Role of β4 integrin phosphorylation in human invasive squamous cell carcinoma: regulation of hemidesmosome stability modulates cell migration

Abstract

Hemidesmosomes (HDs) are multiprotein structures that anchor epithelia to the basement membrane. During squamous cell carcinoma (SCC) invasion, there is a reduction in the number of HDs, which may facilitate dissemination. Mechanisms of HD disassembly are incompletely understood. Previous work has shown that epidermal growth factor (EGF)-induced phosphorylation of the β4 integrin on three of its serines, S(1356)S(1360)S(1364), can induce HD disassembly in normal cells. Here, we examine the role of β4 integrin serine phosphorylation in SCC. We have found that around 60% of invasive cutaneous SCC show increased β4 phosphorylation on S(1356) when compared with carcinoma in situ or normal tissue. To assess the mechanisms by which SCC increases β4 phosphorylation, we performed in vitro analyses. Compared with keratinocytes, SCC cells showed increased levels of S(1356) phosphorylation in the absence of EGF, correlating with reduced HD-like structures. In addition, phospho-S(1356) signal was largely segregated from other HD components. Epidermal growth factor receptor and PKC inhibitors inhibited basal levels of S(1356) phosphorylation in SCC, suggesting that cells use intrinsic mechanisms to activate the EGF signaling pathway to induce β4 phosphorylation. Moreover, these inhibitors stabilized HD-like structures in SCC cells and reduced their migratory ability. Mutation of S(1356)S(1360)S(1364) in SCC cells to non-phosphorylatable alanines stabilized HD-like structures and substantially reduced migration, while mutation into phosphorylation mimicking aspartate reduced HD-like structures but had no effect on migration, suggesting that serine phosphorylation function is releasing anchorage rather than promoting migration. Altogether these results suggest that β4 serine phosphorylation may have an important role during SCC invasion by destabilizing HDs and facilitating migration.

Figure 1. Regulation of β4 phosphorylation (phospho-S₁₃₅₆) by EGF in HaCaT keratinocytes

A. Anti-phospho-S₁₃₅₆ Ab specificity. Lysates obtained from EGF-stimulated Cos 7 cells transfected with wt β4 or mutant β4 A₁₃₅₆ were probed with Abs against β4 and phospho-S₁₃₅₆. B. Time course of S₁₃₅₆ phosphorylation in HaCaT keratinocytes. Cells were serum-starved and EGF-stimulated (50ng/ml) for different times, and analyzed by Western blotting using phospho-S₁₃₅₆ and β4 Abs. C. S₁₃₅₆ phosphorylation is PKC-dependent. HaCaT keratinocytes were EGF-stimulated in the presence or absence of conventional PKC inhibitor Go6976, or with PMA, then analyzed by Western blotting using phospho-S₁₃₅₆ and β4 Abs.

Figure 2. Phosphorylation of the β4 integrin is increased in human SCC

A–H. FFPE sections stained with β4 integrin (A,C,E,G) and phospho-S₁₃₅₆ (B,D,F,H). Abs were revealed by IHC in normal skin (A,B), carcinoma in situ (C,D), and well differentiated (E,F) or poorly differentiated (G,H) invasive cutaneous SCC. Bar=100um. I. Frequency plots of predominant IHC score for β4 and phospho-S₁₃₅₆ in tissue sections. FFPE (left panels) or frozen sections (right panels) were scored based on the observed staining intensity of indicated Abs, using a scale of 0 to 4. Each symbol represents a separate section and the horizontal line represents the mean score for each category. *p<0.05.

Figure 3. Distribution pattern of β4 phosphorylation in human SCC frozen sections: discontinuous patches that can exclude other HD components

Frozen sections from skin (A–C) or invasive SCC (D–L) were dual-immunostained using Abs against (indicated within image): phospho-S₁₃₅₆ (red); and β4, Laminin-332 or BPAG1 (green). Colocalization in the third column was determined by threshold correlation analysis (22), which shows correlating areas above threshold in white (colocalizing) or below threshold in red or green (non-colocalizing). e, epidermis; d, dermis; bm, basement membrane; t, tumor; s, stroma. Bar=100um. Notice that β4 phosphorylation may range from mildly discontinuous (E) to highly discontinuous (H). Laminin-332 is mostly continuous and does not follow phospho-S₁₃₅₆ gaps (G–I). While both BPAG1 and phospho-S₁₃₅₆ phosphorylation are discontinuous they show segregation more frequently (J–L).

Figure 4. Increased basal levels of β4 phosphorylation on S₁₃₅₆ in SCC cells correlates with a reduction of HD-like structures

A. Cell lysates from serum-starved HaCaT keratinocytes or SCC cells (A431, SCC-25, Colo-16) were analyzed by Western blotting using phospho-S₁₃₅₆ and β4 Abs. B. Bands were quantified by densitometry. C. IF analysis of total β4 and phospho-S₁₃₅₆ spatial distribution in HaCaT keratinocytes and SCC cells. Cells grown on coverslips were serum-starved and detergent-extracted to identify HD-associated β4 (“extracted”), or not to assess total β4 (“non-extracted”), then fixed and stained using phospho-S₁₃₅₆ and β4 Abs. HaCat keratinocytes show characteristic HD-like structures identified by β4 staining on the basal aspect of the cell (thin arrows). Phospho-S₁₃₅₆ signal can be observed in SCC cells (A431, SCC-25 and Colo-16) in HD-like structures (thin arrows) and retraction fibers (arrowheads). Bar=10um. D. Dual immunostaining analysis of detergent-extracted or non-extracted Hacat cells using anti-β4 (green) and anti-plectin antibodies (red). After detergent extraction most of the detergent-resistant β4 colocalizes with plectin in HD-like structures. Bar=10um. E. Percentage of detergent-resistant β4 in HD-like structures: Using IF analysis, the integrated fluorescence density for β4 was calculated for cells extracted or not with detergent buffer before fixation as described in Methods, and expressed as % of detergent-resistant β4 / total β4. Data shown are means±SE of >200 cells. *p<0.05.

Figure 5. Phospho-S₁₃₅₆ is partly segregated from other HD components in SCC cells

SCC-25 cells were grown in coverslips, fixed and stained for IF analysis using the indicated Ab. The third column shows colocalization in yellow. β4 and phospho-S₁₃₅₆ colocalize in retraction fibers and a portion of the HD-like structures (A–C). While HD component plectin colocalizes well with total β4 in HD-like structures (D–F), it is partially segregated from the phospho-S₁₃₅₆ signal (G–I). Notice inverse gradients between phospho-S₁₃₅₆ and plectin signals in colocalized areas. HD component BPAG1 colocalizes well with β4 (J–L) whereas it is highly segregated from phospho-S₁₃₅₆ signal (M–O). Bar= 10um.

Figure 6. EGFR and PKC inhibitors reduce S₁₃₅₆ basal phosphorylation in SCC cells, stabilizing HD-like structures and inhibiting cell migration

A. Serum-starved SCC cells were treated or not with EGFR inhibitor Gefitinib (10uM, upper panel) or conventional PKC inhibitor Go6976 (2uM, lower panel) for one hour, then lysed and analyzed by Western blotting using phospho-S₁₃₅₆ and β4 Abs. B. Effects of Gefitinib and Go6976 on HD-like structures stability. SCC cells were grown on coverslips and treated or not with inhibitor for 1h, then detergent-extracted, fixed and processed for IF using anti-β4 Ab. The integrated fluorescence density for the HD-like structures was quantified as described in Methods, and expressed in arbitrary units per cell. Data shown are means±SE of >200 cells. *p<0.05. C. Effects of Gefitinib and Go6976 on SCC cell migration using in vitro wound healing assay. Confluent SCC cells were scratched with a yellow tip. Inhibitors were added or not and the wound was allowed to close. Images captured at the start and end of the experiment were used to quantify wound closure (%). Data shown are means±SE of three independent experiments *p<0.05.

Figure 7. Abrogation of β4 phosphorylation sites S₁₃₅₆S₁₃₆₀S₁₃₆₄ in SCC cells increases HD-like structure stability and hinders cell migration

Wild type β4 myc or triple mutants containing either a Ser→Ala (β4-AAA-myc) or Ser→Asp (β4-DDD-myc) substitutions on S₁₃₅₆S₁₃₆₀S₁₃₆₄, were stably expressed in β4 shRNA-silenced A431 cells. A. Cells expressing the β4 constructs were analyzed by Western blotting using anti-myc Ab, showing similar level of expression. B. Analysis of HD-like structures stability in β4 phosphorylation mutants. Cells grown on coverslips were detergent-extracted or not before fixation, and processed for IF using anti-myc Ab. Notice that only wtβ4-myc and β4-AAA-myc mutant were substantially retained in HD-like structures after extraction. C. Resistance of β4 phosphorylation mutants to EGF-induced HD-like structures disassembly. Cells on coverslips were treated or not with EGF for 30 minutes before being detergent-extracted, fixed and processed for IF using anti-myc Ab to analyze incorporation into HD–like structures. Quantitation of HD-like structures by digital image analysis is expressed in arbitrary units of fluorescence integrated density per cell. *p<0.05. D. Effect of β4 mutants on cell migration using in vitro wound healing assay. Cells were assayed as in previous figure.