The laminin-binding integrins are mechanosensory receptors critical for cell adhesion and structural organization that link the extracellular matrix (ECM) to the cytoskeleton. Integrin α6β1 is associated with prostate cancer (PCa) invasion, metastasis and decreased cancer-specific survival. Kindlins are integrin adaptors and mechanosensory focal adhesion (FA) proteins that activate and cluster integrins in response to structural ECM alterations in the tumor microenvironment. Hypoxia is a well-known inducer of tumor migration and invasion, results in ECM remodeling, and is physiologically relevant for PCa. The study's objective was to determine if integrin-kindlin-2 adhesion complexes were responsive to hypoxia and the dynamic distribution of integrin-kindlin-2 complexes using immunofluorescence microscopy (IFM). The methods included a combination of biochemical analysis of kindlin-2 adhesion complexes, qRT-PCR, immunoblotting, and immunocytochemistry. DU145 cells were analyzed in HEPES-containing media with 10% FBS under hypoxic conditions (1% O2) continuously exposed for 4, 8, 12, and 16 hours. The results showed that hypoxia increased the mRNA expression of CAIX, a well-known HIF-1α target gene, within 4 hours, resulting in 7-fold maximum expression. Similarly, VEGF-A mRNA was responsive to hypoxia as expected. In contrast, HIF-1α, α6 integrin, and kindlin-2 mRNA levels remained unchanged as compared to normoxia. Strikingly, we found that kindlin-2-containing adhesion complexes increased under hypoxia conditions as compared to normoxia and detected by immunoprecipitation, the complexes contained α6β1 integrin. It is important to note that the constitutive levels of either α6 integrin or kindlin-2 were not altered by hypoxia. Using IFM under normoxic conditions, we confirmed the location of kindlin-2 within phosphorylated (Y31) paxillin (pPXN)-containing FAs and within extended plasma membrane domains exclusive of pPXN. However, under hypoxic conditions, an increased reorganization of pPXN-containing kindlin-2 complexes occurred within 4 hours, was continuously changing up to 16 hours, with increasing fibrillar forms of FAs. In addition, kindlin-2 was observed as increased in plasma membrane protrusions devoid pPXN. The dynamic nature of the hypoxia-driven FAs was observed by a time course analysis and indicated the apparent assembly and disassembly of the structures during 16-hours of PCa cells exposed to hypoxia. We conclude that kindlin-2-integrin complexes are responsive to hypoxia and contain the unexpected α6β1 integrin in the membrane exclusive of FAs.
(Partially supported by NIH grants CA P30 23074, DOD W81XWH-19-1-0455, and NCI R01 CA242226).
Citation Format: Daniel Hernandez-Cortes, Beatrice S. Knudsen, Noel A. Warfel, Anne E. Cress. Dynamic kindlin-2 complexes containing a laminin-binding integrin are responsive to hypoxia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB256.