Research ArticleExperimental Studies
Open Access

Interaction of Integrin αvβ8 With Type I Collagen Promotes Squamous Cell Carcinoma Cell Motility via RAC1 Activation

YASUTAKA ISHIDA, TOMOAKI SHINTANI, TADAYOSHI NOBUMOTO, SHIGERU SAKURAI, TOMOAKI HAMANA, SOUICHI YANAMOTO and YASUTAKA HAYASHIDO
Anticancer Research November 2023, 43 (11) 4833-4841; DOI: https://doi.org/10.21873/anticanres.16680

Abstract

Background/Aim: The interaction of integrin αvβ8 with type I collagen was shown to promote oral squamous cell carcinoma (SCC) cell proliferation via the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway. However, the role of integrin αvβ8 in SCC progression remains poorly understood. In this study, the role of integrin αvβ8 in oral SCC progression was therefore investigated. Materials and Methods: Integrin αv and β8 protein expression in oral SCC cells was examined by western blotting. Oral SCC cell motility was investigated using modified Boyden chamber assays. Behavior of oral SCC cells was examined in three-dimensional culture using type I collagen gel. Ras homolog family member A (RHOA), Ras-related C3 botulinum toxin substrate 1 (RAC1), and cell division control protein 42 homolog (CDC42) activity of oral SCC cells was analyzed by pull-down assays. Results: SCC cells with high integrin αvβ8 expression levels had a high ability to migrate on type I collagen and exhibited enhanced invasion into type I collagen gel. In SCC cells with high integrin αvβ8 expression level, cultivation on type I collagen induced RAC1 activation. Treatment with RAC1 inhibitor reduced type I collagen-induced motility of SCC cells. Down-regulation of integrin β8 by specific antisense oligonucleotide reduced type I collagen-induced RAC1 activation and suppressed cell motility and invasion into type I collagen gel. Conclusion: The interaction of integrin αvβ8 with type I collagen facilitates SCC cell motility and invasion via RAC1 activation. Therefore, integrin αvβ8 and RAC1 may represent new targets for inhibiting metastasis and invasion in patients with oral SCC.

Key Words:

A lethal property of malignant tumors, including squamous cell carcinoma (SCC), is the ability to invade surrounding tissues and form metastatic foci at distant sites (1, 2). Tumor invasion and metastasis are mediated by the surrounding microenvironment, including extracellular matrix (ECM) proteins and stromal cells (3-5). Integrins are heterodimeric transmembrane receptors composed of α and β subunits, and bind a wide range of ligands, including ECM proteins and cell-surface proteins (6, 7). Integrin–ECM protein interactions induce various signal-transduction pathways, which regulate cell proliferation, motility, and differentiation (8, 9). Integrins therefore contribute to tumor progression by facilitating the proliferation, proteolytic activity, motility, and survival of tumor cells. Several immunohistochemical studies have reported altered integrin expression in malignant tumors. Specifically, overexpression of the integrin αv subfamily, such as αvβ1, αvβ3, αvβ5 and αvβ6, has been correlated with poor prognosis in ovarian, lung, colorectal, and gastric cancer (10-13). Integrin αvβ3 is a cell-surface receptor for active matrix metalloproteinase-2, indicating that integrin αvβ3 regulates tumor invasion and metastasis by increasing pericellular proteolysis (14). Our previous study revealed that the interaction of integrin αvβ8 with type I collagen promotes oral SCC cell proliferation via the mitogen-activated protein kinase/extracellular signal-regulated kinase pathway (15). However, the biological role and function of integrin αvβ8 in cancer progression remains poorly understood.

In the present study, the effect of integrin αvβ8 on oral SCC cell motility, as well as the induced signal transduction pathways regulating oral SCC cell motility, were investigated.

Materials and Methods

Cells and culture. Oral SCC cell lines, Ca9-22 (16), KO (17, 18), SCCKN (15), and ZA (19) were used. Ca9-22, SCCKN, and ZA cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and RPMI 1640 medium (RD medium) (20) supplemented with 10% fetal bovine serum (FBS) at 37°C with 5% CO2. KO cells were cultured in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F-12 nutrient mixture (DF medium) (21) supplemented with 10% FBS at 37°C with 5% CO2.

Western blotting. To detect integrin αv and β8 proteins, western blotting analysis was performed. Detailed methods for western blotting analysis have been described previously (22). Mouse monoclonal antibody to integrin αv (sc-9969; Santa Cruz Biotechnology, Dallas, TX, USA) or mouse monoclonal antibody to integrin β8 (CL7290; Thermo Fischer Scientific, Waltham, MA, USA) was used as primary antibody. Mouse monoclonal antibody to β-actin (AM4302; Thermo Fischer Scientific) was used as the internal control. Protein expression levels were compared by densitometry using Fiji/ImageJ software (NIH, Bethesda, MD, USA). Western blot quantification was normalized with β-actin signal and expressed relative to ZA cells.

Cell motility assay. Cell motility was analyzed with modified Boyden chamber assays using Chemotaxicell chambers with 8 μm pores (Kurabo, Osaka, Japan), and determining the number of cells migrating from the upper chamber to the bottom plate as described elsewhere (22, 23). ECM proteins and poly-L-lysine were purchased from Sigma–Aldrich (St. Louis, MO, USA). Filters were precoated with type I collagen, fibronectin, laminin, vitronectin or type IV collagen (all at a concentration of 100 μg/ml) by incubation overnight at 4°C. Poly-L-lysine (100 μg/ml) was used as a non-integrin-dependent adhesion substrate. Oral SCC cells were harvested with EDTA-trypsin and suspended for 1 h in RD or DF medium containing 10% FBS to recover. The cells (3×105) were subsequently resuspended in RD or DF medium containing 0.1% bovine serum albumin and added to the upper compartment of each Chemotaxicell chamber. After incubation for 24 h at 37°C, cells were fixed and counted.

To investigate the movement of oral SCC cells under RAC1 inhibition, the cells were cultured in the presence of RAC1 inhibitor NSC23766 (range=1-20 μmol/l; Calbiochem, La Jolla, CA, USA).

Collagen gel culture. The effect of type I collagen on SCC cell behavior was investigated by three-dimensional culture using type I collagen gel as previously described (15).

Rho GTPase activity assay. Oral SCC cell RHOA, RAC1, and CDC42 activity was analyzed by pull-down assays using RHOA/RAC1/CDC42 Activation Assay Combo Biochem Kits (Cytoskeleton, Denver, CO, USA). Cells suspended in serum-free medium were seeded on culture dishes coated with 100 μg/ml type I collagen or poly-L-lysine. After incubation for 15 min, the cells were lysed with ice-cold cell lysis buffer (50 mM Tris pH 7.5, 10 mM MgCl2, 0.5 M NaCl, 2% (GEPAL® CA-630; Sigma–Aldrich) for 10 min. The cell lysates were centrifuged at 14,000 × g for 5 min at 4°C and the supernatant (500 μg total cell protein) was incubated with 20 μg Rhotekin RBD- or PAK PBD-agarose beads on a rotator for 1 h at 4°C. The beads were then washed three times with washing buffer (25 mM Tris pH 7.5, 30 mM MgCl2, 40 mM NaCl). Protein samples were eluted with 2× Laemmli sample buffer and analyzed by immunoblotting with anti-RHOA (ARH03; Cytoskeleton), anti-RAC1 (ARC03; Cytoskeleton) or anti-CDC42 (ACD03; Cytoskeleton). Total RHOA, RAC1, and CDC42 levels were determined by western blotting analysis of whole-cell lysates. Protein expression levels were compared using densitometry using Fiji/ImageJ software. Western blot quantification was normalized using the GTP RAC1 signal and relative to a standard control morpholino oligonucleotide transfected cells.

Down-regulation using integrin β8-specific morpholino antisense oligonucleotide. A morpholino antisense oligonucleotide specific for integrin β8 (GeneTools, Philomath, OR, USA) was used to down-regulate integrin β8. The antisense oligonucleotide sequence was 5′-AAGCCAGGGCCGAGCCGCACATAAT-3′. A standard control morpholino oligonucleotide (5′-CCTCTTACCTCAGTTACAAT TTATA-3′) was used as a negative control. Oligonucleotides were delivered into the cells according to the GeneTools protocol. Briefly, 80-100% confluent Ca9-22 and KO cells were treated with 10 μM morpholino antisense oligonucleotide or the standard control oligonucleotide and 6 μM Endo-Porter reagent (GeneTools) for 24 h. The band intensity was normalized to total RAC1 by western blotting analysis of whole cell lysates.

Statistical analysis. Statistical analysis was performed with EZR (Saitama Medical Center, Jichi Medical University, Saitama, Japan) (24). All data are presented as means±SD of at least three independent experiments. Student’s t-test or Dunnett’s test were used to compare differences between groups, and statistical significance was set at p<0.05.

Results

Integrin αvβ8 expression in oral SCC cells and oral SCC cell motility on ECM proteins. Western blotting indicated that Ca9-22 and KO cells expressed high levels of integrin αv and β8 proteins. Compared with Ca9-22 and KO cells, SCCKN and ZA cells expressed lower levels of integrin αv and β8 proteins (Figure 1A). The modified Boyden chamber assays revealed that the migration of SCCKN and ZA cells, with low integrin αvβ8 expression levels, was low on all ECM proteins evaluated. Ca9-22 and KO cells, with high integrin αvβ8 expression levels, exhibited enhanced migratory ability compared with SCCKN and ZA cells. Type I collagen markedly enhanced Ca9-22 and KO cell migration (Figure 1B). Three-dimensional culture of Ca9-22, KO, SCCKN, and ZA cells in type I collagen gel revealed that SCCKN and ZA cells formed small, spherical colonies, whereas Ca9-22 and KO cells form dilated colonies with irregular margins, and many cells migrating into the surrounding collagen gel (Figure 1C).

Figure 1.
Figure 1.

Effect of integrin αvβ8 on oral squamous cell carcinoma cell motility and invasion. A: Western blot analysis of integrin αv and β8 protein expression levels in Ca9-22, KO, SCCKN, and ZA cells. Significantly different from ZA cells at: *p<0.05 and **p<0.01 (Dunnett’s test). B: Modified Boyden chamber assay of SCC cell motility. Results from counting cells in eight optical fields are given as mean±SD of at least three independent experiments. C: Ca9-22, KO, SCCKN, and ZA cell morphology in type I collagen gel. Scale bar=100 μm.

RAC1 activation in oral SCC cells by interaction with type I collagen. To elucidate the role of RHOA, CDC42 and RAC1 in type I collagen-induced cell motility, Ca9-22, KO, SCCKN, and ZA cells were replated onto type I collagen or poly-L-lysine gels, and RHOA, CDC42, and RAC1 activation was investigated by pull-down assay. Activated RHOA and CDC42 were observed in all cell lines cultured on the poly-L-lysine control, and cultivation on type I collagen did not alter RHOA (Figure 2A) or CDC42 (Figure 2B) activation. In contrast, RAC1 activation was reduced in all cell lines cultured on poly-L-lysine, and no activated RAC1 was observed in SCCKN or ZA cells. Cultivation on type I collagen led to RAC1 activation in Ca9-22 and KO cells but not in SCCKN or ZA cells (Figure 2C).

Figure 2.
Figure 2.

Ras homolog family member A (RHOA), Ras-related C3 botulinum toxin substrate 1 (RAC1), and cell division control protein 42 homolog (CDC42) activation in oral squamous cell carcinoma cells on type I collagen gels. Pull-down assays and western blots of GTP activated RHOA (A), CDC42 (B), and RAC1 (C).

The role of RAC1 in oral SCC cell motility and invasion. As type I collagen induced RAC1 activation and Ca9-22 and KO cell migration, the effect of RAC1 inhibition on type I collagen-induced cell motility was examined. Treatment with RAC1 inhibitor NSC23766 reduced Ca9-22 and KO cell motility in a concentration-dependent manner. Treatment with 20 μM NSC23766 significantly reduced Ca9-22 and KO cell migration on type I collagen to 15% and 56%, respectively (Figure 3A), and Ca9-22 and KO cells embedded in type I collagen gel formed small, spherical colonies in the presence of NSC23766 (Figure 3B).

Figure 3.
Figure 3.

Effect of Ras-related C3 botulinum toxin substrate 1 (RAC1) inhibition in oral squamous cell carcinoma cell motility and invasion. A: The cells were resuspended in culture medium with different concentrations of RAC1 inhibitor NSC23766 and added to each well of a Chemotaxicell chamber with type I collagen coated filters. After incubation for 24 h at 37°C, the chambers were fixed. Motility Results are represented as percentage of the untreated control, and the mean±SD of triplicate determinations. Significantly different from the untreated control at: *p<0.05 and **p<0.01 (Dunnett’s test). B: Ca9-22 and KO cell morphology in the presence of 10 μM NSC23766 for 12 days in type I collagen gel. Scale bar=100 μm.

The role of integrin β8 in oral SCC cell motility via RAC1 activation. To examine the effect of integrin β8 suppression on SCC cell motility on type I collagen, cells were transfected with a morpholino antisense oligonucleotide targeting the integrin β8 subunit. Transfection with the antisense oligonucleotide targeting the integrin β8 subunit reduced Ca9-22 and KO cell motility on type I collagen to 70% and 68%, respectively (Figure 4A), and strongly suppressed RAC1 activation in Ca9-22 cells on type I collagen. Suppression of RAC1 activation was also observed in KO cells cultured on type I collagen (Figure 4B). We next examined the effect of integrin β8 suppression on Ca9-22 and KO cell morphology in type I collagen gels and found that Ca9-22 and KO cells transfected with the control oligonucleotide formed dilated colonies with irregular margins and most cells disseminated into the surrounding collagen gel, whereas Ca9-22 and KO cells transfected with the antisense oligonucleotide formed small, spherical colonies and dissemination was suppressed (Figure 4C).

Figure 4.
Figure 4.

Suppression of integrin β8 reduces type I collagen-induced motility and activation of Ras-related C3 botulinum toxin substrate 1 (RAC1) in squamous cell carcinoma cells. A: β8 Antisense oligo- or control oligo-transfected Ca9-22 and -KO cells were subsequently added to each Chemotaxicell chamber with type I collagen coated-filters. After incubation for 24 h at 37°C, the cells were fixed and counted. Motility results are represented as percentage of the number of migrating Ca9-22 or KO cells transfected with control oligonucleotide, and the mean±SD of triplicate determinations. **Significantly different at p<0.01 (Student’s t-test). B: Ca9-22 and KO cells transfected with β8 antisense oligo- or control oligo were plated onto type I collagen-coated dishes. The cells were lysed 15 min after plating, and the activation of RAC1 was analyzed by pull-down assay. The band intensity of activated RAC1 was normalized to total RAC1. Error bars indicate SD. **Significantly different at p<0.01 (Student’s t-test). C: Morphology of β8 antisense oligo- and control oligo- transfected Ca9-22 and -KO cells in type I collagen gel. Scale bar=100 μm.

Discussion

Integrin αv subunits heterodimerize with β1, β3, β5, β6 or β8 subunits and regulate various biological processes, such as cell adhesion, proliferation, and differentiation (25-27). Several studies have shown that integrin αvβ3, αvβ5, and αvβ6 are implicated in carcinogenesis, tumor invasion, and metastasis (28-30). Our previous study revealed that the interaction of integrin αvβ8 with type I collagen mediates oral SCC cell proliferation via the MAPK/ERK signaling pathway, suggesting that integrin αvβ8 may play a significant role in oral SCC progression (15).

In the present study, we investigated the role of integrin αvβ8 in oral SCC cell motility. Type I collagen markedly enhanced the motility of Ca9-22 and KO cells expressing high levels of integrin αvβ8. In contrast, the motility of SCCKN and ZA cells expressing low levels of integrin αvβ8 was not greatly altered by type I collagen. Three-dimensional type I collagen gel culture revealed that SCCKN and ZA cells embedded in type I collagen gel formed small, spherical colonies, whereas Ca9-22 and KO cells formed dilated colonies with irregular margins and most cells disseminated towards the surrounding gel, which suggests that the interaction of integrin αvβ8 with type I collagen facilitates Ca9-22 and KO cell invasion by enhancing cell motility.

The binding of integrins to ECM proteins leads to the activation of several signaling pathways, including AKT serine/threonine kinase 1/phosphatidylinositol-4,5-bisphosphate 3-kinase kinase, mitogen-activated protein kinase, and Rho family GTPase signaling (31-33). Integrin-mediated cell migration is largely regulated by Rho family GTPases, including RHOA, CDC42 and RAC1 (33-36). We therefore investigated the role of Rho family GTPase signaling in type I collagen-induced motility of oral SCC cells. CDC42 and RHOA activation were observed in Ca9-19, KO, SCCKN, and ZA cells cultured on poly-L-lysine, a non-integrin-dependent adhesion substrate, and cultivation on type I collagen had no effect on CDC42 and RHOA activation. RAC1 activation was drastically reduced in all cell lines cultured on poly-L-lysine. There are reports of RAC1 activation when human vascular smooth muscle cells and fibroblasts were cultured on type 1 collagen, but there are no reports of this occurring in oral SCC cells (37, 38). Cultivation on type I collagen led to RAC1 activation in Ca9-19 and KO cells but not in SCCKN or ZA cells. Although it has already been shown that integrin-mediated cell adhesion results in tyrosine phosphorylation of intracellular proteins and subsequent activation of mitogen-activated protein kinase, phosphatidylinositol-4,5-bisphosphate 3-kinase, and G protein of the low-molecular-weight RHO family, there are few reports of type I collagen-induced RAC1 activation in oral SCC cells (39, 40). Furthermore, no activation of RAC1 was observed in SCCKN and ZA cells, and the reasons for this are unclear and require further investigation. Treatment with RAC1 inhibitor NSC23766 suppressed type I collagen-induced motility of Ca9-22 and KO cells, and also reduced Ca9-19 and KO cell invasion into type I collagen. These results indicate that activation of RAC1 is one of the factors that regulate type I collagen-induced oral SCC cell motility.

The integrin αv subfamily consists of five distinct heterodimers, namely αvβ1, αvβ3, αvβ5, αvβ6, and αvβ8, which recognize specific ligands. Some studies indicated that only integrin αvβ8 is a potential collagen receptor (41, 42) and our previous study revealed that the interaction of integrin αvβ8 with type I collagen promotes SCC cell proliferation (15). In the present study, we found that type I collagen induces RAC1 activation and enhances the motility of oral SCC cells expressing high integrin αvβ8 levels. Formation of α/β heterodimers is essential for integrin expression and function, and although integrin αv subunits can dimerize with β1, β3, β5, β6 or β8 subunits, the β8 subunit dimerizes only with the αv subunit. We therefore suppressed the integrin β8 subunit of Ca9-22 and KO cells to examine the role of integrin αvβ8 in type I collagen-induced motility of SCC cells. Suppression of integrin β8 by its antisense oligonucleotide reduced Ca9-22 and KO cell motility on type I collagen. However, Ca9-22 and KO cell motility on poly-L lysine was not altered by integrin β8 suppression. In addition, down-regulation of integrin β8 suppressed invasion by Ca9-22 and KO cells into type I collagen gel. There have been several reports on RAC1 and integrins. RAC1 was reported to participate in both FcγR-mediated phagocytosis in the mouse macrophage cells and in integrin αvβ3-mediated apoptotic cell uptake in primary murine bone marrow-derived macrophages (43, 44). Furthermore, milk fat globule-epidermal growth factor–factor VIII-induced activation of RAC1 involved in phagocytosis of apoptotic cells was shown to occur in an integrin αvβ5-dependent manner (45, 46). A recent report showed that oral SCC cells phagocytose apoptotic oral SCC cells in a RAC1-dependent manner and that phagocytosis promotes SCC cell migration (39). However, as far as we are aware, there have been no reports of integrin β8 being involved in type I collagen-induced activation of RAC1 in oral SCC cells, hence the results of this study are novel. Our findings suggest that the interaction of integrin αvβ8 with type I collagen induces SCC cell motility and dissemination via RAC1 activation.

Conclusion

The interaction of integrin αvβ8 with type I collagen activates RAC1 in oral SCC cells, thereby enhancing cell motility and invasion. This interaction of integrin αvβ8 with type I collagen and RAC1 may provide potential targets for treatment of oral SCC.

Footnotes

  • Authors’ Contributions

    Yasutaka Ishida: Conceptualization, methodology, investigation, data curation, visualization. Tomoaki Shintani: Investigation, writing – review and editing. Tadayoshi Nobumoto: Investigation. Shigeru Sakurai: Investigation. Tomoaki Hamana: Investigation. Souichi Yanamoto: Supervision. Yasutaka Hayashido: Conceptualization, methodology, investigation, writing – original draft.

  • Conflicts of Interest

    The Authors have no conflicts of interest to declare.

  • Received August 20, 2023.
  • Revision received September 27, 2023.
  • Accepted October 2, 2023.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

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Interaction of Integrin αvβ8 With Type I Collagen Promotes Squamous Cell Carcinoma Cell Motility via RAC1 Activation
YASUTAKA ISHIDA, TOMOAKI SHINTANI, TADAYOSHI NOBUMOTO, SHIGERU SAKURAI, TOMOAKI HAMANA, SOUICHI YANAMOTO, YASUTAKA HAYASHIDO
Anticancer Research Nov 2023, 43 (11) 4833-4841; DOI: 10.21873/anticanres.16680
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