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miR-148b reduces liver and lung metastasis formation

Supplementary Methods - PDF file 95K, Additional information regarding all reagents, antibodies, vectors and primers employed in this study, including experimental procedures

Analysis of miR-214 and miR-148b sponge efficacy

miR-214 and miR-148b affect migration and invasion ability

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.

miR-148b expression modulations in normal or tumor breast cells

miR-214 expression modulations in normal or tumor breast cells

miR-148b target genes analysis

miR-214 and miR-148b sponge sequences

ITGA5 and ALCAM control transendothelial migration in vitro and extravasation in mice

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. Cancer Res; 73(13); 4098–111. ©2013 AACR.

miR-214 and miR-148b coordinate lung metastasis formation in an opposite manner

miR-214 inhibition and miR-148b overexpression impair melanoma cell extravasation

Supplementary Figure and Table Legends - PDF file 74K, Legend for Supplemental Table S1 and Supplemental Figures S1-S4

Supplementary tables and figure legends

Supplementary Table S1 - PDF file 98K, miR-214 and ALCAM promote melanoma metastasis independently of BRAFV600E mutation status

Supplementary Figures S1-S4 - PDF file 482K, miR-214, miR-148b and ALCAM expression modulations (S1); ALCAM is upregulated by miR-214 and controls cell movement but not proliferation (S2); ALCAM and miR-148b regulations (S3); ALCAM, miR-214, miR-148b and TFAP2 expression in human melanoma samples (S4)

miR-148b expression modulations in melanoma cells

miR-214 expression modulations in melanoma cells

miR-214 and miR-148b do not significantly affect proliferation

Background Tumor progression is based on a close interaction between cancer cells and Tumor MicroEnvironment (TME). Here, we focus on the role that Cancer Associated Fibroblasts (CAFs), Mesenchymal Stem Cells (MSCs) and microRNAs (miRs) play in breast cancer and melanoma malignancy. Methods We used public databases to investigate miR-214 expression in the stroma compartment of primary human samples and evaluated tumor formation and dissemination following tumor cell injections in miR-214 overexpressing (miR-214over) and knock out (miR-214ko) mice. In addition, we dissected the impact of Conditioned Medium (CM) or Extracellular Vesicles (EVs) derived from miR-214-rich or depleted stroma cells on cell metastatic traits. Results We evidence that the expression of miR-214 in human cancer or metastasis samples mostly correlates with stroma components and, in particular, with CAFs and MSCs. We present data revealing that the injection of tumor cells in miR-214over mice leads to increased extravasation and metastasis formation. In line, treatment of cancer cells with CM or EVs derived from miR-214-enriched stroma cells potentiate cancer cell migration/invasion in vitro. Conversely, dissemination from tumors grown in miR-214ko mice is impaired and metastatic traits significantly decreased when CM or EVs from miR-214-depleted stroma cells are used to treat cells in culture. Instead, extravasation and metastasis formation are fully re-established when miR-214ko mice are pretreated with miR-214-rich EVs of stroma origin. Mechanistically, we also show that tumor cells are able to induce miR-214 production in stroma cells, following the activation of IL-6/STAT3 signaling, which is then released via EVs subsequently up-taken by cancer cells. Here, a miR-214-dependent pro-metastatic program becomes activated. Conclusions Our findings highlight the relevance of stroma-derived miR-214 and its release in EVs for tumor dissemination, which paves the way for miR-214-based therapeutic interventions targeting not only tumor cells but also the TME.

An interactive crosstalk between tumor and stroma cells is essential for metastatic melanoma progression. We evidenced that ESDN/DCBLD2/CLCP1 plays a crucial role in endothelial cells during the spread of melanoma. Precisely, increased extravasation and metastasis formation were revealed in ESDN-null mice injected with melanoma cells, even if the primary tumor growth, vessel permeability, and angiogenesis were not enhanced. Interestingly, improved adhesion of melanoma cells to ESDN-depleted endothelial cells was observed, due to the presence of higher levels of E-selectin transcripts/proteins in ESDN-defective cells. In accordance with these results, anticorrelation was observed between ESDN and E-selectin in human endothelial cells. Most importantly, our data revealed that cimetidine, an E-selectin inhibitor, was able to block cell adhesion, extravasation, and metastasis formation in ESDN-null mice, underlying a major role of ESDN in E-selectin transcription upregulation, which according to our data, may presumably be linked to STAT3. Based on our results, we propose a protective role for ESDN during the spread of melanoma and reveal its therapeutic potential. ispartof: CANCER LETTERS vol:510 pages:13-23 ispartof: location:Ireland status: published

MicroRNAs (miRNAs) are small, non-coding RNAs about 22 nucleotides in length that regulate the expression of target genes post-transcriptionally, and are highly involved in cancer progression. They are able to impact a variety of cell processes such as proliferation, apoptosis and differentiation and can consequently control tumor initiation, tumor progression and metastasis formation. miRNAs can regulate, at the same time, metabolic gene expression which, in turn, influences relevant traits of malignancy such as cell adhesion, migration and invasion. Since the interaction between metabolism and adhesion or cell movement has not, to date, been well understood, in this review, we will specifically focus on miRNA alterations that can interfere with some metabolic processes leading to the modulation of cancer cell movement. In addition, we will analyze the signaling pathways connecting metabolism and adhesion/migration, alterations that often affect cancer cell dissemination and metastasis formation.

microRNAs (miRNAs) are small non-coding RNAs acting as negative regulators of gene expression and involved in tumor progression. We recently showed that inhibition of the pro-metastatic miR-214 and simultaneous overexpression of its downstream player, the anti-metastatic miR-148b, strongly reduced metastasis formation. To explore the therapeutic potential of miR-148b, we generated a conjugated molecule aimed to target miR-148b expression selectively to tumor cells. Precisely, we linked miR-148b to GL21.T, an aptamer able to specifically bind to AXL, an oncogenic tyrosine kinase receptor highly expressed on cancer cells. Axl-148b conjugate was able to inhibit migration and invasion of AXL-positive, but not AXL-negative, cancer cells, demonstrating high efficacy and selectivity in vitro. In parallel, expression of ALCAM and ITGA5, two miR-148b direct targets, was reduced. More importantly, axl-148b chimeric aptamers were able to inhibit formation and growth of 3D-mammospheres, to induce necrosis and apoptosis of treated xenotransplants, as well as to block breast cancer and melanoma dissemination and metastatization in mice. Relevantly, axl aptamer acted as specific delivery tool for miR-148b, but it also actively contributed to inhibit metastasis formation, together with miR-148b. In conclusion, our data show that axl-148b conjugate is able to inhibit tumor progression in an axl- and miR-148b-dependent manner, suggesting its potential development as therapeutic molecule.

Attenuated Listeria monocytogenes (Lmat-LLO) represents a valuable anticancer vaccine and drug delivery platform. Here we show that in vitro Lmat-LLO causes ROS production and, in turn, apoptotic killing of a wide variety of melanoma cells, irrespectively of their stage, mutational status, sensitivity to BRAF inhibitors or degree of stemness. We also show that, when administered in the therapeutic setting to Braf/Pten genetically engineered mice, Lmat-LLO causes a strong decrease in the size and volume of primary melanoma tumors, as well as a reduction of the metastatic burden. At the molecular level, we confirm that the anti-melanoma activity exerted in vivo by Lmat-LLO depends also on its ability to potentiate the immune response of the organism against the infected tumor. Our data pave the way to the preclinical testing of listeria-based immunotherapeutic strategies against metastatic melanoma, using a genetically engineered mouse rather than xenograft models.

We previously demonstrated that miR-214 is upregulated in malignant melanomas and triple-negative breast tumors and promotes metastatic dissemination by affecting a complex pathway including the anti-metastatic miR-148b. Importantly, tumor dissemination could be reduced by blocking miR-214 function or increasing miR-148b expression or by simultaneous interventions. Based on this evidence, with the intent to explore the role of miR-214 as a target for therapy, we evaluated the capability of new chemically modified anti-miR-214, R97/R98, to inhibit miR-214 coordinated metastatic traits. Relevantly, when melanoma or breast cancer cells were transfected with R97/R98, anti-miR-214 reduced miR-214 expression and impaired transendothelial migration were observed. Noteworthy, when the same cells were injected in the tail vein of mice, cell extravasation and metastatic nodule formation in lungs were strongly reduced. Thus, suggesting that R97/R98 anti-miR-214 oligonucleotides were able to inhibit tumor cell escaping through the endothelium. More importantly, when R97/R98 anti-miR-214 compounds were systemically delivered to mice carrying melanomas or breast or neuroendocrine pancreatic cancers, a reduced number of circulating tumor cells and lung or lymph node metastasis formation were detected. Similar results were also obtained when AAV8-miR-214 sponges were used in neuroendocrine pancreatic tumors. Based on this evidence, we propose miR-214 as a promising target for anti-metastatic therapies.

In the last decades, progresses in medical oncology have ameliorated the treatment of patients and their outcome. However, further improvements are still necessary, in particular for certain types of tumors such as pancreatic, gastric, and lung cancer as well as acute myeloid leukemia where early detection and monitoring of the disease are crucial for final patient outcome. Liquid biopsy represents a great advance in the field because it is less invasive, less time-consuming, and safer compared to classical biopsies and it can be useful to monitor the evolution of the disease as well as the response of patients to therapy. Liquid biopsy allows the detection of circulating tumor cells, nucleic acids, and exosomes not only in blood but also in different biological fluids: urine, saliva, pleural effusions, cerebrospinal fluid, and stool. Among the potential biomarkers detectable in liquid biopsies, microRNAs (miRNAs) are gaining more and more attention, since they are easily detectable, quite stable in biological fluids, and show high sensitivity. Many data demonstrate that miRNAs alone or in combination with other biomarkers could improve the diagnostic and prognostic power for many different tumors. Despite this, standardization of methods, sample preparation, and analysis remain challenging and a huge effort should be made to address these issues before miRNA biomarkers can enter the clinic. This review summarizes the main findings in the field of circulating miRNAs in both solid and hematological tumors.

Cancer is a multistep disease based on crucial interactions between tumor cells and the microenvironment (extracellular matrix and stroma/immune cells). In fact, during dissemination, tumor cells have to escape from the primary tumor mass, cross the basal membrane, interact with endothelial cells to enter blood vessels (intravasation), survive in the bloodstream, get in contact with endothelial cells again to exit the bloodstream (extravasation) and seed in distant organs. Interactions between tumor and stroma cells are strongly coordinated by microRNAs (miRNAs), small non-coding RNAs able to silence protein coding genes by binding to specific recognition sites, mostly located at the 3' UTR of mature mRNAs. Relevantly, miRNA expression is often altered (overexpression or downregulation) in tumor cells and influenced by stroma cells. At the same time, miRNAs are abundant and essential in stroma cells during tumor cell dissemination and their expression is influenced by tumor cells. In fact, for instance, conditional ablation of Dicer in the endothelium of tumor bearing-mice leads to reduced tumor growth and microvessel density. In this review, we specifically focus on the role of miRNAs in endothelial cells regarding their positive or negative intervention on tumor angiogenesis or lymphoangiogenesis or when tumor cells detach from the tumor mass and intravasate or extravasate in/out of the blood vessels. Examples of pro-angiogenic miRNAs are miR-9 or miR-494, often overexpressed in tumors, which accumulate in tumor cell microvescicles and, therefore, get transferred to endothelial cells where they induce migration and angiogenesis. Differently, miR-200 and miR-128 are often downregulated in tumors and inhibit angiogenesis and lymphoangiogenesis. Instead, miR-126 controls intravasation while miR-146a, miR-214, miR-148b govern extravasation, in a positive or negative manner. Finally, at the end, we summarize opportunities for therapeutic interventions based on miRNAs acting on endothelial cells.

About 90% of breast cancer mortalities are due to the spread of breast cancer cells (BCCs) from a primary tumor to distant organs, a process known as metastasis. However, the molecular mechanisms underlying metastasis remain poorly understood. Substantial evidence now supports a major role for the tumor microenvironment (TME) in catalyzing breast cancer metastasis. Indeed, observations indicate that proximal interactions between BCCs and cells of the TME induce altered gene expression programs in BCCs, allowing for the navigation of the various steps of the metastatic cascade. Our group and others observed that breast tumors recruit mesenchymal stem cells (MSCs): multipotent fibroblasts that normally exert tissue maintenance functions. We and others have observed that physical interactions of MSCs with BCCs are sufficient to drive their metastatic dissemination in murine xenograft models, via the induction of epithelial-mesenchymal transition (EMT) and dedifferentiation into stem cell-like states (cancer stem cells, or CSCs), states tightly associated with the capacity to seed new tumors (for example in foreign tissues) and with chemotherapeutic resistance. However, the TME-induced molecular pathways regulating such mechanisms remain poorly understood. MicroRNAs (miRNAs, miRs) are small noncoding RNAs that regulate gene expression via base-pair interactions with messenger RNAs (mRNAs), resulting in mRNA degradation or translational inhibition. Due to their ability to interact with large numbers of target mRNAs simultaneously, miRNAs are major regulators of cell identity, and thereby serve critical roles in metastasis. We performed miRnome-wide screening of MSC-stimulated BCCs to determine if TME interactions might contribute to BCC metastasis via the deregulation of miRNAs. We observed that proximal MSCs induce aberrant expression of a specific set of miRNAs in BCCs, which had not been previously implicated in breast cancer pathogenesis. These miRNAs, led by the transcriptionally co-regulated miR-199a-3p and miR-214, were sufficient to actuate the metastasis of weakly metastatic human BCCs in xenograft models. We observed that exogenous expression of the miRNAs provided BCCs with phenotypes and gene markers characteristic of CSCs, including enhanced tumor initiation capacities. Interestingly, we found that the MSC-induced miRNAs function as an interrelated network, and converge upon a common novel target: the speech associated gene FOXP2. Knockdown of FOXP2 phenocopied the metastatic phenotypes observed in MSC-induced miRNA expressing BCCs. Importantly, elevated levels of the MSC-induced miRNAs or depressed levels of FOXP2 could predict patient prognosis in the clinic. Altogether, our results incriminate FOXP2 and it’s MSC-induced miRNA regulatory network as novel determinants of breast cancer metastasis. Citation Format: Benjamin G Cuiffo, Antoine Campagne, George W Bell, Antonio Lembo, Francesca Orso, Evan Lien, Manoj K Bhasin, Monica Raimo, Summer E Hanson, Andriy Marusyk, Peiman Hematti, Kornelia Polyak, Odette Mariani, Stefano Volinia, Anne Vincent-Salomon, Daniela Taverna, Antoine E Karnoub. Mesenchymal stem cell regulated microRNAs converge on the speech gene FOXP2 and regulate breast cancer metastasis [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-07-02.

BRAF inhibitors (BRAFi) and the combination therapy of BRAF and MEK inhibitors (MEKi) were recently approved for therapy of metastatic melanomas harbouring the oncogenic BRAFV600 mutation. Although these therapies have shown pronounced therapeutic efficacy, the limited durability of the response indicates an acquired drug resistance that still remains mechanistically poorly understood at the molecular level. We conducted transcriptome gene profiling in BRAFi-treated melanoma cells and identified that Mer tyrosine kinase (MerTK) is specifically upregulated. MerTK overexpression was demonstrated not only in melanomas resistant to BRAFi monotherapy (5 out of 10 samples from melanoma patients) but also in melanoma resistant to BRAFi+MEKi (1 out of 3), although MEKi alone does not affect MerTK. Mechanistically, BRAFi-induced activation of Zeb2 stimulates MerTK in BRAFV600 melanoma through mTORC1-triggered activation of autophagy. Co-targeting MerTK and BRAFV600 significantly reduced tumour burden in xenografted mice, which was pheno-copied by co-inhibition of autophagy and mutant BRAFV600.

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.

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.

Introduction Morgana is a chaperone protein encoded by the CHORDC1 gene. Its deletion is embryonic lethal due to apoptosis of the cells of the inner cell mass. We recently characterised the role of Morgana in myeloid malignancies, as the haploinsufficiency of the protein in mice is able to induce a fatal and transplantable myeloproliferative disease resembling human Atypical Chronic Myeloid Leukaemia (aCML). 5 out of 5 aCML patients and 16% of Philadelphia-positive CML patients express low/undetectable levels of Morgana in their bone marrow. As we never found mutations or deletions of CHORDC1 gene, we decided to investigate if Morgana can be targeted by miRNAs. Five miRNAs are predicted to target Morgana: miR-15a/b and miR-16 sharing the same seed sequence and miR-26a/b. Material and methods HEK-293T cells were used to overexpress miRNAs predicted to target Morgana mRNA. The level of miRNAs overexpression and Morgana mRNA was assessed with qRT-PCR and Morgana protein level with Western Blot at different time points. The seed sequences for the selected miRNAs in the 3’UTR of CHORDC1 gene were than mutagenized to validate the specificity of the binding. Bioinformatic analysis were used to correlate miRNAs and Morgana expression levels in leukaemia and lymphoma. Results and discussions We demonstrated that miRNA-15a/b and miRNA-26a/b are able to bind to Morgana 3’-UTR and, in this way, mediate its mRNA deregulation leading to a reduction of Morgana, both at mRNA and protein level. We were able to highlight an anti-correlation between Morgana and miRNAs expression in haematological tumours: in particular miR-15b in Chronic Lymphocytic Leukaemia and Lymphomas, miR-15a in aCML and CML and miR-26a and miR-16 in Lymphomas. Conclusion Morgana is able to act both as proto-oncogene and as oncosuppressor depending on tissue type and levels of expression as it is frequently found both overexpressed and downregulated. Different approaches to elucidate its mechanisms of regulation failed and we believe that miRNAs are just one of them. Further investigations are needed to clirify how Morgana expression is regulated in different type of tumours.

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.

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.

The activator protein 2 (AP-2) transcription factors are essential proteins for oestrogenic repression of the ERBB2 proto-oncogene in breast cancer cells. In the present study, we have examined the possible oestrogenic regulation of AP-2 genes themselves in breast-tumour-derived lines. As early as 1 h after oestrogen treatment, AP-2gamma mRNA was markedly increased, whereas AP-2alpha was down-regulated, but with slower kinetics, and AP-2beta was not affected at all. Addition of anti-oestrogens ablated these effects. Modulation of the protein levels corresponded to changes in the transcript levels, thus suggesting that in oestrogen-treated cells, an inversion of the balance between AP-2alpha and AP-2gamma isoforms occurs. The 5'-untranslated region (5'-UTR) of the human AP-2gamma gene contains one consensus and one degenerate oestrogen-responsive element (ERE). Reporter constructs carrying the AP-2gamma promoter and the 5'-UTR were up-regulated by oestrogens in transient transfection assays. Deletion of the most conserved (but not of the degenerate) ERE from reporter constructs abrogated the oestrogenic response, although both ERE-containing segments were footprinted in DNaseI protection assays. In vitro binding assays demonstrated the ability of oestrogen receptor alpha (ERalpha) to bind to this site, and chromatin immunoprecipitation analysis of the endogenous gene showed that ERalpha occupies this region in response to oestrogens. We conclude that AP-2gamma is a primary oestrogen-responsive gene and suggest that AP-2 proteins may mediate some oestrogenic responses.

Molecular biology experiments allow to obtain reliable data about the expression of different classes of molecules involved in several cellular processes. This information is mostly static and does not give much clue about the causal relationships (i.e., regulation) among the different molecules. A typical scenario is the presence of a set of modulated mRNAs (up or down regulated) along with an over expression of one or more small non-coding RNAs molecules like miRNAs. To computationally identify the presence of transcriptional or post-transcriptional regulatory modules between one or more miRNAs and a set of target modulated genes, we propose a computational pipeline designed to integrate data from multiple online data repositories. The pipeline produces a set of three types of putative regulatory motifs involving coding genes, intronic miRNAs, and transcription factors. We used this pipeline to analyze the results of a set of expression experiments on a melanoma cell line that showed an over expression of miR-214 along with the modulation of a set of 73 other genes. The results suggest the presence of 27 putative regulatory modules involving miR-214, NFKB1, SREBPF2, miR-33a and 9 out of the 73 miR-214 modulated genes (ALCAM, POSTN, TFAP2A, ADAM9, NCAM1, SEMA3A, PVRL2, JAG1, EGFR1). As a preliminary experimental validation we focused on 9 out of the 27 identified regulatory modules that involve miR-33a and SREBF2. The results confirm the importance of the predictions obtained with the presented computational approach.

Nuclear receptors regulate target gene expression in response to steroid and thyroid hormones, retinoids, vitamin D and other ligands. These ligand-dependent transcription factors function by contacting various nuclear cooperating proteins, called coactivators and corepressors, which mediate local chromatin remodeling as well as communication with the basal transcriptional apparatus. Nuclear receptors and their coregulatory proteins play a role in cancer and other diseases, one leading example being the estrogen receptor pathway in breast cancer. Coregulators are often present in limiting amounts in cell nuclei and modifications of their level of expression and/or structure lead to alterations in nuclear receptor functioning, which may be as pronounced as a complete inversion of signaling, i.e. from stimulating to repressing certain genes in response to an identical stimulus. In addition, hemizygous knock-out of certain coactivator genes has been demonstrated to produce cancer-prone phenotypes in mice. Thus, assessment of coactivator and corepressor expression and structure in tumors may turn out to be essential to determine the role of nuclear receptors in cancer and to predict prognosis and response to therapy.

Highlights • miR-21 targets KRIT1. • miR-21 and KRIT1 expression anticorrelate in human breast tumors. • KRIT1 is involved in miR-21-mediated tumor cell growth., miR-21 is overexpressed in tumors and it displays oncogenic activity. Here, we show that expression of miR-21 in primary tumors anticorrelates with KRIT1/CCM1, an interacting partner of the Ras-like GTPase Rap1, involved in Cerebral Cavernous Malformations (CCM). We present evidences that miR-21 silences KRIT1 by targeting its mRNA 3′UTR and that this interaction is involved in tumor growth control. In fact, miR-21 over-expression or KRIT1 knock-down promote anchorage independent tumor cell growth compared to controls, whereas the opposite is observed when anti-miR-21 or KRIT1 overexpression are employed. Our findings suggest that miR-21 promotes tumor cell growth, at least in part, by down-modulating the potential tumor suppressor KRIT1.

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. Cancer Res; 73(13); 4098–111. ©2013 AACR.

Introduction: The therapy of metastatic melanoma (MM) was radically changed by the introduction of BRAF inhibitors (BRAFi). Even if highly effective in the short term, patients invariably develop resistance in the long term. For this reason other inhibitors as well as alternative or complementary therapeutic strategies are being tested in these patients. Among the immune checkpoint targets of clinical importance is PD-1, which is expressed by T cells and which binds to the PD-L1 ligand, which may be expressed by melanoma cells. We and others showed that PD-L1 is an independent negative prognostic marker for patients with MM. Methods: BRAFi-/MEKi-resistant melanoma cell lines were generated by treating cells with increasing concentrations of BRAFi or MEKi. Resistance, viability and aggressiveness were analyzed by MTT, migration and wound healing assays. Results were confirmed using xenograft models. Resistant cell lines were compared using RNAseq to identify enriched genetic pathways involved in the resistance. Luciferase reporter assay analysis was used to study the direct interactions between the PD-L1 and miR-17-5p. Results: By comparing responses to BRAFi in PD-L1+ and PD-L1- variants of the A375 cell line, we found that PD-L1 expression conferred resistance to BRAFi or MEKi. Conversely, silencing of the molecule restored sensitivity to these drugs. Resistant melanoma cell lines acquired PD-L1 expression and were characterized by a specific genetic profile, with the modulation of genes controlling cell movement and immune responses. Consistently, these cells showed a more aggressive behavior both in vitro and in xenograft models. PD-L1 silencing in resistant cells decreased invasive properties and restored expression of HLA-II molecules. PD-L1 up-regulation was only partly dependent on the paradoxical activation of the MAPK pathway, which characterized resistant cells. In addition, we found that resistance to BRAFi and MEKi down-modulated miR-17-5p, which showed an inverse correlation with PD-L1. Transfection of miR-17-5p into BRAFi-resistant cell lines induced the down-modulation of PD-L1, reduced the aggressive behavior of the cells and partially restored sensitivity to BRAFi. Finally, in the plasma of patients with MM, miR-17-5p was inversely correlated with expression of PD-L1 in the tumor tissue. Conclusions: These data demonstrate a direct link between expression of PD-L1 and resistance to BRAFi, as well as to a more aggressive behavior of melanoma cells. Furthermore, we define a novel post-transcriptional circuit responsible for PD-L1 up-regulation, based on a direct interaction between miR-17-5p and PD-L1 mRNA. Lastly, miR-17-5p plasmatic levels show an inverse correlation with PD-L1 expression by tumor cells, suggesting that they may be useful in monitoring disease outcome and drug sensitivity. Citation Format: Davide Brusa, Aureliano Stingi, Valentina Audrito, Francesca Orso, Sara Serra, Roberta Buonincontri, Francesco Neri, Gianna Baroni, Barbara Merelli, Romina Nassini, Daniela Massi, Salvatore Oliviero, Daniela Taverna, Mario Mandalà, Silvia Deaglio. Up-regulation of PD-L1 in melanoma determines resistance to BRAF and MEK inhibitors, induces a more aggressive phenotype and is partially mediated through post-transcriptional mechanisms involving miR-17-5p. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 241.

Review on TFAP2A (transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha)), with data on DNA, on the protein encoded, and where the gene is implicated.

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.

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 (alpha, beta, gamma, delta, and epsilon) that play relevant roles in embryonic development, as demonstrated by the phenotypes of the corresponding knockout mice. Here, we show that AP-2alpha and AP-2gamma 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 xenotransplant 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-2alpha knockdown cells and observed that AP-2alpha 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.

AP-2 proteins are a family of developmentally-regulated transcription factors. They are encoded by five different genes (alpha, beta, gamma, delta, and epsilon) but they share a common structure. AP-2 plays relevant roles in growth, differentiation, and adhesion by controlling the transcription of specific genes. Evidence shows that the AP-2 genes are involved in tumorigenesis and for instance, they act as tumor suppressors in melanomas and mammary carcinomas. Here we investigated the function of the AP-2alpha protein in cancer formation and progression focusing on apoptosis and migration. We introduced AP-2alpha-specific siRNA (as oligos or in retroviruses) in HeLa or MCF-7 human tumor cells and obtained a pronounced down-modulation of AP-2a mRNA and protein levels. In these cells, we observed a significant reduction of chemotherapy-induced apoptosis, migration, and motility and an increase in adhesion suggesting a major role of AP-2a during cancer treatment and progression (migration and invasion). We have data suggesting that migration is, at least in part, regulated by secreted factors. By performing a whole genome microarray analysis of the tumor cells expressing AP-2alpha siRNA, we identified several AP-2alpha-regulated genes involved in apoptosis and migration such as FAST kinase, osteopontin, caspase 9, members of the TNF family, laminin alpha 1, collagen type XII, alpha 1, and adam.

In the complex world of post-transcriptional regulation, miR-214 is known to control in vitro tumor cell move- ment and survival to anoikis, as well as in vivo malignant cell extravasation from blood vessels and lung metastasis formation. miR-214 has also been found to be highly expressed in human melanomas, and to directly and indirectly regulate several genes involved in tumor progression and in the establishment of dis- tant metastases (Penna et al., 2011). In this work, we exploit a computational pipeline integrating data from multiple online data repositories to identify the presence of transcriptional or post-transcriptional regulatory modules involving miR-214 and a set of 73 previously identified miR-214 regulated genes. We identified 27 putative regulatory modules involving miR-214, NFKB1, SREBPF2, miR-33a and 9 out of the 73 miR-214 modulated genes (ALCAM, POSTN, TFAP2A, ADAM9, NCAM1, SEMA3A, PVRL2, JAG1, EGFR1). As a pre- liminary experimental validation we focused on 9 out of the 27 identified regulatory modules that involve two main players, miR-33a and SREBF2. The results confirm the importance of the predictions obtained with the presented computational approach