CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance (2021)
Figure 5

Figure 5. Functional Validation of Genes Involved in BRAFi-Resistance and Relapse

(A) PCA analysis of melanoma cell lines in function of their dedifferentiation states (generated by the webtool http://systems.crump.ucla.edu/dediff/index.php).

(B) SMAD3 expression increases with melanoma cell dedifferentiation. PCA analysis of SMAD3 expression in melanoma cell lines in function of their dedifferentiation states. Scale: red color corresponds to a high SMAD3 expression level (unit of the scale : Log2 FPKM).

(C) SMAD3 expression in these four subtypes of melanoma cells (U, undifferentiated; NC, neural crest-like; T, transitory; M, melanocytic). Number in each group: U = 10, NC = 14, T = 12, M = 17. Error bars reflect mean + s.d.. Multiple comparisons have been done using ordinary one-way ANOVA **: p<0.01, ***p: <0.001.

(D) SMAD3 gain-of-function increases BRAFi-resistance. Determination of BRAFi half maximal inhibitory concentrations (IC50 values) for 501Mel cell lines (Log[Vem] (nM)). Parental 501Mel cells (n=5), 501Mel cells expressing dCas9 and HSF1-p65-MS2 (named here 501Mel 2+, n=5), the 501Mel 2+ cells expressing a control guide (backbone, n=5) and the 501Mel 2+cells overexpressing SMAD3 (n=3 biologically independent experiments). A representative experiment has been chosen. Dotted line is used to determine the IC50 values.

(E) SMAD3 and SLC9A5 gain-of-function increases invasion capability of melanoma cells. Invasion assays for engineered cells lines (melanoma spheroids): CTR; 501Mel 2+, SMAD3; 501Mel 2+cells overexpressing SMAD3. Two other cell lines overexpressing SLC9A5 or BIRC3 have been tested. Explanation for ratio calculation is detailed in Fig. EV3A. Results obtained from two biologically independent experiments. (n=5, 4, 6 and 3 spheroids, respectively). Error bars reflect mean + s.d.. Multiple comparisons have been done using ordinary one-way ANOVA, *: p<0.05, ***p: <0.001.

(F) Characterization of SKMel28 sensitive- and resistant- cell lines (SKMel28S and SKMel28R). Determination of BRAFi half maximal inhibitory concentrations (IC50 values) (Log[Vem] (nM)). A representative experiment has been chosen among two experiments. Dotted line is used to determine the IC50 values.

(G) SMAD3 depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib) on BRAFi-resistant cells (SKMel28R). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib; BRAFi). n=3 biologically independent experiments. Each histogram represents the mean + s.d. ; Multiple comparisons have been done using ordinary one-way ANOVA, **: p<0.01.

(H) SMAD3 depletion (siRNA#1 & #2) decreased cell density and increased BRAFi effect (vemurafenib, BRAFi) on BRAFi-resistant cells (Me1402). CTR for non-targeting siRNA. DMSO for dimethylsulfoxide (solvent of vemurafenib). n=3 biologically independent experiments. Each histogram represents the mean + s.d. ; Multiple comparisons have been done using ordinary one-way ANOVA, *: p<0.05, **: p<0.01.

(I) Validation of SMAD3 knock-down by western-blot experiments in Me1402 cells exposed or not to vemurafenib (BRAFi (Vem) 5µM, 2 days). Cells were exposed to BRAFi 24hr after siRNA transfection. Vemurafenib inhibitory effect on mutated BRAF was evaluated by analysing the phospho-ERK1/2 levels. ERK and HSC70 serves as loading control.

(J) Validation of SMAD3 inhibitor (SIS3, SMAD3i). Effect of SMAD3i (10µM) on the level of phospho-SMAD3 Ser423/425 in response to TGFβ (2ng/mL, 1hr) (or solvent: HCl 4mM + Bovine serum albumin 1mg/mL) in SKMel28R cells (expressing a high endogenous level of SMAD3 mRNA). Serum starved cells (500,000 per well) were pretreated with SMAD3i 10µM (or control solvent) during 2hr before TGFβ addition. SMAD3 and HSC70 serve as loading control for western-blot experiments.

(K) Inhibitory effect of SMAD3i (SIS3) on the SMAD-responsive luciferase activity. Vector encodes the Firefly luciferase reporter gene under the control of a minimal (m)CMV promoter and tandem repeats of the SMAD Binding Element (SBE). Cells (10,000 per well) were pretreated with SMAD3i 10µM or control solvent during 1.5hr, and next cells were exposed to TGFβ 10ng/mL (or solvent: HCl 4mM + Bovine serum albumin 1mg/mL) for 6hr. n=3 biologically independent experiments. Each histogram represents the mean + s.d. ; Bilateral Student test (with non-equivalent variances); TGFβ vs TGFβ+SMAD3i *: p<0.05, **: p<0.01

(L) SMAD3, Phospho-SMAD3 Ser423/425 and BIRC3 expression levels in melanoma cell lines. Four subtypes of melanoma cells (U, undifferentiated; NC, neural crest-like; T, transitory; M, melanocytic) have been compared. 501Mel and M249 cells are melanocytic cells in contrast to dedifferentiated BRAFi-resistant cells (R). Three couples of melanoma cell lines (Sensitive (S) and (R)) have been used to illustrate the SMAD3 and BIRC3 increases in BRAFi-resistant cell lines. HEK293 cells are used as control (kidney). HSC70 serves as loading control for western-blot experiments. Each antibody has been evaluated on individual membrane (explaining the three HSC70, loading controls).

(M) The vemurafenib decreased melanoma cell density. Cell lines have been exposed to Vem (1 or 5 µM, 84h) to define two groups of cell lines (sensitive (blue) and "resistant" (red) cell lines). Normal human melanocytes (NEHM) have been used to evaluate the effect of Vem on normal cells (mean of 3 independent donors). Representative values (mean of biological triplicates) of n=3 biologically independent experiments.

(N) The SMAD3 inhibitor decreased melanoma cell density. Cell lines have been exposed to SMAD3i (0, 3, 10, 15 or 20 µM, 84h). The cell lines defined as S and R to BRAFi in the item 5M are indicated in blue and red. Normal human melanocytes (NEHM) have been used to evaluate the effect of SMAD3i on normal cells (n=3 independent donors). The SMAD3i effect on NHEMs is weak and manageable for these normal cells (NHEM). Representative values (mean of biological triplicates) of n=2 biologically independent experiments.

(O) The chemical inhibition of SMAD3 by SIS3 (SMAD3i) improved current therapy effect (BRAFi+MEKi; Vem 5µM and Cobi 1µM) on BRAFi-resistant cells (SKMel28R, M229R & M238R). Cells have been treated as detailed for Fig. 5M. Representative values (mean of biological triplicates) of n=2 biologically independent experiments.

Data information: Western-blot results are representative of at least two independent experiments. Source data and unprocessed original blots are available in Figure appendix S2 source data.

See also Fig. EV3.