Increased invasive behaviour in cutaneous squamous cell carcinoma with loss of basement-membrane type VII collagen

Abstract

Type VII collagen (ColVII) is the main component of anchoring fibrils, attachment structures within the lamina densa of the basement membrane that are responsible for attachment of the epidermis to the dermis in skin. Mutations in the human ColVII gene, COL7A1, cause the severe inherited blistering disorder recessive dystrophic epidermolysis bullosa (RDEB) affecting skin and mucosae, associated with a greatly increased risk of skin cancer. In this study, we examined the effect of loss of ColVII on squamous cell carcinoma (SCC) tumourigenesis using RNAi in a 3D organotypic skin model. Our findings suggest that loss of ColVII promotes SCC migration and invasion as well as regulating cell differentiation with evidence for concomitant promotion of epithelial-mesenchymal transition (EMT). Immunostaining of RDEB skin and a tissue array of sporadic cutaneous SCCs confirmed that loss of ColVII correlates with decreased involucrin expression in vivo. Gene-expression-array data and immunostaining demonstrated that loss of ColVII increases expression of the chemokine ligand-receptor CXCL10-CXCR3 and downstream-associated PLC signalling, which might contribute to the increased metastatic potential of SCCs with reduced or absent ColVII expression. Together, these findings may explain the aggressive behaviour of SCCs in RDEB patients and may also be relevant to non-RDEB skin cancer, as well as other tumours from organs where ColVII is expressed.

Fig. 1.

Specific knockdown of ColVII in cutaneous SCC by siRNA. (A) WB analysis of ColVII from conditioned media of MET1 (left panel) and SCC-IC1 (right panel) cells, 4 days post-transfection with a pool of siRNAs targeting ColVII mRNA (siCOL7) or a pool of non-targeting siRNAs (siC). Laminin 332 (Lam332) WB was performed to demonstrate equal protein loading. (B) Timecourse expression of ColVII over a period of 10 days after siRNA transfection in MET1 cells. The upper panel shows a WB of conditioned media and cell extracts from siC (lane 1) and siCOL7 (lane 2) cells at days 2 to 10 post-transfection. Lam332 and GAPDH WBs were included as internal controls. The bottom panel shows densitometry analysis after normalisation against the respective loading controls, demonstrating that a significant ColVII knockdown is maintained up to day 8 in cells, or day 10 in conditioned media, post-transfection. (C) The upper panel shows a WB of conditioned media from siC, siCOL7, si1, si2, si3 and si4 MET1 cells at day 4 post-transfection. The lower panel shows densitometry analysis after normalisation against the loading control, demonstrating that all the siRNAs from the pool induce significant ColVII knockdown. (D) WB analysis of conditioned media from siC and siCOL7 MET1 cells transfected with 20 to 2.5 nM final concentration of siRNA (lanes si20, si12.5, si7.5 and si2.5, respectively). The position of the bands corresponding to full-length 290 kDa ColVII (ColVII-FL), 145 kDa NC1 domain of ColVII (ColVII-NC1) and the 160 kDa reduced (cleaved) α3 chain (α3 chain-r) of Lam332 is indicated.

Fig. 2.

Loss of ColVII in cutaneous SCC increases in vitro cell migration, which can be rescued by secreted ColVII. (A) The upper panel shows representative pictures of an in vitro scratch assay using siC and siCOL7 MET1 cells, taken before (t0) and 36 hours (t36) after migration. The lower panel shows the percentage change in migration in an in vitro scratch assay calculated using ImageJ. Data represent the mean ± s.d. (n=8). The experiment was repeated three times with similar results. (B) Computer-generated migration values of an in vitro scratch assay of siC and siCOL7 MET1 cells incubated with conditioned media from both siC and siCOL7 cells. Conditioned media were harvested 24 hours after incubation and incubated with the respective cells for 36 hours. A twofold decrease in cell migration was observed in siCOL7 cells when incubated with siC-conditioned media. Data represent the mean ± s.d. (n=8). (C) Computer-generated migration values of an in vitro scratch assay for 36 hours of siC and siCOL7 MET1 cells incubated with recombinant full-length ColVII or the recombinant NC1 domain. Recombinant proteins were added at a concentration of 5 μg/ml in media. BSA was used as a control. Both recombinant proteins partially reduced cell migration of siCOL7 cells. Data represent the mean ± s.d. (n=6). (D) An in vitro scratch assay for 36 hours showing that in RDEB SCC keratinocytes (Ks), which have no detectable ColVII expression, siRNAs for ColVII did not have an effect on cell migration. Data represent the mean ± s.d. (n=6). All experiments were performed at day 4 post-transfection. *, ** and *** indicate P<0.05, P<0.01 and P<0.001, respectively. Scale bars: 100 μm.

Fig. 3.

Loss of ColVII in cutaneous SCC increases invasion, accompanied by increased MMP2 expression/activity and altered expression of EMT markers. (A) Organotypic cultures of siCOL7 and siC MET1 cells on collagen:matrigel gels with incorporated fibroblasts (top panel) immunostained with a pan-cytokeratin antibody and visualised with DAB. siCOL7 cells displayed increased invasion into the gel when compared with siC cells. The lower panel shows quantification of the depth of invasion. ***P<0.001. (B) Upper and lower panels show a WB and a gelatin zymogram, respectively, of MMP2 from conditioned media of siC and siCOL7 MET1 (left panel) as well as SCC-IC1 (right panel) cells. siCOL7 cells showed increased expression and activity of MMP2. A duplicate blot was probed with a Lam332 antibody. The position of the bands corresponding to 92 kDa pro-MMP9, 72 kDa pro-MMP2 and 64 kDa MMP2, as well as the 160 kDa reduced α3 chain (α3 chain-r) of Lam332, is indicated. (C) Detail of siC and siCOL7 MET1 cells at the migrating front in an in vitro scratch assay after staining with a MMP2 antibody (green). (D) Immunofluorescence staining of sections of siC and siCOL7 MET1 organotypic cultures on DED with antibodies to ColVII (red) and the EMT markers, E-cadherin (red), MMP2 (green) and vimentin (green) showing decreased expression of membranous E-cadherin and increased expression of MMP2 and vimentin-positive cells in the papillary dermis in siCOL7 sections. Experiments were performed at day 4 post-transfection in B and C. DAPI (blue) was used as a nuclear stain. Scale bars: 50 μm.

Fig. 4.

Expression of vimentin and αvβ3 integrin is increased in invading siCOL7 cells, and αvβ3 integrin expression inversely correlates with ColVII expression in vivo. (A) IF staining of sections of siC and siCOL7 MET1 organotypic cultures on collagen:matrigel gels stained with antibodies to vimentin (green) (two top panels) and αvβ3 integrin (green, bottom panel) showing increased expression of both markers in siCOL7 invading cells. (B) IF staining with antibodies to ColVII (red) and αvβ3 integrin (green) in normal skin and in peritumoural skin of two unrelated RDEB patients with differing levels of ColVII expression (decreased, patient A/absent, patient B). αvβ3 integrin expression in vivo, which was not detected in normal skin, was observed in basal and immediate suprabasal layers in RDEB skin and inversely correlated with ColVII expression. DAPI (blue) was used as a nuclear stain. Scale bars: 50 μm.

Fig. 5.

Loss of ColVII in cutaneous SCC is associated with disorganised epithelial differentiation. IF staining of sections of siC and siCOL7 MET1 organotypic cultures on DED with antibodies to the early differentiation marker, keratin 10 (red), the hyperproliferation marker, keratin 6 (green), and the later differentiation markers, keratin 2e (red), involucrin (red) and transglutaminase I (TGM I, red). There was a significant downregulation of keratin 10 and upregulation of keratin 6 in siCOL7 sections. Keratin 2e and involucrin showed an earlier and disorganised pattern of expression, and TGM I was downregulated in siCOL7 sections. DAPI (blue) was used as a nuclear stain. Scale bars: 50 μm.

Fig. 6.

Expression of ColVII positively correlates with involucrin expression in cutaneous SCC in vivo. (A) IF staining with an antibody to involucrin (green) in normal skin and in peritumoural skin of two unrelated RDEB patients (patient A and B). Reduced and disorganised expression of involucrin was observed in vivo in skin sections from RDEB patients who have decreased (patient A) or absent (patient B) ColVII expression, when compared to sections of normal skin. (B) IF double-labelling with antibodies to ColVII (green) and the differentiation marker, involucrin (red), in a skin SCC tissue array (SK802). Co-expression of the two proteins is shown in orange. Pictures are representative of a panel of 74 skin SCC sections where a positive correlation between ColVII and involucrin expression was observed. Tumour cells from a well-differentiated SCC (SCCI) show strong involucrin expression, which co-localises with ColVII (orange). Tumour cells at the edge of islands of the SCC show moderate expression of ColVII. In a moderately differentiated SCC (SCCII), there is reduced involucrin and ColVII expression. A poorly differentiated SCC (SCCIII) shows absent expression of both proteins. DAPI (blue) was used as a nuclear stain. Scale bars: 50 μm.

Fig. 7.

Recombinant ColVII protein injection corrects loss of terminal epithelial differentiation and EMT. IF double-staining with antibodies to ColVII (green) and involucrin (red) (A), and to ColVII (green) and E-cadherin (red) (B), in siC and siCOL7 MET1 sections, with (+ColVII, bottom panel) and without (top panel) injection of recombinant ColVII protein. Organotypic cultures were incubated for a total period of 14 days, and injected with recombinant full-length ColVII (10 μg in PBS) 7 days after starting the experiment. Control organotypic cultures were injected with PBS only. DAPI (blue) was used as a nuclear stain. Scale bars: 50 μm.

Fig. 8.

ColVII regulates expression of the chemokine ligand-receptor CXCL10-CXCR3 and PLC-β4. (A) Summary of the CXCL and PLC genes, which were differentially expressed (+, upregulated; –, downregulated) in siCOL7 compared with siC MET1 cells in the whole-genome Illumina expression arrays. (B) IF staining with antibodies to CXCL10, its CXCR3 receptor and PLC-β4, in sections of siCOL7 and siC MET1 organotypic cultures on collagen:matrigel gels. There was a significant increase in expression of CXCL10 and CXCR3, as well as PLC-β4, in the invading siCOL7 cells compared with siC cells, suggesting that loss of ColVII induces a chemokine gradient. (C) Computer-generated migration values of an in vitro scratch assay for 12 hours of siC and siCOL7 MET1 cells in the presence of a CXCR3 neutralising antibody or an isotype control (mouse IgG). Addition of a CXCR3 neutralising antibody (10 μg/ml) reduced siCOL7 cell migration to control levels. The experiment was performed at day 4 post-transfection. Data represent the mean ± s.d. (n=6). *P<0.05. (D) IF staining with a CXCR3 (green) antibody in peritumoural skin of two unrelated RDEB patients. In normal skin, expression of CXCR3 is mainly cytoplasmic and restricted to the upper spinous and granular layers of the epidermis. Patient A, with reduced expression of ColVII, showed a pattern of CXCR3 expression similar to the one observed in normal skin. In a patient with absent ColVII (patient B), CXCR3 expression was membranous and present in the cells of the basal and immediate suprabasal layers of the epidermis. DAPI (blue) was used as a nuclear stain. Scale bars: 50 μm.

Fig. 9.

Ingenuity-generated network suggests that loss of ColVII affects TGFβ and/or NFκB signalling. (A) Network showing connections between ColVII and CXCL10-CXCR3 through TGFβ and NFκB signalling in siCOL7 cells. The differentially upregulated (red) and downregulated (green) genes in siCOL7 MET1 cells shown in the whole-genome Illumina expression arrays were mapped in the Ingenuity database. A signalling network generated by the Ingenuity Pathway Analysis software showed that relevant genes such as MMPs (including MMP2), integrin αV (ITGAV), vimentin (VIM) and CXCLs, as well as TGFβ-related genes, were linked through TGFβ and NFκB. The network shows direct (full arrows) and indirect (dotted arrows) interactions of TGFβ and NFκB with the up- and downregulated genes, which include CXCL10, CXCR3 and PLCB4. The colour intensity in the genes relates to the degree of fold change, with darker red/green corresponding to a higher-fold change. (B) IF staining with an antibody for p-Smad2/3 (green) in sections of siCOL7 and siC MET1 organotypic cultures on collagen:matrigel gels. The plasma membrane was stained with biotin-labelled lectin PNA (red). Top and bottom panels (confocal microscopy) show different magnifications of the same section. There was a significant increase in expression of p-Smad2/3 in the invading siCOL7 cells compared to siC cells. DAPI (blue) was used as a nuclear stain. Scale bars: 50 μm in the top panels, B; 15 μm in the bottom panels, B.