The actin modulator hMENA regulates GAS6‐AXL axis and pro‐tumor cancer/stromal cell cooperation

Abstract The dynamic interplay between cancer cells and cancer‐associated fibroblasts (CAFs) is regulated by multiple signaling pathways, which can lead to cancer progression and therapy resistance. We have previously demonstrated that hMENA, a member of the actin regulatory protein of Ena/VASP family, and its tissue‐specific isoforms influence a number of intracellular signaling pathways related to cancer progression. Here, we report a novel function of hMENA/hMENAΔv6 isoforms in tumor‐promoting CAFs and in the modulation of pro‐tumoral cancer cell/CAF crosstalk via GAS6/AXL axis regulation. LC‐MS/MS proteomic analysis reveals that CAFs that overexpress hMENAΔv6 secrete the AXL ligand GAS6, favoring the invasiveness of AXL‐expressing pancreatic ductal adenocarcinoma (PDAC) and non‐small cell lung cancer (NSCLC) cells. Reciprocally, hMENA/hMENAΔv6 regulates AXL expression in tumor cells, thus sustaining GAS6‐AXL axis, reported as crucial in EMT, immune evasion, and drug resistance. Clinically, we found that a high hMENA/GAS6/AXL gene expression signature is associated with a poor prognosis in PDAC and NSCLC. We propose that hMENA contributes to cancer progression through paracrine tumor–stroma crosstalk, with far‐reaching prognostic and therapeutic implications for NSCLC and PDAC.

. Clinical characteristics of NSCLC tumor samples relative to L-CAFs primary cultures

Appendix Figure Legends
Appendix Figure S1 A. Representative images of IF analysis of P-CAFs, L-CAF and L-NFs stained with FAP Ab (green). Nuclei were stained with DAPI. Scale bar: 50 μm B,C. Real time qRT-PCR analysis of PDGFRβ (B) and EpCAM (C) in P-CAFs and L-CAFs and in cancer cell lines (PANC-1, A549, H1650).
D. Real time qRT-PCR analysis of ENAH (hMENA) expression in in P-CAFs and L-CAFs.
Appendix Figure S2 A C. Representative immunoblot analysis of EpPDAC and CAF PDAC with E-cadherin, Pan-hMENA and hMENA isoform specific antibodies hMENA 11a and hMENAΔv6. Actin was used as a loading control.
Appendix Figure S3 A. Representative immunoblot of hMENA/hMENAv6 expression levels in fibroblasts from "non tumoral" lung tissue obtained from patient #17, (L#17 DFs) and paired CAFs obtained from lung cancer tissue (L#17 CAFs) (left panel). Quantified data by densitometry represented as fold change of hMENAv6/ACTIN ratio in three different paired DF and CAF samples preparations (CAFs: #13,17,20) (right panel). Data are expressed as mean ± SD. P values were calculated by two-sided Student's t-test. *P< 0.05.
B. Immunoblot of hMENAv6 expression levels in L-CAFs (n=17). Patients from whom CAF were derived is indicated (#). HSP70 was used as loading control.
C. Representative IHC images of primary NSCLC tissues of patients #9 and #20 stained with Pan-hMENA mAb. Insets are higher magnification images of the indicated area showing that hMENA(t), is heterogeneously expressed in stromal cells with a morphology compatible with CAF as indicated by the arrows. Scale bar: 100 μm, inset: 50 μM.
Appendix Figure S4 Top. Shown are the correlations of hMENA (ENAH) mRNA expression withα a-SMA (ACTA2) and FAP mRNA expression in primary NSCLC fibroblast cells (n=15). Pearson's rho values are shown.
Bottom. Dotplots showing the mRNA expression of a-SMA (ACTA2) and FAP in primary NSCLC fibroblast cells (n=15). The samples were stratified into two groups on the basis of the hMENA mRNA levels, using as cut-off the median hMENA expression value as follows: low, below the median value; high, above the median value. Shown in each dotplot are the mean value (red dot), and standard deviations (vertical line). Statistical significance was calculated by two-tailed Student's t-test. P values are shown.
Appendix Figure S5 A. Representative immunoblot analysis of FAP expression in P-NFs and P-CAFs. TUBULIN was used as a loading control.
B. Representative images of collagen gel contraction of P-and L-NF and P-#138 and L-#189 CAFs.
C. Representative image of gel zymography of MMP2 activity in P-NF and P-CAFs #36. Similar results were obtained in two independent experiments. D. Quantification by ELISA of MMP-2 secreted level in the CM of pancreatic normal fibroblasts P-NF), and PDAC CAFs (P-CAF). Representative results from 2 independent experiments are shown. Data are expressed as mean ± SD. P value was calculated by two-sided Student's t-test. **P< 0.01.
C. Representative images and quantification of gel zymography of P-and L-NFs, P-CAFs (#110) and L-CAFs (#400) transfected with control or hMENA v6 expressing vectors, showing an increased MMP2 activity in fibroblasts overexpressing hMENAv6 isoform compared to control cells (CNT, set as 100).