Involvement of Kindlin-1 in cutaneous squamous cell carcinoma

Abstract

Kindler syndrome (KS) is a rare genodermatosis resulting from loss-of-function mutations in FERMT1, the gene that encodes Kindlin-1. KS patients have a high propensity to develop aggressive and metastatic cutaneous squamous cell carcinoma (cSCC). Here we show in non-KS-associated patients that elevation of FERMT1 expression is increased in actinic keratoses compared to normal skin, with a further increase in cSCC supporting a pro-tumorigenic role in this population. In contrast, we show that loss of Kindlin-1 leads to increased SCC tumor growth in vivo and in 3D spheroids, which was associated with the development of a hypoxic tumor environment and increased glycolysis. The metalloproteinase Mmp13 was upregulated in Kindlin-1-depleted tumors, and increased expression of MMP13 was responsible for driving increased invasion of the Kindlin-1-depleted SCC cells. These results provide evidence that Kindlin-1 loss in SCC can promote invasion through the upregulation of MMP13, and offer novel insights into how Kindlin-1 loss leads to the development of a hypoxic environment that is permissive for tumor growth.

Fig. 1. FERMT1 expression in sporadic human cSCC.

Analysis of bulk RNAseq data indicates that FERMT1 levels increase with disease state (A) and DvP quartile (B). FERMT1 RNA levels do not significantly correlate with primary tumour differentiation status (C) (WD well differentiated, MD moderately differentiated, PD poorly differentiated). FERMT1 expression levels do not correlate with primary tumour depth (D) or diameter (E). FERMT1 expression levels are not significantly altered in tumours with annotated perineural or lymphovascular invasion (InV n = 7) when compared to those with these parameters not recorded on the diagnostic pathology records (NR, n = 59) (F). Boxplots are annotated by two-sided Wilcoxon rank-sum test or Kruksal-Wallis test as indicated with P values <= 0.05 indicating a significant difference between sample groups. P-values are not adjusted for multiple testing. The Boxes visualize the interquartile range (IQR) and median, while whiskers show largest and smallest values within 1.5* IQR from upper and lower quartiles. Data beyond the whiskers are deemed outliers.

Fig. 2. Loss of Kindlin-1 leads to increased cSCC invasion and 3D growth.

A 2D SCC cell viability over time assessed with AlamarBlue™. B Viable cells from cSCC spheroids stained with Calcein-AM at end point. C Area of cSCC spheroids after 7 days in culture. D Quantification of percentage of Ki67-positive cells in cSCC spheroids. E cSCC cell adhesion to fibronectin after 2 h. Data obtained from n = 3 (mean ± S. D.). p-values were obtained from two-way (A, B) and one-way ANOVA test (C-E) followed by Tukey post-hoc test; ****p < 0.0001, **p < 0.01 and *p < 0.05.

Fig. 3. Loss of Kindlin-1 leads to increased tumor growth.

A In vivo tumor growth rate (tumor volume/initial tumor volume) following subcutaneous injection of cSCC cell lines. p-values were obtained from two-way ANOVA test followed by Tukey post-hoc test; ****p < 0.0001, ***p < 0.001. B Quantification of percentage of Ki67-positive cells in cSCC tumors. p-values were obtained from one-way ANOVA test followed by Tukey post-hoc test; **p < 0.01 and *p < 0.05. Over-representation of biological processes in the sets of genes significantly downregulated (C) and upregulated (D) in Kin1^−/− tumors.

Fig. 4. Loss of Kindlin-1 increases gene expression related to hypoxia in cSCC.

A mRNA expression of hypoxic markers (Hif1a, Ldha, Egln1, Egln3) relative to Actb in SCC tumors (n = 9). B Quantification of percentage of CAIX-positive cells in cSCC tumors (n = 7). C Quantification of HypoxiTRAK™ staining in cSCC spheroids over time (n = 3). D Quantification of percentage of CAIX-positive cells in cSCC spheroids (n = 5). E mRNA expression of hypoxic markers (Hif1a, Ldha, Egln1, Egln3) relative to Actb in cSCC spheroids (n = 3). F Mitochondria membrane potential in cSCC cells. The values represent the mean±S. D. p-values were obtained from two-tailed unpaired t-test and from two-way ANOVA test followed by Tukey post-hoc test (C only); **p < 0.01 and *p < 0.05.

Fig. 5. Kindlin-1 loss alters matrix metalloproteinase expression.

A Quantification of collagen proportionate area (left) with representative SHG images (right) in Kin1-WT and Kin1^−/− tumors. Green, GFP; cyan, SHG. Scale bar = 250 µm. p-value obtained from two-tailed t-test; ****p < 0.0001 from n = 21 Kin1-WT and n = 23 Kin1^−/− tumors. B–G Expression levels of Mmp1, Mmp2, Mmp3, Mmp7, Mmp9 and Mmp13 in cSCC spheroids relative to Actb, normalized to the average expression of 2D Kin1-WT. H cSCC spheroid invasion in collagen type I, with representative images after 7 days (bottom panel). Scale bar = 400 µm. I Area of cSCC cell degradation of fluorescent gelatin, with representative images (bottom panel) showing intact fluorescent gelatin (green), phalloidin (red) and nuclei (Hoechst). Scale bar = 40 µm. Data obtained from n = 3 (mean±S. D.). p-values proportionate area (left) with representative were obtained from one-way ANOVA test followed by Tukey post-hoc test (B–G) and from two-tailed t-test (H, I); ****p < 0.0001, ***p < 0.001, **p < 0.01 and *p < 0.05.

Fig. 6. Kindlin-1-dependent regulation of MMP13 controls SCC invasion.

A Mmp13 mRNA expression relative to Actb (left panel) and percentage of MMP13-positive cells (right panel) in cSCC tumors (n = 9). B Increased MMP13 expression in cSCC cell lines exposed to hypoxia. Effects of treatment with MMP13 inhibitor WAY 170523 (C) or shRNA knockdown of MMP13 (E) in SCC spheroid invasion in collagen type I, representative images after 7 days. Scale bar = 400 µm. D Mmp13 mRNA expression in shRNA MMP13 SCC cell lines. Data obtained from n = 3 (mean±S. D.). p-values were obtained from one-way ANOVA test followed by Tukey post-hoc test; ***p < 0.001, **p < 0.01 and *p < 0.05.