DUSP1 and SOX2 expression determine squamous cell carcinoma of the salivary gland progression

Abstract

Salivary gland squamous cell carcinomas (SG-SCCs) constitute a rare type of head and neck cancer which is linked to poor prognosis. Due to their low frequency, the molecular mechanisms responsible for their aggressiveness are poorly understood. In this work we studied the role of the phosphatase DUSP1, a negative regulator of MAPK activity, in controlling SG-SCC progression. We generated DUSP1 KO clones in A253 human cells. These clones showed a reduced ability to grow in 2D, self-renew in ECM matrices and to form tumors in immunodeficient mice. This was caused by an overactivation of the stress and apoptosis kinase JNK1/2 in DUSP1^-/+ clones. Interestingly, RNAseq analysis revealed that the expression of SOX2, a well-known self-renewal gene was decreased at the mRNA and protein levels in DUSP1^-/+ cells. Unexpectedly, CRISPR-KO of SOX2 did not recapitulate DUSP1^-/+ phenotype, and SOX2-null cells had an enhanced ability to self-renew and to form tumors in mice. Gene expression analysis demonstrated that SOX2-null cells have a decreased squamous differentiation profile -losing TP63 expression- and an increased migratory phenotype, with an enhanced epithelial to mesenchymal transition signature. In summary, our data indicates that DUSP1 and SOX2 have opposite functions in SG-SCC, being DUSP1 necessary for tumor growth and SOX2 dispensable showing a tumor suppressor function. Our data suggest that the combined expression of SOX2 and DUSP1 could be a useful biomarker to predict progression in patients with SG-SCCs.

Figure 1

DUSP1 loss decreases cell growth and self-renewal in SG-SCC. (A) Western blot illustrating DUSP1 expression in A253 control cells and DUSP1^−/+ pool of cells and clones (Cl1, 5, 9 and 10). (B) Left, Western blot illustrating ERK and JNK phosphorylation in A253 control and DUSP1 KO clones in basal medium (0.5% FBS) or stimulated 30 min with 10% FBS. Right, Bar graphs showing pERK/ERK and pJNK/JNK ratio quantifications (n = 3, t-test). (C) Graph illustrating the fold change growth of control (DUSP1^+/+) and DUSP-1KO clones (n = 3, Two-way ANOVA). (D) Brightfield (upper panel) or H&E stains micrographs of organoid sections (lower panel) illustrating the size and shape of DUSP1^+/+ and DUSP1^−/+ clones after 7 days growing in BME. (E) Violine plot illustrating organoid size (Left) and boxplot illustrating organoids/field in DUSP1^+/+ and DUSP1^−/+ clones (One-way ANOVA). (F) Representative micrographs of organoid sections stained with KI67 (red, upper panel) or Casp3 (lower panel) of DUSP1^+/+ and DUSP1^−/+ clones. (F’) Scatter plot and box plot illustrating the percentage of KI67 or Caspase 3 positive cells per organoid (one-way ANOVA). (G) Graph illustrating the percentage of apoptotic of live cells in organoids (*p = 0.05–0.01; **p < 0.01; ***p < 0.001 and ****p < 0.0001; n = 3, two-way ANOVA). (H) Graph illustrating the percentage of cell through the cell cycle (*p = 0.05–0.01; **p < 0.01; ***p < 0.001 and ****p < 0.0001; n = 3, two-way ANOVA). (I) Representative western blot illustrating the activation of pERK and pJNK in response to BCI (5 uM) over time. (J) Left, Representative brightfield micrographs illustrating A253 organoids treated with DMSO or BCI (5 uM) for 7 days. Right, Violine plot illustrating organoid size (upper) and boxplot illustrating organoids/field (lower) in DMSO of BCI treat A253 cells (n = 3, t-test). Scale bars, 50 µm.

Figure 2

DUSP1 loss impairs tumor growth and triggers squamous differentiation in SG-SCCs. (A) Tumor growth rates after intradermal injection of 100,000 control (black) and DUSP1^−/+ (orange) human A253 cells (mean ± SEM, n = 6 transplants, two-way ANOVA). (B) H&E stains of sections from control of DUSP1^−/+ tumors. (C) Confocal micrographs illustrating Caspase 3 positivity (red) within a6 integrin positive (white) tumor cells. (D) Violine plot illustrating the number of Caspase 3 positive cells per tumor area in DUSP1^+/+ and DUSP1^−/+ tumors (n = 3, One-way ANOVA). (E) Heatmap illustration the DEG between DUSP1^+/+ and DUSP1^−/+ cells. (F) Graph showing the pathways (left) or gene ontology biological processes (right) enriched or depleted in DUSP1^−/+ cells. In bold are highlighted the most relevant findings. (G) Bar graphs showing the increased (upper) expression (Normalized counts) of squamous genes and decreased expression (lower) of mesenchymal genes in DUSP1^−/+ cells. (H) Confocal micrographs illustrating KRT10 positivity (red) within a6 integrin positive (white) tumor cells. (H’) Violin plots quantifying the intensity (right panel) or area (right panel) of KRT10 on DUSP1^+/+ and DUSP1^−/+ tumors (One-way ANOVA). (I) Brightfield (left) and (J) confocal (right) micrographs of Vimentin (brown or red) staining on organoid or tumor sections respectively from DUSP1^+/+ and DUSP1^−/+ cells. a6 integrin staining (white) demarcates the boundary between tumor epithelial cells and tumor stroma (Str). Scale bars, H = 50 µm.

Figure 3

SOX2 expression is lost in DUSP1^−/+ but its over-expression cannot rescue DUSP1^−/+ phenotype. (A) Gene expression of SOX2 in DUSP1^+/+ and DUSP1^−/+ cells. (B) Western blot showing SOX2 protein expression in DUSP1^+/+ and DUSP1^−/+ cells. (C) Confocal micrographs showing SOX2 expression (red) within a6-integrin (white) positive cancer cells. (D) Western blot illustrating the over expression of SOX2. (E) Violine plot illustrating organoid size in DUSP1^+/+ and DUSP1^−/+ clone with or without SOX2 OE (One-way ANOVA). (F) Western blot from DUSP1^+/+ and DUSP1^−/+ organoids with or without SOX2 OE illustrating JNK activity. Scale bars, 100 µm.

Figure 4

SOX2 loss drives tumor growth in SG-SCCs. (A) Western blot showing SOX2 protein expression in control (sgTomato) and SOX2 KO cells (sgSOX2.1 and sgSOX2.2). (B) Graph illustrating the fold change growth of sgTomato and sgSOX2 cells (n = 3, Two-way ANOVA). (C) Brightfield (upper panel) or H&E stains micrographs of organoid sections (lower panel) illustrating the size and shape of sgTomato and sgSOX2 cells after 7 days growing in BME. (D) Violine plot illustrating organoid size (Left) and boxplot illustrating organoids/field in sgTomato and sgSOX2 A253 cells (one-way ANOVA). (E) Confocal micrographs of organoid sections stained with KI67 (red) in organoids grown from sgTomato and sgSOX2 A253 cells. Ecadherin (ECAD, white) delimits the organoid). (E’) Scatter plot illustrating the percentage of KI67 positive cells per organoid (one-way ANOVA). (F) Tumor growth rates after intradermal injection of 100,000 sgTomato (black) and sgSOX2 (green) human A253 cells (mean ± SEM, n = 6 transplants, two-way ANOVA). (G) Confocal micrographs illustrating SOX2 positivity (red) within a6 integrin positive (white) tumor cells (upper). Lower panels illustrate the remaining SOX2 expression (white) in sgSOX2.1 tumors. (H) Left panel, confocal micrographs illustrating pH3 positive cells (red) within a6 integrin positive (white) tumor cells. Right panel, Violin plots representing the number of pH3 positive cells per tumor area in sgTomato and sgSOX2 tumors. (one-way ANOVA). Scale bars, 50 µm.

Figure 5

SOX2 KO SG-SCC suffer an epithelial to mesenchymal transition. (A) Heatmap illustration the DEG between sgTomato and sgSOX2 A253 cells. (B) Graph showing the gene ontology biological processes (left) or pathways (right) enriched or depleted in sgSOX2.2 cells. In bold are highlighted the most relevant findings. (C) Bar graphs showing the decreased expression (Normalized counts) of squamous differentiation genes in sgSOX2.2 cells. (D) Left panel, Confocal micrographs illustrating KRT10 positivity (red) within a6 integrin positive (white) tumor cells. Right panel, Violin plots quantifying the intensity (right panel) or area (right panel) of KRT10 on sgTomato and sgSOX2 tumors (One-way ANOVA). (E) Bar graphs showing the increased expression (Normalized counts) of mesenchymal genes in sgSOX2.2 cells. (F) Micrographs showing vimentin staining in sgTomato and sgSOX2 organoids. (G) Box plot illustrating the % of wound closure in sgTomato and sgSOX2 cells (n = 3, t-test). (H) Left, Confocal micrographs demonstrating a decreased TP63 expression (red) in sgSOX2.2 grown tumors in comparison with sgTomato controls. a6-integrin (white) marks cancer cells; right, Violin plots representing the measurement of TP63 nuclear intensity within the a6-integrin positive cancer cells (one-way ANOVA). I. Bar graphs showing the increased expression (Normalized counts) of self-renewal transcription factors in sgSOX2.2 cells. Scale bars, 50 µm.