Claudia Piovan

microRNA-205 Regulates HER3 in Human Breast Cancer

An increasing amount of experimental evidence shows that microRNAs can have a causal role in breast cancer tumorigenesis as a novel class of oncogenes or tumor suppressor genes, depending on the targets they regulate. HER2 overexpression is a hallmark of a particularly aggressive subset of breast tumors, and its activation is strictly dependent on the trans-interaction with other members of HER family; in particular, the activation of the PI3K/Akt survival pathway, so critically important in tumorigenesis, is predominantly driven through phosphorylation of the kinase-inactive member HER3. Here, we show that miR-205, down-modulated in breast tumors compared with normal breast tissue, directly targets HER3 receptor, and inhibits the activation of the downstream mediator Akt. The reintroduction of miR-205 in SKBr3 cells inhibits their clonogenic potential and increases the responsiveness to tyrosine-kinase inhibitors Gefitinib and Lapatinib, abrogating the HER3-mediated resistance and restoring a potent proapoptotic activity. Our data describe miR-205 as a new oncosuppressor gene in breast cancer, able to interfere with the proliferative pathway mediated by HER receptor family. Our study also provides experimental evidence suggesting that miR-205 can improve the responsiveness to specific anticancer therapies.

MicroRNA Cluster 221-222 and Estrogen Receptor α Interactions in Breast Cancer

BACKGROUND Several lines of evidence have suggested that estrogen receptor alpha (ERalpha)-negative breast tumors, which are highly aggressive and nonresponsive to hormonal therapy, arise from ERalpha-positive precursors through different molecular pathways. Because microRNAs (miRNAs) modulate gene expression, we hypothesized that they may have a role in ER-negative tumor formation. METHODS Gene expression profiles were used to highlight the global changes induced by miRNA modulation of ERalpha protein. miRNA transfection and luciferase assays enabled us to identify new targets of miRNA 206 (miR-206) and miRNA cluster 221-222 (miR-221-222). Northern blot, luciferase assays, estradiol treatment, and chromatin immunoprecipitation were performed to identify the miR-221-222 transcription unit and the mechanism implicated in its regulation. RESULTS Different global changes in gene expression were induced by overexpression of miR-221-222 and miR-206 in ER-positive cells. miR-221 and -222 increased proliferation of ERalpha-positive cells, whereas miR-206 had an inhibitory effect (mean absorbance units [AU]: miR-206: 500 AU, 95% confidence interval [CI]) = 480 to 520; miR-221: 850 AU, 95% CI = 810 to 873; miR-222: 879 AU, 95% CI = 850 to 893; P < .05). We identified hepatocyte growth factor receptor and forkhead box O3 as new targets of miR-206 and miR-221-222, respectively. We demonstrated that ERalpha negatively modulates miR-221 and -222 through the recruitment of transcriptional corepressor partners: nuclear receptor corepressor and silencing mediator of retinoic acid and thyroid hormone receptor. CONCLUSIONS These findings suggest that the negative regulatory loop involving miR-221-222 and ERalpha may confer proliferative advantage and migratory activity to breast cancer cells and promote the transition from ER-positive to ER-negative tumors.

Interplay between microRNAs and the epigenetic machinery: An intricate network

microRNAs take their place into the epigenetic world revealing a complicated network of reciprocal interconnections: not only they are able to control gene expression at a post-transcriptional level, thus representing a new important class of regulatory molecules, but they are also directly connected to the epigenetic machinery through a regulatory loop. Indeed, if epigenetic modifications, such as DNA methylation or histone acetylation, have been demonstrated to affect microRNA expression, and to be potentially responsible for the aberrant miRNA regulation observed in cancer, the other side of the coin is represented by the capacity of microRNAs to control the epigenetic machinery directly targeting its enzymatic components. This review will analyze and describe the regulatory loop interconnecting microRNAs and epigenetics, describing either how epigenetics can affect the miRNome, as well as how epi-miRNAs can control the epigenome, particularly focusing on the alterations observed in human cancer.

Estrogen Mediated-Activation of miR-191/425 Cluster Modulates Tumorigenicity of Breast Cancer Cells Depending on Estrogen Receptor Status

MicroRNAs (miRNAs), single-stranded non-coding RNAs, influence myriad biological processes that can contribute to cancer. Although tumor-suppressive and oncogenic functions have been characterized for some miRNAs, the majority of microRNAs have not been investigated for their ability to promote and modulate tumorigenesis. Here, we established that the miR-191/425 cluster is transcriptionally dependent on the host gene, DALRD3, and that the hormone 17β-estradiol (estrogen or E2) controls expression of both miR-191/425 and DALRD3. MiR-191/425 locus characterization revealed that the recruitment of estrogen receptor α (ERα) to the regulatory region of the miR-191/425-DALRD3 unit resulted in the accumulation of miR-191 and miR-425 and subsequent decrease in DALRD3 expression levels. We demonstrated that miR-191 protects ERα positive breast cancer cells from hormone starvation-induced apoptosis through the suppression of tumor-suppressor EGR1. Furthermore, enforced expression of the miR-191/425 cluster in aggressive breast cancer cells altered global gene expression profiles and enabled us to identify important tumor promoting genes, including SATB1, CCND2, and FSCN1, as targets of miR-191 and miR-425. Finally, in vitro and in vivo experiments demonstrated that miR-191 and miR-425 reduced proliferation, impaired tumorigenesis and metastasis, and increased expression of epithelial markers in aggressive breast cancer cells. Our data provide compelling evidence for the transcriptional regulation of the miR-191/425 cluster and for its context-specific biological determinants in breast cancers. Importantly, we demonstrated that the miR-191/425 cluster, by reducing the expression of an extensive network of genes, has a fundamental impact on cancer initiation and progression of breast cancer cells.

Oncosuppressive role of p53-induced miR-205 in triple negative breast cancer

An increasing body of evidence highlights an intriguing interaction between microRNAs and transcriptional factors involved in determining cell fate, including the well known “genome guardian” p53. Here we show that miR‐205, oncosuppressive microRNA lost in breast cancer, is directly transactivated by oncosuppressor p53.

UCbase & miRfunc: a database of ultraconserved sequences and microRNA function

Four hundred and eighty-one ultraconserved sequences (UCRs) longer than 200 bases were discovered in the genomes of human, mouse and rat. These are DNA sequences showing 100% identity among the three species. UCRs are frequently located at genomic regions involved in cancer, differentially expressed in human leukemias and carcinomas and in some instances regulated by microRNAs (miRNAs). Here we present UCbase & miRfunc, the first database which provides ultraconserved sequences data and shows miRNA function. Also, it links UCRs and miRNAs with the related human disorders and genomic properties. The current release contains over 2000 sequences from three species (human, mouse and rat). As a web application, UCbase & miRfunc is platform independent and it is accessible at http://microrna.osu.edu/.UCbase4.

miR-205 Hinders the Malignant Interplay Between Prostate Cancer Cells and Associated Fibroblasts

AIMS Tumor microenvironment is a strong determinant for the acquisition of metastatic potential of cancer cells. We have recently demonstrated that cancer-associated fibroblasts (CAFs) elicit a redox-dependent epithelial-mesenchymal transition (EMT) in prostate cancer (PCa) cells, driven by cycloxygenase-2/hypoxia-inducible factor-1 (HIF-1)/nuclear factor-κB pathway and enhancing tumor aggressiveness. Here, we investigated the involvement of microRNAs (miRNAs) in tumor-stroma interplay to identify possible tools to counteract oxidative stress and metastasis dissemination. RESULTS We found that miR-205 is the most downmodulated miRNA in PCa cells upon CAF stimulation, due to direct transcriptional repression by HIF-1, a known redox-sensitive transcription factor. Rescue experiments demonstrated that ectopic miR-205 overexpression in PCa cells counteracts CAF-induced EMT, thus impairing enhancement of cell invasion, acquisition of stem cell traits, tumorigenicity, and metastatic dissemination. In addition, miR-205 blocks tumor-driven activation of surrounding fibroblasts by reducing pro-inflammatory cytokine secretion. INNOVATION Overall, such findings suggest miR-205 as a brake against PCa metastasis by blocking both the afferent and efferent arms of the circuit between tumor cells and associated fibroblasts, thus interrupting the pro-oxidant and pro-inflammatory circuitries engaged by reactive stroma. CONCLUSION The evidence that miR-205 replacement in PCa cells is able not only to prevent but also to revert the oxidative/pro-inflammatory axis leading to EMT induced by CAFs sets the rationale for developing miRNA-based approaches to prevent and treat metastatic disease.

A set of NF-κB–regulated microRNAs induces acquired TRAIL resistance in Lung cancer

Significance TRAIL (TNF-related apoptosis-inducing ligand) is a promising antitumor agent effective in a very small subset of lung cancer patients with low toxicity. However, the majority of lung tumors are TRAIL-resistant and very little is known about how tumor cells acquire resistance to TRAIL. Here, we show that continuous exposure to subtoxic concentrations of TRAIL induces NF-κB–dependent up-regulation of miR-21, miR-30c, and miR-100, which by silencing caspase-8, caspase-3, TRAF7, and FoxO3a further strengthens the NF-κB signaling, inducing acquired TRAIL resistance. Our findings imply that combinatory therapies of NF-κB inhibitors and TRAIL might be a useful therapy to improve the response of lung cancer to TRAIL. TRAIL (TNF-related apoptosis-inducing ligand) is a promising anticancer agent that can be potentially used as an alternative or complementary therapy because of its specific antitumor activity. However, TRAIL can also stimulate the proliferation of cancer cells through the activation of NF-κB, but the exact mechanism is still poorly understood. In this study, we show that chronic exposure to subtoxic concentrations of TRAIL results in acquired resistance. This resistance is associated with the increase in miR-21, miR-30c, and miR-100 expression, which target tumor-suppressor genes fundamental in the response to TRAIL. Importantly, down-regulation of caspase-8 by miR-21 blocks receptor interacting protein-1 cleavage and induces the activation of NF-κB, which regulates these miRNAs. Thus, TRAIL activates a positive feedback loop that sustains the acquired resistance and causes an aggressive phenotype. Finally, we prove that combinatory treatment of NF-κB inhibitors and TRAIL is able to revert resistance and reduce tumor growth, with important consequences for the clinical practice.

miR-9 and miR-200 regulate PDGFRβ-mediated endothelial differentiation of tumor cells in triple-negative breast cancer

Organization of cancer cells into endothelial-like cell-lined structures to support neovascularization and to fuel solid tumors is a hallmark of progression and poor outcome. In triple-negative breast cancer (TNBC), PDGFRβ has been identified as a key player of this process and is considered a promising target for breast cancer therapy. Thus, we aimed at investigating the role of miRNAs as a therapeutic approach to inhibit PDGFRβ-mediated vasculogenic properties of TNBC, focusing on miR-9 and miR-200. In MDA-MB-231 and MDA-MB-157 TNBC cell lines, miR-9 and miR-200 promoted and inhibited, respectively, the formation of vascular-like structures in vitro Induction of endogenous miR-9 expression, upon ligand-dependent stimulation of PDGFRβ signaling, promoted significant vascular sprouting of TNBC cells, in part, by direct repression of STARD13. Conversely, ectopic expression of miR-200 inhibited this sprouting by indirectly reducing the protein levels of PDGFRβ through the direct suppression of ZEB1. Notably, in vivo miR-9 inhibition or miR-200c restoration, through either the generation of MDA-MB-231-stable clones or peritumoral delivery in MDA-MB-231 xenografted mice, strongly decreased the number of vascular lacunae. Finally, IHC and immunofluorescence analyses in TNBC specimens indicated that PDGFRβ expression marked tumor cells engaged in vascular lacunae. In conclusion, our results demonstrate that miR-9 and miR-200 play opposite roles in the regulation of the vasculogenic ability of TNBC, acting as facilitator and suppressor of PDGFRβ, respectively. Moreover, our data support the possibility to therapeutically exploit miR-9 and miR-200 to inhibit the process of vascular lacunae formation in TNBC. Cancer Res; 76(18); 5562-72. ©2016 AACR.

Human anti-nucleolin recombinant immunoagent for cancer therapy

Significance Because of its selective expression on the surface of a variety of different cancer cells, but not on their normal counterparts, nucleolin (NCL) represents an attractive target for antineoplastic treatments. However, previously described NCL-targeting molecules, although promising, still suffer from intrinsic limitations. Here, we describe the identification of the first fully human anti-NCL immune-based agent displaying antineoplastic activity against solid tumors, both in vitro and in vivo. This molecule could represent the prototype of a novel class of NCL-targeting drugs with enormous clinical potential as tools for the diagnosis and therapy of a wide range of human cancers. Nucleolin (NCL) is a nucleocytoplasmic protein involved in many biological processes, such as ribosomal assembly, rRNA processing, and mRNA stabilization. NCL also regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and aggressiveness. Interestingly, NCL is expressed on the surface of actively proliferating cancer cells, but not on their normal counterparts. Therefore, NCL is an attractive target for antineoplastic treatments. Taking advantage of phage-display technology, we engineered a fully human single-chain fragment variable, named 4LB5. This immunoagent binds NCL on the cell surface, it is translocated into the cytoplasm of target cells, and it abrogates the biogenesis of NCL-dependent miRNAs. Binding of 4LB5 to NCL on the cell surface of a variety of breast cancer and hepatocellular carcinoma cell lines, but not to normal-like MCF-10a breast cells, dramatically reduces cancer cell viability and proliferation. Finally, in orthotopic breast cancer mouse models, 4LB5 administration results in a significant reduction of the tumor volume without evident side effects. In summary, here we describe, to our knowledge, the first anti-NCL single-chain fragment variable displaying antineoplastic activity against established solid tumors, which could represent the prototype of novel immune-based NCL-targeting drugs with clinical potential as diagnostic and therapeutic tools in a wide variety of human cancers.