The lncRNAMALAT1-WTAP axis: a novel layer of EMT regulation in hypoxic triple-negative breast cancer.

Early metastatic disease development is one characteristic that defines triple-negative breast cancer (TNBC) as the most aggressive breast cancer (BC) subtype. Numerous studies have identified long non-coding RNAs (lncRNA) as critical players in regulating tumor progression and metastasis formation. Here, we show that MALAT1, a long non-coding RNA known to promote various features of BC malignancy, such as migration and neo angiogenesis, regulates TNBC cell response to hypoxia. By profiling MALAT1-associated transcripts, we discovered that lncRNA MALAT1 interacts with the mRNA encoding WTAP protein, previously reported as a component of the N6-methyladenosine (m6A) modification writer complex. In hypoxic conditions, MALAT1 positively regulates WTAP protein expression, which influences the response to hypoxia by favoring the transcription of the master regulators HIF1α and HIF1ß. Furthermore, WTAP stimulates BC cell migratory ability and the expression of N-Cadherin and Vimentin, hallmarks of epithelial-to-mesenchymal transition (EMT). In conclusion, this study highlights the functional axis comprising MALAT1 and WTAP as a novel prognostic marker of TNBC progression and as a potential target for the development of therapeutic approaches for TNBC treatment.

ID4-dependent secretion of VEGFA enhances the invasion capability of breast cancer cells and activates YAP/TAZ via integrin ß3-VEGFR2 interaction.

Understanding the mechanisms of breast cancer cell communication underlying cell spreading and metastasis formation is fundamental for developing new therapies. ID4 is a proto-oncogene overexpressed in the basal-like subtype of triple-negative breast cancer (TNBC), where it promotes angiogenesis, cancer stem cells, and BRACA1 misfunction. Here, we show that ID4 expression in BC cells correlates with the activation of motility pathways and promotes the production of VEGFA, which stimulates the interaction of VEGFR2 and integrin ß3 in a paracrine fashion. This interaction induces the downstream focal adhesion pathway favoring migration, invasion, and stress fiber formation. Furthermore, ID4/ VEGFA/ VEGFR2/ integrin ß3 signaling stimulates the nuclear translocation and activation of the Hippo pathway member's YAP and TAZ, two critical executors for cancer initiation and progression. Our study provides new insights into the oncogenic roles of ID4 in tumor cell migration and YAP/TAZ pathway activation, suggesting VEGFA/ VEGFR2/ integrin ß3 axis as a potential target for BC treatment.

Non-Coding RNAs in the Crosstalk between Breast Cancer Cells and Tumor-Associated Macrophages.

Non-coding RNAs (ncRNAs) play a pivotal role in regulating the tumor microenvironment (TME) by controlling gene expression at multiple levels. In tumors, ncRNAs can mediate the crosstalk between cancer cells and other cells in the TME, such as immune cells, stromal cells, and endothelial cells, influencing tumor development and progression. Tumor-associated macrophages (TAMs) are among the most abundant inflammatory cells infiltrating solid cancers that promote tumorigenesis, and their infiltration correlates with a poor prognosis in many tumors. Cancer cells produce different ncRNAs that orchestrate TAM recruitment and polarization toward a tumor-promoting phenotype. Tumor-reprogrammed macrophages shape the TME by promoting angiogenesis and tissue remodeling, and suppressing the anti-tumor activity of adaptive immune cells. TAMs can also produce ncRNA molecules that boost cancer cell proliferation and direct their phenotype and metabolic changes facilitating cancer progression and metastasis. This review will focus on the crosstalk between cancer cells and TAMs mediated by microRNAs and long non-coding RNAs during breast cancer (BC) initiation and progression.

Secreted Non-Coding RNAs: Functional Impact on the Tumor Microenvironment and Clinical Relevance in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is a subtype of breast carcinoma characterized by poor prognosis and high rate of metastasis. Current treatment is based on chemo- and/or radiotherapy and surgery. TNBC is devoid of estrogen, progesterone and HER2 receptors. Although precision medicine has come a long way to ameliorate breast cancer disease management, targeted therapies for the treatment of TNBC patients are still limited. Mounting evidence has shown that non-coding RNAs (ncRNAs) drive many oncogenic processes at the basis of increased proliferation, invasion and angiogenesis in TNBC, strongly contributing to tumor progression and resistance to treatments. Many of these ncRNAs are secreted in the tumor microenvironment (TME) and impinge on the activity of the diverse immune and stromal cell types infiltrating the TME. Importantly, secreted ncRNAs may be detected as circulating molecules in serum/plasma from cancer patients and are emerging a promising diagnostic/therapeutic tools in TNBC. This review aims to discuss novel insights about the role of secreted circulating ncRNAs in the intercellular communication in the tumor microenvironment and their potential clinical use as diagnostic and prognostic non-invasive biomarkers in TNBC.

MALAT1-dependent hsa_circ_0076611 regulates translation rate in triple-negative breast cancer.

Vascular Endothelial Growth Factor A (VEGFA) is the most commonly expressed angiogenic growth factor in solid tumors and is generated as multiple isoforms through alternative mRNA splicing. Here, we show that lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) and ID4 (inhibitor of DNA-binding 4) protein, previously referred to as regulators of linear isoforms of VEGFA, induce back-splicing of VEGFA exon 7, producing circular RNA circ_0076611. Circ_0076611 is detectable in triple-negative breast cancer (TNBC) cells and tissues, in exosomes released from TNBC cells and in the serum of breast cancer patients. Circ_0076611 interacts with a variety of proliferation-related transcripts, included MYC and VEGFA mRNAs, and increases cell proliferation and migration of TNBC cells. Mechanistically, circ_0076611 favors the expression of its target mRNAs by facilitating their interaction with components of the translation initiation machinery. These results add further complexity to the multiple VEGFA isoforms expressed in cancer cells and highlight the relevance of post-transcriptional regulation of VEGFA expression in TNBC cells.

New Molecular Mechanisms and Clinical Impact of circRNAs in Human Cancer.

Next generation RNA sequencing techniques, implemented in the recent years, have allowed us to identify circular RNAs (circRNAs), covalently closed loop structures resulting in RNA molecules that are more stable than linear RNAs. This class of non-coding RNA is emerging to be involved in a variety of cell functions during development, differentiation, and in many diseases, including cancer. Among the described biological activities, circRNAs have been implicated in microRNA (miRNA) sequestration, modulation of protein-protein interactions and regulation of mRNA transcription. In human cancer, circRNAs were implicated in the control of oncogenic activities such as tumor cell proliferation, epithelial-mesenchymal transition, invasion, metastasis and chemoresistance. The most widely described mechanism of action of circRNAs is their ability to act as competing endogenous RNAs (ceRNAs) for miRNAs, lncRNAs and mRNAs, thus impacting along their axis, despite the fact that a variety of additional mechanisms of action are emerging, representing an open and expanding field of study. Furthermore, research is currently focusing on understanding the possible implications of circRNAs in diagnostics, prognosis prediction, effectiveness of therapies and, eventually, therapeutic intervention in human cancer. The purpose of this review is to discuss new knowledge on the mechanisms of circRNA action, beyond ceRNA, their impact on human cancer and to dissect their potential value as biomarkers and therapeutic targets.

miR-15/107 microRNA Gene Group: Characteristics and Functional Implications in Cancer.

The miR-15/107 group of microRNAs (miRNAs) encloses 10 annotated human members and is defined based on the presence of the sequence AGCAGC near the mature miRNAs' 5' end. Members of the miR-15/107 group expressed in humans are highly evolutionarily conserved, and seven of these miRNAs are widespread in vertebrate species. Contrary to the majority of miRNAs, which recognize complementary sequences on the 3'UTR region, some members of the miR-15/107 group are peculiarly characterized by the ability to target the coding sequence (CDS) of their target mRNAs, inhibiting translation without strongly affecting their mRNA levels. There is compelling evidence that different members of the miR-15/107 group regulate overlapping lists of mRNA targets but also show target specificity. The ubiquitously expressed miR-15/107 gene group controls several human cellular pathways, such as proliferation, angiogenesis, and lipid metabolism, and might be altered in various diseases, such as neurodegenerative diseases and cancer. Intriguingly, despite sharing the same seed sequence, different members of this family of miRNAs may behave as oncomiRs or as tumor suppressor miRNAs in the context of cancer cells. This review discusses the regulation and functional contribution of the miR-15/107 group to the control of gene expression. Moreover, we particularly focus on the contribution of specific miR-15/107 group members as tumor suppressors in breast cancer, reviewing literature reporting their ability to function as major controllers of a variety of cell pathways and to act as powerful biomarkers in this disease.

Paracrine Signaling from Breast Cancer Cells Causes Activation of ID4 Expression in Tumor-Associated Macrophages.

BACKGROUND: Tumor-associated macrophages (TAMs) constitute a major portion of the leukocyte infiltrate found in breast cancer (BC). BC cells may reprogram TAMs in a pro-angiogenic and immunosuppressive sense. We previously showed that high expression of the ID4 protein in triple-negative BC cells leads to the induction of a proangiogenic program in TAMs also through the downregulation of miR-107. Here, we investigated the expression and function of the ID4 protein in TAMs. METHODS: Human macrophages obtained from peripheral blood-derived monocytes (PBDM) and mouse RAW264.7 cells were used as macrophage experimental systems. ID4-correlated mRNAs of the TCGA and E-GEOD-18295 datasets were analyzed. RESULTS: We observed that BC cells determine a paracrine induction of ID4 expression and activation of the ID4 promoter in neighboring macrophages. Interestingly, ID4 expression is higher in macrophages associated with invasive tumor cells compared to general TAMs, and ID4-correlated mRNAs are involved in various pathways that were previously reported as relevant for TAM functions. Selective depletion of ID4 expression in macrophages enabled validation of the ability of ID4 to control the expression of YAP1 and of its downstream targets CTGF and CYR61. CONCLUSION: Collectively, our results show that activation of ID4 expression in TAMs is observed as a consequence of BC cell paracrine activity and could participate in macrophage reprogramming in BC.

PI3K Inhibitors Curtail MYC-Dependent Mutant p53 Gain-of-Function in Head and Neck Squamous Cell Carcinoma.

PURPOSE: Mutation of TP53 gene is a hallmark of head and neck squamous cell carcinoma (HNSCC) not yet exploited therapeutically. TP53 mutation frequently leads to the synthesis of mutant p53 proteins with gain-of-function activity, associated with radioresistance and high incidence of local recurrences in HNSCC. EXPERIMENTAL DESIGN: Mutant p53-associated functions were investigated through gene set enrichment analysis in the Cancer Genome Atlas cohort of HNSCC and in a panel of 22 HNSCC cell lines. Mutant p53-dependent transcripts were analyzed in HNSCC cell line Cal27, carrying mutant p53H193L; FaDu, carrying p53R248L; and Detroit 562, carrying p53R175H. Drugs impinging on mutant p53-MYC-dependent signature were identified interrogating Connectivity Map (https://clue.io) derived from the Library of Integrated Network-based Cellular Signatures (LINCS) database (http://lincs.hms.harvard.edu/) and analyzed in HNSCC cell lines and patient-derived xenografts (PDX) models. RESULTS: We identified a signature of transcripts directly controlled by gain-of-function mutant p53 protein and prognostic in HNSCC, which is highly enriched of MYC targets. Specifically, both in PDX and cell lines of HNSCC treated with the PI3Kα-selective inhibitor BYL719 (alpelisib) the downregulation of mutant p53/MYC-dependent signature correlates with response to this compound. Mechanistically, mutant p53 favors the binding of MYC to its target promoters and enhances MYC protein stability. Treatment with BYL719 disrupts the interaction of MYC, mutant p53, and YAP proteins with MYC target promoters. Of note, depletion of MYC, mutant p53, or YAP potentiates the effectiveness of BYL719 treatment. CONCLUSIONS: Collectively, the blocking of this transcriptional network is an important determinant for the response to BYL719 in HNSCC.