Biochemical and Biophysical Research Communications
Integrin αβ1, αvβ, α6β effectors p130Cas, Src and talin regulate carcinoma invasion and chemoresistance
Research highlights
► Proteomics of clustered integrin αβ1, αvβ, α6β receptors in oral carcinoma. ► p130Cas, Dek, Src and talin regulate oral carcinoma invasion. ► p130Cas, talin, Src and zyxin regulate oral carcinoma resistance to cisplatin.
Introduction
Patients with stages III and IV oral carcinoma have poor survival rates that have not significantly changed over four decades due, in part, to frequent metastasis and resistance to both radiation and chemotherapy of these tumors [1]. The role of the tumor stroma in chemoresistance has been an area of intense study over the last decade. Tumor–stromal fibroblasts form a dense or rigid stroma that may function as a barrier to prevent the penetration of therapeutics into tumor tissue [2], [3]. Adhesion of breast tumor cell lines to matrix secreted by stromal fibroblasts is sufficient to confer tumor cell resistance to chemotherapeutics [4], [5]. In our laboratory, we found that adhesion to the matrix secreted by oral carcinoma is sufficient to induce chemoresistance (A. Berrier, submitted manuscript). Adhesions within the tumor matrix comprised of collagens, fibronectins and laminins mediated by engagement of integrin αβ1, αvβ and α6β receptors regulates invasion and chemoresistance in a variety of tumors [6], [7], [8]. Even though integrins can regulate tumor invasion and chemoresistance, it is currently not known whether effectors downstream of integrins coordinate invasive potential with resistance to cisplatin.
We used a proteomics approach to identify integrin effectors in oral carcinoma. Recruitment to integrin cytoplasmic complexes was induced by clustering integrin receptors at the cell surface with anti-integrin antibodies immobilized on magnetic beads. The integrin-associated complexes were biochemically isolated, and the resulting tryptic peptides were analyzed by mass spectrometry. We characterized roles for a subset of the identified integrin effectors in oral carcinoma invasion and resistance to cisplatin.
Section snippets
Integrin receptor clustering
HN4 and HN12 cells were isolated from the primary tumor lesion and lymph node, respectively, of a patient with tongue cancer [9]. HN12 and HN4 cells were cultured in DMEM supplemented with fetal bovine serum, penicillin/streptomycin and hydrocortisone in 10% CO2 [10]. Cells were placed in suspension at a density of 2 × 106 cells/ml in DMEM supplemented with 1% bovine calf serum and penicillin/streptomycin. The cells were incubated with magnetic beads (4.5 μm diameter) conjugated with the following
The selective recruitment of proteins to integrin αβ1, αVβ or α6β complexes in oral carcinoma
We hypothesized that specific integrin effectors critical for invasion and chemoresistance are recruited to integrin αvβ, α6β and αβ1 receptors in oral carcinoma [17], [18], [19]. To test this notion, we first determined whether clustering integrin receptors with αv, α6 or β1 subunits in HN12 cells resulted in the specific recruitment of distinct effectors to each receptor. HN12 cells are an oral squamous cell carcinoma (OSCC) metastatic line that retains their invasive phenotype in vitro [10].
Discussion
Integrin mediated adhesions to extracellular matrix within the tumor microenvironment regulate both tumor invasion and resistance to chemotherapeutics [6], [7], [8]. Whether integrin-associated proteins coordinate invasive potential with chemoresistance is poorly understood. Proteomics analysis of clustered integrin αvβ, α6β or αβ1 receptors was performed to identify candidate integrin effectors in oral carcinomas. One outcome of the proteomics analysis of integrin complexes was the
Acknowledgments
The authors thank the Katrina Visiting Faculty Program sponsored by the NCMHD/NIDCR/NIH, the NIDCR intramural research program and LSUHSC-NO School of Dentistry for support, and the Yates Laboratory (NIH Grant P41 RR011823) for the proteomics. The expertise provided by Hynda Kleinman and Deborah Philp in invasion assays and Becky Worthylake in microscopy are greatly appreciated. The authors also thank the Department of Oral and Craniofacial Biology for their core facilities.
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