Abstract 3834: Assessing the role of extracellular matrix-integrins in metastatic squamous cell carcinoma
Authors: Aleman John , Nguyen Khoa A. , Ke Yao , Young Christian D. , Wang Xiao-Jing 
Published: June 2022

Purpose: Patients with head and neck squamous cell carcinoma (HNSCC) have a dismal survival rate. The effects of the extracellular matrix (ECM) on cancer progression have been long studied, but the roles of specific integrins in the process of HNSCC metastasis remain to be dissected. This study aims to determine how HNSCC cells affect the production of laminin-binding integrins, and how these integrins participate in the ECM interactions necessary for a metastatic phenotype.

Experimental design: Our laboratory has produced syngeneic mouse SCC cell lines, P029 and A223, derived from Keratin15+ stem cells with Smad4 loss and KrasG12D mutation. In syngeneic recipients, SCCs derived from P029 cells transplanted to the flank mouse skin produced spontaneous metastases to the lung while SCCs derived from A223 cells did not form metastases. Having the same genotype, these cell lines serve as models to examine cancer cell interactions with the ECM and resulting effects on invasion and metastasis. Bulk RNA sequencing (RNAseq) was performed to compare cultured metastatic P029 cells versus non-metastatic A223 cells and identify differentially expressed genes that regulate SCC cells and ECM interactions. Immunoassays and functional invasion assays were performed to evaluate ECM-cancer cell signaling and influence on cancer cell invasion in these two cell lines.

Results: RNAseq analysis revealed that, relative to A223 cells, P029 cells have increased levels of integrins, the main mechanoreceptors for numerous ECM ligands and matrix proteins. Laminin-binding integrins, including integrins α4 and β6, were greater at the RNA and protein levels. Additionally, P029 cells expressed higher levels of fibronectin and laminin coding genes compared to non-metastatic A223 cells. Correlatively, high expression of these matrix proteins is associated with worse patient survival. Additionally, P029 SCC cells displayed differential gene expression of tight and apical junctions, PI3K signaling components, and regulators of cytoskeletal remodeling. Furthermore, ELISA and western blot analysis revealed that P029 has aberrant TGFβ-Smad signaling as indicated by the elevated release of TGFβ-1 protein and higher levels of phosphorylated Smad2 and Smad3 relative to non-metastatic A223. Treating P029 cells with TGFβ-1 significantly increased their motility and invasion. Conversely, migration and motility of P029 cells were radically reduced by the TGFβ inhibitor galunisertib. Additionally, the mouse cytokine array revealed that mouse plasma bearing P029 tumors had a greater circulation of CXCL16, MMP-9, proliferin, and serpin E1. These proteins are associated with ECM remodeling and metastasis in HNSCC.

Conclusions: SCC cells with metastatic properties upregulate integrins which bind to non-collagen matrix proteins and have elevated invasive and migratory capacity contributed from activated TGFβ signaling.

Citation Format: John Aleman, Khoa A. Nguyen, Yao Ke, Christian D. Young, Xiao-Jing Wang. Assessing the role of extracellular matrix-integrins in metastatic squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3834.