Role of exosome-associated miR-939 in breast cancer metastatic processes 1Di Modica M., 1Casalini P., 1Regondi V., 1Sandri M., 2Baroni S., 2Iorio M.V., 3Zanetti A., 1Tagliabue E. and 1Triulzi T.

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1Molecular Targeting Unit, 2Start Up Unit, Dept. of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori of Milan, Italy. 3Dept. of Biochemestry and Molecular Farmacology, Istituto di Ricerche Farmacologiche "Mario Negri" Milan, Italy.

Background •Metastasis is the leading cause of mortality in cancer patients. During the metastatic spreading, cancer cells acquire the ability to transmigrate through blood vessels by inducing changes and disrupting the endothelial cells junctions. VE-cadherin has emerged as a critical player in the maintenance of endothelial barrier stability and integrity, controlling endothelial cell-cell contacts and, in turn, vascular permeability. Loss of VE-cadherin in human umbilical vein endothelial cells (HUVECs) is induced by invasive breast cancer to increase their transendothelial migration (Hadari M, 2012). •miR-939 is located on chr. 8q in an intronic region of CPSF1 gene; its expression promotes the proliferation and anchorage-independent growth of ovarian cancer cells (Ying X, 2015). miR-939 was found at higher level in the bloodstream of patients with lung adenocarcinomas compared to healthy subjects (Rani S, 2013). Furthermore, miR-939 expression levels have been found to be up-modulated in the endothelial cells co-cultured with colon cancer cells (Zhuang G, 2012). •Triple negative breast cancer (TNBC) represents the most aggressive subtype with the worst prognosis. The high rates of recurrence might be explained by different mechanisms and routes in metastatic spreading as the entrance in the bloodstream rather than in the lymphatic system (Dent R, 2009; Yaman S, 2012).

Since there are evidences of secreted miRNAs, mainly associated with exosomes, able to modulate adhesion molecules favoring the metastatic process (Zhou W, 2014), we hypothesized a possible extracellular role of miR-939-5p (miR-939) in mediating TNBC metastatic spreading by targeting VE-cadherin in endothelial cells.

AIM Study of exosome-associated-miR-939 role on VE-cadherin expression and endothelial barrier integrity

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Conclusions

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2- Effects of VE-cadherin inhibition in HUVECs

VE-cadherin down modulation inhibits tube formation ability and increased permeability of HUVEC monolayers.

miR-939 disrupts endothelial monolayers miR-939 inhibits tube formation

Tube formation assay. Evaluation of HUVECs tube formation on Matrigel after transfection (scr, miR-939) (72h).

Permeability assay. Evaluation of adherens junctions in HUVECs transfected (72h) (scr, miR-939) by confocal microscopy analysis (red: VE-cad, blue: nuclei). Green arrows show loss of VE-cadherin and cell-cell contacts. Scale bars: 10 μm. Monolayer permeability was evaluated by measuring the amount of rhodamine-labeled dextran in the lower chamber of a Boyden chamber with HUVECs monolayer grown on 0.4 um filter transfected with miR-939 mimic or scr. (* p<0.05 by Student's t-test).

• miR-939 efficiently destroys the integrity of endothelial barrier leading to an increased monolayer permeability by targeting VE-cadherin in cellular adherens junctions. • Exosomes-associated miR-939 are absorbed by endothelial cells and the derived disruption of adherens junctions in HUVECs monolayers favours transendothelial migration of BC cells. • In human sample, tumors with high miR-939 levels and positive lymph nodes are 9 times more likely to relapse than negative tumors. Taken together our data identify miR-939 as a key player in the entry of cancer cells into the blood circulation and provide a potential mechanism underlying the higher rates of recurrence and mortality in TNBC.

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VE-cadherin is a direct target of miR-939 miR-939 down modulates VE-cadherin

Five miR-target prediction tools (DIANAmt, miRanda, miRDB, miRWalk, TargetScan) in the miR-Walk database assigned to VE-cadherin the highest prediction score as miR-939 putative target.

miR-939 regulates VE-cadherin expression in HUVECs by direct interaction with VE-cadherin-3’UTR.

1- Target validation

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VE-cadherin evaluation in HUVECs transfected with miR-939 mimic or scr (72h) by WB and confocal microscopy analysis (red: VE-cad, blue: nuclei). Scale bars: 100 μm.

Luciferase Assay. Relative luciferase activity in HEK293 cells transfected with miR-939 mimic or scr as control and pmiR-GLO containing wt or mutated CDH5-3'UTR. (Data are expressed as ratio on scr. ** p<0.01 by Student’s t-test).

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3- Effects of exosomes-associated miR-939 on endothelial barrier integrity

miR-939 is present in exosomes of transfected MDAMB231 Exosomes uptake in HUVECs

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Uptake of exosomes-associated miR-939 in HUVECs allows BC cells to transmigrate through endothelial barrier as a result of adherens junctions impairment.

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Exosomes-associated miR-939 down modulates VE-cadherin

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miR-939 expression levels were analyzed by qRT-PCR in exosomes purified from transfected MDAMB231 ( *** p<0.001 by Student’s t-test). Exosomes derived from MDAMB231 wt or transfected (scr, miR-939) were characterized by WB. MDAMB231 cell lysate was used as control.

Exosomes uptake was assessed by immunofluorescence confocal acquisition after o.n. treatment of HUVECs with PKH67-labelled exosomes (green). (red: Ve-cad, blue: nuclei). Scale bar: 10 μm. The intracellular levels of miR-939 and pre-miR-939 in HUVECs were analyzed by qRT-PCR after 72h treatment with exosomes (*** p<0.001 by Student’s t-test).

VE-cadherin expression in HUVECs treated with exosomes (48h) was evaluated by WB and confocal microscopy analysis (red: Ve-cad, blue: nuclei). Scale bars: 10 μm.

miR-939 scr miR-939 scr

Transendothelial migration assay. Endothelial barrier integrity of HUVECs monolayers transfected (A) or treated with exosomes containing miR-939 (B) was investigated. MDAMB231-GFP cells (231-GFP) were seeded on HUVECs monolayer, grown on Boyden chamber with 8µm filters, and let migrate for 7h. Results, by IF microscope, are expressed as area occupied by cells on the bottom of the transwell, as evaluated by digital image analysis (ImagePro Plus).

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4- Analysis of miR-939 expression in human samples and prognostic significance

miR-939 expression interacts with lymph node status in predicting bad prognosis in TNBC.

N:513; Basal:98, HER2:57, LumA:231; LumB:127

Basal-like BCs have higher miR-939 levels

miR-939 expression levels were evaluated in samples of the public available dataset The Cancer Genome Atlas (TCGA) according to PAM50 classification. (** p<0.01, *** p<0.001 by Student’s t-test).

miR-939 prognostic significance in relation to lymph node status

Multivariate proportional hazard analysis of disease free survival (DFS).

HR 95% C.I. p-value

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miR-/N+ 6.65 1.12-39.35 0.037

miR+/N- 2.73 0.42-17.31 0.287

miR+/N+ 9.04 1.60-51.09 0.013

Age, >50 0.35 0.98-1.29 0.115

Multifocality, yes 4.30 1.24- 4.94 0.022

Lacunea, yes 4.60 1.21-17.45 0.025

A cohort of 67 TNBCs from patients treated at Fondazione IRCCS Istituto Nazionale dei Tumori of Milan, was analyzed for miR-939 expression by qRT-PCR: • miR-939 expression was not associated with other clinico-pathological variables (lymph node status (N), lacunae, multifocality, age, grade and size). • Patients with high miR-939 expression in their tumors showed a borderline statistical significance towards worse prognosis (DFS, p=0.0934).

Bibliography • Dent R., et al. Pattern of metastatic spread in triple-negative breast cancer. Breast Cancer Res Treat 2009. • Haidari M., et al. Intergin α2β1 mediates tyrosine phosphorylation of vascular endothelial cadherin induced by invasive breast cancer cells. J Biol Chem 2012. • Rani S., et al. Global analysis of serum microRNAs as potential biomarkers for lung adenocarcinomas. Cancer Biol Ther 2013. •Yaman S., et al. Lymphatic and capillary invasion patterns in triple negative breast cancer. Am Surg 2012. • Ying X., et al. MiR-939 promotes the proliferation of human ovarian cancer cells by repressing APC2 expression. Biochim Biophys Acta 2015. • Zhou W., et al. Cancer-secreted miR-105 destroys vascular endothelial barriers to promote metastasis. Cancer Cell 2014. • Zhuang G., et al. Tumor-secreted miR-9 promotes endothelial cell migration and angiogenesis by activating the JAK-STAT pathway. EMBO J 2012.

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Kaplan-Meier analysis. DFS survival was evaluated according to miR-939 expression and N status. p-value by Log-Rank test.

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