CRISPR screens identify tumor-promoting genes conferring melanoma cell plasticity and resistance (2021)
Figure 7

Figure 7. SMAD3 Drives Phenotype Switching and Resistance to Melanoma Therapies

(A) SMAD3 signature has been established by comparing BRAFi-resistance genes and SMAD3-regulated genes identified by chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq) (Ramachandran et al, 2018).

(B) Basal SMAD3-signature in six melanoma cell lines (S for sensitive to BRAFi and R for Resistant). n=2 biologically independent experiments. Each histogram represents the median with interquartile range.

(C) Inducibility of SMAD3-signature in six melanoma cell lines exposed to TGF-β (10 ng/mL, 48h). Data were normalized to cell lines exposed to solvent (4mM HCl + 1mg/mL human BSA). Values obtained with the TGF-β stimulated M238R cell line have been set to 1. n=2 biologically independent experiments. Each histogram represents the median with interquartile range.

(D) SMAD3-signature discriminates differentiation states of 53 melanoma cell lines (Tsoi et al, 2018). The SMAD3-signature in four subgroups of melanoma cell lines. Each point represents a cell line (n= 17, 12, 14 and 10 respectively for melanocytic, transitory, neural crest-like and undifferentiated cell lines); Each histogram represents the median with interquartile range; Multiple comparisons have been done using ordinary one-way ANOVA; **: p<0.01, ***: p<0.001.

(E) Heatmap depicting mRNA levels of SMAD3-signature in BRAF(V600E) non-treated melanoma patients (dataset from TCGA; SKCM, BRAF(V600E) mutated: n=118). Three pigmentation genes (MITF, MLANA and TYR) have been added to highlight the differentiation states of tumors. ~20% of tumors are considered as dedifferentiated tumors (red box) with a high SMAD3-signature. Scale corresponds to Z scores.

(F) SMAD3-signature in pre-treatment biopsies from responders and non-responders to anti-PD-1 treatment (from the Hugo's cutaneous melanoma cohort, n=15 and 13 respectively) (Hugo et al, 2016). Each histogram represents the median with interquartile range, one tailed Mann-Whitney test; *: p=0.0324.

(G) SMAD3-signature in two groups (before BRAFi treatment or during relapse) of V600E patients from the Rizos's cohort (Rizos et al, 2014). (14 on 16 patients displayed an upregulation of SMAD3- signature during relapse).

(H) SMAD3 depletion decreases expression of SMAD3-regulated genes. SMAD3 knock-down by siRNA decreased mRNA expression of BRAFi-resistance genes in SKMel28R and Me1402. NRP1 mRNA was not detected in our experimental conditions. n=3 biologically independent experiments. Each histogram represents the mean + s.d. ; Bilateral Student test (with non-equivalent variances) *: p<0.05, **: p<0.01, ***: p<0.001. Dotted line highlights the value of 1.

(I) SMAD3 signature overlapped with mesenchymal signature in melanoma tumors (TCGA, n=459) (Akbani et al, 2015). Melanocytic signature highlights the differentiation states of tumors (Corre et al, 2018). SMAD3 signature and mesenchymal signature correlate in melanoma tumors (TCGA, n=459) (Mak et al, 2016). Scale corresponds to Z scores.

(J) The SMAD3 signature overlaps with the glioblastoma mesenchymal subtype (Jin et al, 2017). Scale corresponds to Z scores.

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