Long non-coding RNA TINCR suppresses metastatic melanoma dissemination by preventing ATF4 translation (2020)
Figure 4

Figure 4. TINCR re-expression inhibits migration in vitro, tumor growth and metastasis spreading in vivo and overcomes resistance to MEK inhibitors of metastatic melanoma PDXs.

A qRT-PCR analysis of TINCR expression (left) of MM13 and MM2 PDX cells infected with lentiviral vectors expressing full-length TINCR cDNA. PDX cells infected with empty vector (Empty) were used as control. TINCR RNA levels relative to L32P housekeeping gene and control (mean±sd) from three biological replicates are shown. Statistical analyses were performed using Student t -test (***: p<0.001).

B In vitro relative migration (mean±sd) from three independent experiments (biological replicates) is shown in MM13 and MM2 TINCR overexpressing (TINCR) compared to control (Empty) cells. Statistical analyses were performed using Student t -test (***: p<0.001).

C In vitro cell proliferation was assayed 72 hours post infection in Empty and TINCR MM13 and MM2 PDX cells at the indicated time points (1-5 days) using CellTiter-Glo viability assay. Relative proliferation rate (mean±sd) of three independent experiments are shown.

D Drug-response curves showing sensitization to MEK inhibitor Trametinib of MM13 (NRAS-mutant PDX) and to Trametinib and BRAF inhibitor Vemurafenib of TINCR MM2 cells (BRAF-mutant PDX). The Empty (control) and TINCR expressing MM13 cells were treated with increasing concentrations of drug (ranging from 5 to 5000 nM) and cell viability was assayed after 72 hours by CellTiter-Glo viability assay. Error bars represent s.e.m. of three biological replicates. The dose response curve was fit with nonlinear regression (GraphPad Prism).

E Western blot analysis of the expression of the invasive and proliferative phenotype markers EPHA2, EGFR, AXL, PDGFRβ, SOX10, and MITF in TINCR-overexpressing MM13 and MM2 cells. Total protein lysates were extracted from PDX cells 7 days after infection and analyzed by immunoblot assay using the indicated antibodies. Vinculin was used as loading control. This experiment is representative of three independent experiments showing comparable levels of TINCR overexpression (8-10 folds).

F In vivo growth of Empty vector and TINCR cDNA expressing MM13 cells following intradermal injection into NSG mice. Four animals (200,000 cells/mouse) per group were used in each experiment. Tumor growth (mean±sd) was monitored weekly and tumor volume (mean±sd) measured at 4 weeks for primary tumors (originated in the site of injection, left panel) and at 8 weeks for spontaneous metastases which colonized lymph nodes (right panel). The results of two independent experiments are shown. Statistical analyses of control and TINCR-overexpressing groups from both experiments were performed using Student t -test (**: p<0.01).

G Representative pictures of lymph node metastases derived from Empty and TINCR expressing MM13 PDX cells are shown. Hematoxylin-eosin staining of sections of liver and lung metastases derived from Empty and TINCR MM13 PDX cells. Metastasis formation was evaluated 8 weeks after transplantation into NSG mice. Pictures of representative lungs and livers infiltrated with metastases are shown in the inset. Scale bars: 200μm.