governing its metastatic dissemination are not fully understood. Upregulation of miR-214 and ALCAM and the loss of TFAP2 expression have been implicated in this process, with TFAP2 a direct target of miR-214. Here, we link miR-214 and ALCAM as well as identify a core role for miR-214 in organizing melanoma metastasis. miR-214 upregulated ALCAM, acting transcriptionally through TFAP2 and also posttranscriptionally through miR-148b (itself controlled by TFAP2), both negative regulators of ALCAM. We also identified several miR-214-mediated prometastatic functions directly promoted by ALCAM. Silencing ALCAM in miR-214-overexpressing melanoma cells reduced cell migration and invasion without affecting growth or anoikis in vitro, and it also impaired extravasation and metastasis formation in vivo. Conversely, cell migration and extravasation was reduced in miR-214-overexpressing cells by upregulation of either miR-148b or TFAP2. These findings were consistent with patterns of expression of miR-214, ALCAM, and miR-148b in human melanoma specimens. Overall, our results define a pathway involving miR-214, miR-148b, TFAP2, and ALCAM that is critical for establishing distant metastases in melanoma.

miR-223 is a coordinator of breast cancer progression as revealed by bioinformatics predictions.

MicroRNAs are single-stranded non-coding RNAs that simultaneously down-modulate the expression of multiple genes post-transcriptionally by binding to the 3′UTRs of target mRNAs. Here we used computational methods to predict microRNAs relevant in breast cancer progression. Specifically, we applied different microRNA target prediction algorithms to various groups of differentially expressed protein-coding genes obtained from four breast cancer datasets. Six potential candidates were identified, among them miR-223, previously described to be highly expressed in the tumor microenvironment and known to be actively transferred into breast cancer cells. To investigate the function of miR-223 in tumorigenesis and to define its molecular mechanism, we overexpressed miR-223 in breast cancer cells in a transient or stable manner. Alternatively we overexpressed miR-223 in mouse embryonic fibroblasts or HEK293 cells and used their conditioned medium to treat tumor cells. With both approaches, we obtained elevated levels of miR-223 in tumor cells and observed decreased migration, increased cell death in anoikis conditions and augmented sensitivity to chemotherapy but no effect on adhesion and proliferation. The analysis of miR-223 predicted targets revealed enrichment in cell death and survival-related genes and in pathways frequently altered in breast cancer. Among these genes, we showed that protein levels for STAT5A, ITGA3 and NRAS were modulated by miR-223. In addition, we proved that STAT5A is a direct miR-223 target and highlighted a possible correlation between miR-223 and STAT5A in migration and chemotherapy response. Our investigation revealed that a computational analysis of cancer gene expression datasets can be a relevant tool to identify microRNAs involved in cancer progression and that miR-223 has a prominent role in breast malignancy that could potentially be exploited therapeutically.

T Cell Receptor (TCR) Gene Transfer with Lentiviral Vectors Allows Efficient Redirection of Tumor Specificity in Naive and Memory T Cells Without Prior Stimulation of Endogenous TCR

We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

p130Cas/Cyclooxygenase-2 axis in the control of mesenchymal plasticity of breast cancer cells

IntroductionIntrinsic plasticity of breast carcinoma cells allows them to undergo a transient and reversible conversion into mesenchymal cells to disseminate into distant organs, where they can re-differentiate to an epithelial-like status to form a cohesive secondary mass. The p130Cas scaffold protein is overexpressed in human ER+ and HER2+ breast cancer where it contributes to cancer progression, invasion and resistance to therapy. However, its role in regulating mesenchymal aggressive breast cancer cells remains to be determined. The aim of this study was to investigate the molecular and functional involvement of this adaptor protein in breast cancer cell plasticity.MethodsWe used silencing strategies and rescue experiments to evaluate phenotypic and biochemical changes from mesenchymal to epithelial traits in breast tumor cell lines. In the mouse A17 cell model previously related to mesenchymal cancer stem cells and basal-like breast cancer, we biochemically dissected the signaling pathways involved and performed functional in vivo tumor growth ability assays. The significance of the signaling platform was assessed in a human setting through the use of specific inhibitors in aggressive MDA-MB-231 subpopulation LM2-4175 cells. To evaluate the clinical relevance of the results, we analyzed publicly available microarray data from the Netherlands Cancer Institute and from the Koo Foundation Sun Yat-Sen Cancer Center.ResultsWe show that p130Cas silencing induces loss of mesenchymal features, by downregulating Vimentin, Snail, Slug and Twist transcriptional factors, resulting in the acquirement of epithelial-like traits. Mechanistically, p130Cas controls Cyclooxygenase-2 transcriptional expression, which in turn contributes to p130Cas-dependent maintenance of mesenchymal phenotype. This cascade of events also compromises in vivo tumor growth through inhibition of cell signaling controlling cell cycle progression. c-Src and JNK kinases are sequential players in p130Cas/ Cyclooxygenase-2 axis and their pharmacological inhibition is sufficient to downregulate Cyclooxygenase-2 leading to an epithelial phenotype. Finally, in silico microarray data analysis indicates that p130Cas and Cyclooxygenase-2 concomitant overexpression predicts poor survival and high probability of breast tumor recurrence.ConclusionsOverall, these data identify a new p130Cas/Cyclooxygenase-2 axis as a crucial element in the control of breast tumor plasticity, opening new therapeutic strategies leading to inhibition of these pathways in aggressive breast carcinoma.

miR-214 and miR-148b targeting inhibits dissemination of melanoma and breast cancer

miR-214 and miR-148b have been proposed to antagonize the effects of each other in enabling or blocking metastasis, respectively. In this study, we provide evidence deepening their role and interrelationship in the process of metastatic dissemination. Depleting miR-214 or elevating miR-148b blocked the dissemination of melanoma or breast cancer cells, an effect that could be accentuated by dual alteration. Mechanistic investigations indicated that dual alteration suppressed passage of malignant cells through the blood vessel endothelium by reducing expression of the cell adhesion molecules ITGA5 and ALCAM. Notably, transendothelial migration in vitro and extravasation in vivo impaired by singly alternating miR-214 or miR-148b could be overridden by overexpression of ITGA5 or ALCAM in the same tumor cells. In clinical specimens of primary breast cancer or metastatic melanoma, we found a positive correlation between miR-214 and ITGA5 or ALCAM along with an inverse correlation of miR-214 and miR-148b in the same specimens. Our findings define an antagonistic relationship of miR-214 and miR-148b in determining the dissemination of cancer cells via tumor-endothelial cell interactions, with possible implications for microRNA-mediated therapeutic interventions aimed at blocking cancer extravasation. Cancer Res; 76(17); 5151-62. ©2016 AACR.

Formation of STAT5/PPARγ Transcriptional Complex Modulates Angiogenic Cell Bioavailability in Diabetes

Objective—Circulating angiogenic cells (CACs) expansion is a multistage process requiring sequential activation of transcriptional factors, including STAT5. STAT5, in concert with peroxisome proliferator-activated receptors (PPARs), seems to induce discrete biological responses in different tissues. In the present study we investigated the role of STAT5 and PPARγ in regulating CAC expansion in normal and diabetic settings. Methods and Results—Normal and diabetic CACs were used. siRNA technology, EMSA, and chromatin immunoprecipitation (ChIP) assay as well as site-directed mutagenesis of the STAT5 response element in the PPARγ promoter enabled us to demonstrate that STAT5 transcriptional activity controls PPARγ expression. Moreover, FACS analysis, coimmunoprecipitation experiments, and ChIP assay revealed that a STAT5/PPARγ transcriptional complex controls cyclin D1 expression and CAC progression into the cell-cycle. Conversely, PPARγ agonists, by preventing the expression of STAT5 and the formation of the STAT5/PPARγ heterodimeric complex failed to promote CAC expansion. Finally, we demonstrated that diabetic CAC functional capability can be recovered by molecules able to activate the STAT5/PPARγ transcriptional complex. Conclusions—Our data identify the STAT5/PPARγ heterodimers as landmark of CAC expansion and provide evidences for a mechanism that partially rescues CAC bioavailability in diabetic setting.