Pharmacological targeting Netrin-1 inhibits EMT in cancer [BulkRNA-seq]

Epithelial-to-Mesenchymal transition (EMT) regulates tumour initiation, progression, metastasis and resistance to anti-cancer therapy. Whereas great progress had recently been made in understanding the role and mechanisms that regulate EMT in cancer, no therapeutic strategy to pharmacologically target EMT had been identified so far. Here, we found that Netrin-1 is upregulated in a primary mouse model of skin squamous cell carcinoma (SCCs) presenting spontaneous EMT. Pharmacological inhibition of Netrin-1 by administrating NP137, an anti-Netrin-1 blocking monoclonal antibody currently used in clinical trials in human cancer, decreased the proportion EMT tumour cells in skin SCCs, as well as decreased the number of metastasis and increased the sensitivity of tumour cells to chemotherapy. Single-cell RNA-seq revealed the presence of different EMT states including epithelial, early and late hybrid EMT as well as fully EMT states in control SCCs. In contrast, administration of NP137 prevents the progression of cancer cells towards a late EMT state and sustains tumour epithelial states. ShRNA knockdown (KD) of Netrin-1 and its receptor Unc5b in EPCAM+ tumour cells inhibited EMT in vitro in the absence of stromal cells and regulated a common gene signature promoting tumour epithelial state and restricting EMT. To assess the relevance of these findings to human cancers, we treated mice transplanted with A549 human cancer cell line that undergoes EMT following TGF-b1 administration with NP137. Netrin-1 inhibition decreased EMT in A549 cells in vivo. Altogether, our results identify a new pharmacological strategy to target EMT in cancer opening novel therapeutic interventions for anti-cancer therapy. FACS isolated EPCAM+ Primary mouse skin SCC cell line derived from LKPR tumors was cultured in Modified eagle medium (MEM) (Capricorn scientific, Cat#SP-2002-500ml) supplemented with 10% Foetal bovine serum (FBS) (Serana, Cat#S-FBS- SA-015), 4μg/ml hydrocortisone (Sigma, Cat#H0888) 1% penicillin/streptomycin (100X) (Capricorn 2 scientific, Cat#PS-B), 2mM L-glutamine, 2ml amphotericin B (100X) (Capricorn scientific, Cat#AMP- B) and 500μl T3 (Sigma, Cat#T6397). _x000B_For generation of Netrin-1 and Unc5b Knock-down Epcam+ cell lines, of HEK 293T (Human embryonic kidney 293T) (ATCC) were used as packaging cells. Transfer plasmid pLKO.1-puro, carrying our gene of interest Unc5b or Ntn1 (Sigma), TRC1 as empty vector, PPAX and PMDG packaging plasmids were transfected into the cells with lipofectamine 2000 (Thermo Fisher Scientific) using Opti-Medium (Capricorn scientific). EPCAM+ cell lins was plated and transduced with Opti-medium (Capricorn scientific). Puromycin resistance test has been realized after transduction. For bulk RNA-sequencing experiement, cells were plated 100% Epcam+ and after 6 days, washed in PBS and detached from the cell culture plate with trypsin. Cells were resuspended and washed with PBS supplemented with 2% FBS (Facs buffer) and incubated with BV711- conjugated anti-EPCAM (rat, clone G8.8, BD Bioscience Cat#563134, dilution 1:100) 30min at room temperature and protected from the light. For cell sorting, cells were washed 2 times in Facs buffer and filtered through a 40- um cell trainer (BD Bioscience). Living single YFP+/ EPCAM+ tumour cells were selected by forward and side scatter, doublet discrimination and Hoestch exclusion. Cells were sorted in lysis buffer for RNA extraction from RNeasy micro kit (Qiagen) and RNA quality was evaluated by Bioanalyzer 2100 (Agilent) prior sequencing.