Circulating miRNAs signature on breast cancer: the MCC-Spain project.

PURPOSE: To build models combining circulating microRNAs (miRNAs) able to identify women with breast cancer as well as different types of breast cancer, when comparing with controls without breast cancer. METHOD: miRNAs analysis was performed in two phases: screening phase, with a total n = 40 (10 controls and 30 BC cases) analyzed by Next Generation Sequencing, and validation phase, which included 131 controls and 269 cases. For this second phase, the miRNAs were selected combining the screening phase results and a revision of the literature. They were quantified using RT-PCR. Models were built using logistic regression with LASSO penalization. RESULTS: The model for all cases included seven miRNAs (miR-423-3p, miR-139-5p, miR-324-5p, miR-1299, miR-101-3p, miR-186-5p and miR-29a-3p); which had an area under the ROC curve of 0.73. The model for cases diagnosed via screening only took in one miRNA (miR-101-3p); the area under the ROC curve was 0.63. The model for disease-free cases in the follow-up had five miRNAs (miR-101-3p, miR-186-5p, miR-423-3p, miR-142-3p and miR-1299) and the area under the ROC curve was 0.73. Finally, the model for cases with active disease in the follow-up contained six miRNAs (miR-101-3p, miR-423-3p, miR-139-5p, miR-1307-3p, miR-331-3p and miR-21-3p) and its area under the ROC curve was 0.82. CONCLUSION: We present four models involving eleven miRNAs to differentiate healthy controls from different types of BC cases. Our models scarcely overlap with those previously reported.

Plasma-derived extracellular vesicle analysis and deconvolution enable prediction and tracking of melanoma checkpoint blockade outcome.

Immune checkpoint inhibitors (ICIs) show promise, but most patients do not respond. We identify and validate biomarkers from extracellular vesicles (EVs), allowing non-invasive monitoring of tumor- intrinsic and host immune status, as well as a prediction of ICI response. We undertook transcriptomic profiling of plasma-derived EVs and tumors from 50 patients with metastatic melanoma receiving ICI, and validated with an independent EV-only cohort of 30 patients. Plasma-derived EV and tumor transcriptomes correlate. EV profiles reveal drivers of ICI resistance and melanoma progression, exhibit differentially expressed genes/pathways, and correlate with clinical response to ICI. We created a Bayesian probabilistic deconvolution model to estimate contributions from tumor and non-tumor sources, enabling interpretation of differentially expressed genes/pathways. EV RNA-seq mutations also segregated ICI response. EVs serve as a non-invasive biomarker to jointly probe tumor-intrinsic and immune changes to ICI, function as predictive markers of ICI responsiveness, and monitor tumor persistence and immune activation.

Resistance to MAPK Inhibitors in Melanoma Involves Activation of the IGF1R-MEK5-Erk5 Pathway.

Combined treatment of metastatic melanoma with BRAF and MEK inhibitors has improved survival, but the emergence of resistance represents an important clinical challenge. Targeting ERK is a suitable strategy currently being investigated in melanoma and other cancers. To anticipate possible resistance to ERK inhibitors (ERKi), we used SCH772984 (SCH) as a model ERKi to characterize resistance mechanisms in two BRAF V600E melanoma cell lines. The ERKi-resistant cells were also resistant to vemurafenib (VMF), trametinib (TMT), and combined treatment with either VMF and SCH or TMT and SCH. Resistance to SCH involved stimulation of the IGF1R-MEK5-Erk5 signaling pathway, which counteracted inhibition of Erk1/2 activation and cell growth. Inhibition of IGF1R with linsitinib blocked Erk5 activation in SCH-resistant cells and decreased their growth in 3D spheroid growth assays as well as in NOD scid gamma (NSG) mice. Cells doubly resistant to VMF and TMT or to VMF and SCH also exhibited downregulated Erk1/2 activation linked to stimulation of the IGF1R-MEK5-Erk5 pathway, which accounted for resistance. In addition, we found that the decreased Erk1/2 activation in SCH-resistant cells involved reduced expression and function of TGFα. These data reveal an escape signaling route that melanoma cells use to bypass Erk1/2 blockade during targeted melanoma treatment and offer several possible targets whose disruption may circumvent resistance. SIGNIFICANCE: Activation of the IGF1R-MEK5-Erk5 signaling pathway opposes pharmacologic inhibition of Erk1/2 in melanoma, leading to the reactivation of cell proliferation and acquired resistance.

The good and bad faces of the CXCR4 chemokine receptor.

Chemokines are chemotactic cytokines that promote cell migration and activation under homeostatic and inflammatory conditions. Chemokines bind to seven transmembrane-spanning receptors that are coupled to heterotrimeric guanine nucleotide-binding (G) proteins, which are the responsible for intracellularly transmitting the activating signals for cell migration. Hematopoiesis, vascular development, lymphoid organ morphogenesis, cardiogenesis and neural differentiation are amongst the processes involving chemokine function. In addition, immune cell trafficking from bone marrow to blood circulation, and from blood and lymph to lymphoid and inflamed tissues, is tightly regulated by chemokines both under physiological conditions and also in autoimmune diseases. Furthermore, chemokine binding to their receptors stimulate trafficking to and positioning of cancer cells into target tissues and organs during tumour dissemination. The CXCL12 chemokine (also known as stromal-cell derived factor-1α, SDF-1α) plays key roles in hematopoiesis and lymphoid tissue architecture, in cardiogenesis, vascular formation and neurogenesis, as well as in the trafficking of solid and hematological cancer cell types. CXCL12 binds to the CXCR4 receptor, a multi-facetted molecule which tightly mirrors CXCL12 functions in homeostasis and disease. This review addresses the important roles of the CXCR4-CXCL12 axis in homeostasis, specially focusing in hematopoiesis, as well as it provides a picture of CXCR4 as mediator of cancer cell spreading, and a view of the available CXCR4 antagonists in different cancer types.

miR-204-5p and miR-211-5p Contribute to BRAF Inhibitor Resistance in Melanoma.

Melanoma treatment with the BRAF V600E inhibitor vemurafenib provides therapeutic benefits but the common emergence of drug resistance remains a challenge. We generated A375 melanoma cells resistant to vemurafenib with the goal of investigating changes in miRNA expression patterns that might contribute to resistance. Increased expression of miR-204-5p and miR-211-5p occurring in vemurafenib-resistant cells was determined to impact vemurafenib response. Their expression was rapidly affected by vemurafenib treatment through RNA stabilization. Similar effects were elicited by MEK and ERK inhibitors but not AKT or Rac inhibitors. Ectopic expression of both miRNA in drug-naïve human melanoma cells was sufficient to confer vemurafenib resistance and more robust tumor growth in vivo Conversely, silencing their expression in resistant cells inhibited cell growth. Joint overexpression of miR-204-5p and miR-211-5p durably stimulated Ras and MAPK upregulation after vemurafenib exposure. Overall, our findings show how upregulation of miR-204-5p and miR-211-5p following vemurafenib treatment enables the emergence of resistance, with potential implications for mechanism-based strategies to improve vemurafenib responses.Significance: Identification of miRNAs that enable resistance to BRAF inhibitors in melanoma suggests a mechanism-based strategy to limit resistance and improve clinical outcomes. Cancer Res; 78(4); 1017-30. ©2017 AACR.

MicroRNA-125a promotes resistance to BRAF inhibitors through suppression of the intrinsic apoptotic pathway.

Melanoma patients with BRAFV600E -mutant tumors display striking responses to BRAF inhibitors (BRAFi); however, almost all invariably relapse with drug-resistant disease. Here, we report that microRNA-125a (miR-125a) expression is upregulated in human melanoma cells and patient tissues upon acquisition of BRAFi resistance. We show that miR-125a induction confers resistance to BRAFV600E melanoma cells to BRAFi by directly suppressing pro-apoptotic components of the intrinsic apoptosis pathway, including BAK1 and MLK3. Apoptotic suppression and prolonged survival favor reactivation of the MAPK and AKT pathways by drug-resistant melanoma cells. We demonstrate that miR-125a inhibition suppresses the emergence of resistance to BRAFi and, in a subset of resistant melanoma cell lines, leads to partial drug resensitization. Finally, we show that miR-125a upregulation is mediated by TGFß signaling. In conclusion, the identification of this novel role for miR-125a in BRAFi resistance exposes clinically relevant mechanisms of melanoma cell survival that can be exploited therapeutically.

A Blk-p190RhoGAP signaling module downstream of activated Gα13 functionally opposes CXCL12-stimulated RhoA activation and cell invasion.

Activation of the GTPase RhoA linked to cell invasion can be tightly regulated following Gα13 stimulation. We have used a cellular model displaying Gα13-dependent inhibition of RhoA activation associated with defective cell invasion to the chemokine CXCL12 to characterize the molecular players regulating these processes. Using both RNAi transfection approaches and protein overexpression experiments here we show that the Src kinase Blk is involved in Gα13-activated tyrosine phosphorylation of p190RhoGAP, which causes RhoA inactivation and ultimately leads to deficient cell invasion. Characterization of molecular interplays between Gα13, Blk and p190RhoGAP revealed that Blk binds Gα13, and that Blk-mediated p190RhoGAP phosphorylation upon Gα13 activation correlates with weakening of Gα13-Blk association connected to increased Blk-p190RhoGAP assembly. These results place Blk upstream of the p190RhoGAP-RhoA pathway in Gα13-activated cells, overall representing an opposing signaling module during CXCL12-triggered invasion. In addition, analyses with Blk- or Gα13-knockdown cells indicated that Blk can also mediate CXCL12-triggered phosphorylation of p190RhoGAP independently of Gα13. However, even if CXCL12 induces the Blk-mediated GAP phosphorylation, the simultaneous stimulation of the guanine-nucleotide exchange factor Vav1 by the chemokine, as earlier reported, leads to a net increase in RhoA activation. Therefore, when Gα13 is concurrently stimulated with CXCL12 there appears to be sufficient Blk activity to promote adequate levels of p190RhoGAP tyrosine phosphorylation to inactivate RhoA and to impair cell invasiveness.