Integrin alpha 6 and calpain 2 are mechanosensitive proteins in breast cancer

摘要

Introduction: Breast cancer metastasizes specifically to the bone, brain and lung, each of which has a distinct stiffness range. These mechanical cues alter gene expression, protein levels and activation, and cell phenotype, all of which impact the ability of cells to migrate, survive, and proliferate at the secondary tissue site. We hypothesize that in breast cancer, this mechanotransduction informs the tissue specificity of survival and growth at a secondary tissue site. We used tunable biomaterials that allow us to isolate stiffness dependent signals to verify the role of stiffness in metastatic cell genotype and phenotype. Materials and Methods: 2D biomaterial platform: Poly(ethylene glycol) dimethacrylate gels were made as previously described. RNA-Seq: We isolated total RNA from MDA-MB-231 cancer cells at 0,1 and 6 days on hydrogels of stiffnesses 1,4,10, and 50 kpa coated with 10μg/cm~2 of collagen 1. The Illumina TRUseq RNA kit was used to generate the cDNA library and samples were run on the MiSeq platform. Differential expression analysis was done using the Tuxedo Suite. Functional Assays: Cell adhesion to surfaces of varying stiffness was quantified, with or without the binding of integrin alpha 6. Migration assays were used to quantify calpain 2 dependent motility. Results and Discussion: The mechanical range of this system allowed us to mimic tissue specific forces in order to understand the underlying mechanobiology of breast cancer metastasis. On these surfaces, RNAseq implicated integrin a6 and calpain 2 as possible novel mechanosensitive genes, and we examined their effects on stiffness dependent cell adhesion and motility. Integrin α6 is a surface receptor that binds specifically to laminin, which increases both the cell spreading rate and maximum area, as compared to collagen 1 alone, when on the soft brain mimic hydrogels. Gene expression levels show much higher expression of the B isoform at low stiffnesses, which has a splicing modification in the cytoplasmic domain that is unique to a mesenchymal, stem-like population. Downstream of focal adhesion initiation, the protease calpain 2 influences migration by controlling membrane protrusions and the disassembly rate of focal adhesions by cleaving structural proteins, such as talinPl. Inhibition of Calpain 2 decreases motility because cells are unable to release focal adhesions at the rear of the cell during migration. Low ECM density increases active calpain, and is accentuated by growth factor receptor activation. Conclusions: We have isolated mechanical cues on 2D PEG-PC hydrogels to understand the mechanisms by which tissue- specific physical environments initiate downstream signaling via integrin alpha 6B and calpain 2. Coupling genetic, protein and functional changes across these tissue-mimic surfaces allows us to connect the signaling roles of stiffness, EGF and integrin binding to understand mechanosensitive cell adhesion and migration.