(D-127) A Versatile, Reductionist Platform for Studying Integrin-EGFR Interactions in Colorectal Cancer

Introduction:: Colorectal cancer (CRC), despite marked improvements in screening and treatment, remains one of the top causes of death in the United States. One of the main contributors to tumor development and progression is the extracellular matrix (ECM), which differs in biochemical composition between tissues: native colon tissue is comprised primarily of collagens, whereas liver—the most common metastatic site—displays elevated levels of fibronectin. Signaling from the ECM into the cell via focal adhesions is well established as a mechanism governing signaling pathways responsible for growth and proliferation; one pathway of pharmaceutical interest is the epidermal growth factor receptor (EGFR) signaling cascade. While treatment with anti-EGFR monoclonal antibodies and small molecules has shown great success in vitro, clinical applications for liver metastatic CRC have been limited, even in wild-type KRAS allele carriers, due to drug resistance still not well understood. Previous work has demonstrated synergistic signaling of integrins and EGFR in other cell types and has been thought to increase downstream receptor tyrosine kinase (RTK) functions, such as proliferation and survival. As such, we hypothesized that these clinical outcomes could be partially attributed to the biochemical differences between colon and liver tissue, influencing downstream EGFR signaling.


Materials and Methods:: We developed a poly(ethylene glycol) (PEG)-peptide-based hydrogel model of the colon or liver ECM using peptide sequences for integrin binding sites of collagen and fibronectin domains found in CRC primary and metastatic tumor microenvironments. We employed the DGEA peptide sequence from type I collagen, which is recognized by the α2β1 integrin, and the RGDS peptide sequence derived from fibronectin, recognized by various integrins including α5β1 and α5β3. To encode for rapid (< 20 mins) hydrogel dissolution, we developed sortase-sensitive crosslinkers that formed stable hydrogels with multifunctional strained alkyne-bearing PEG via bioorthogonal strain-promoted azide-alkyne cycloaddition (SPAAC). Engineered sortase variants—bacterial transpeptidases—have been evolved to display improved catalytic efficiency and high specificity for their peptide recognition sequences; here, we utilized the eSrt(4S-9) variant that recognizes the LPESG motif and has been shown to be “biologically invisible” by our group via global transcriptomic RNA-sequencing. Our model enables interrogation of the interactions of various ECM adhesion components with signaling pathways via immunofluorescence, Western blotting and RT-qPCR.

Results, Conclusions, and Discussions:: CACO2 colorectal adenocarcinoma cells with no BRAF or KRAS mutations were cultured in gels containing RGDS, DGEA, both, or scrambled peptide sequences. After a week of culture, we stimulated cells with EGF, analyzed the amount and rate of ERK1/2 phosphorylation via immunofluorescent staining, and confirmed and additionally verified Akt phosphorylation via Western blotting. Both assays revealed a suppressive role of DGEA on EGFR signaling cascades; conversely, RGDS-modified gels displayed robust ERK1/2 activation. Additionally, the rapid kinetics of EGFR signaling were confirmed in our system: a spike in phosphorylation levels occurred at 20 mins. RT-qPCR identified the tyrosine kinase PTPN2 as upregulated in cells in the collagen-mimetic hydrogels; however, various integrin expression levels were not significantly different amongst the various conditions, implying that the levels of integrin expression did not necessarily change in the various formulations, yet their interactions with EGFR are highly dependent on the microenvironment. Blocking PTPN2 with the tyrosine kinase inhibitor BVT948, thus circumventing the integrin binding interactions, returned ERK1/2 phosphorylation levels to those of cells in RGD-modified gels.

As the search for novel druggable targets in oncology continues, the role of the ECM must be considered, with many recent publications suggesting targeting this area may be beneficial in conjunction with other current therapies. We have presented a simplified 3D model to study ECM-EGFR cross-communication in a controlled manner in CRC more akin to an in vivo setting. Our method is very versatile and allows for the substitution of any ECM adhesion sequence; we expect this modality will prove beneficial in high-throughput screening of cellular interactions with other ligands. Our results demonstrate that the composition of the ECM has the potential to activate and influence the downstream functions of RTKs by changing the localization or accessibility of the receptor. We demonstrated that adhesion with the fibronectin-binding domain synergizes with EGF stimulation to promote a robust response, whereas association with the collagen-adhesion site dampens downstream EGFR activation via PTPN2. Through reductionist interrogation of matrix adhesion and its influence on biochemical signaling in engineered microenvironments, we can gain a better understanding of the complex interplay between ECM and cancer progression.


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