Abstract
It is increasingly appreciated that 3D cultures are more predictive of in vivo therapeutic efficacy than 2D cultures. Using in vitro 3D type I collagen cultures of human colorectal cancer (CRC) cell line HCA-7 derivatives CC, SC, and CC-CR, we previously identified that activation of receptor tyrosine kinases (RTKs) MET and RON contributed to resistance to the EGF receptor (EGFR)-directed therapeutic antibody cetuximab. The de novo mode of cetuximab resistance in SC cells could be overcome by crizotinib, a multi-RTK inhibitor that also targets MET and RON. We now show that crizotinib also overcomes acquired cetuximab resistance in CC-CR cells. Phospho-RTK array analysis showed increased phosphorylation of several RTKs, including MET and RON, in SC and CC-CR cells compared to cetuximab-sensitive CC counterparts. Furthermore, other multi-RTK inhibitors cabozantinib and BMS-777607 helped overcome cetuximab resistance, as measured by 3D colony growth and activation state of key signaling molecules. Conversely, addition of RTK ligands HGF and NRG1 induced cetuximab resistance in CC cells, which could be blocked by addition of crizotinib. We further determined the mechanism of the cooperative effect of cetuximab and crizotinib by FACS analysis and observed increased cell cycle arrest in G1 phase in cetuximab-resistant CRC 3D cultures. Finally, we show that crizotinib overcomes cetuximab resistance in vivo in SC nude mice xenografts. Thus, our work shows that multi-RTK inhibition strategy is a potent, broadly applicable strategy to overcome resistance to EGFR-targeted therapeutics in CRC and highlights the relevance of 3D cultures in these studies.Statement of implication: Using in vitro 3D CRC cultures and in vivo CRC xenografts, we show that parallel inhibition of multiple RTKs with small molecule inhibitors overcomes de novo and acquired resistance to EGFR-directed therapies in CRC.
Highlights
Receptor tyrosine kinase (RTK) signaling is one of the major dysregulated pathways in cancer that contributes to transformation and is a major therapeutic target [1,2,3]
Using in vitro 3D type I collagen cultures of human colorectal cancer (CRC) cell line HCA-7 derivatives CC, SC, and CC-CR, we previously identified that activation of receptor tyrosine kinases (RTKs) MET and RON contributed to resistance to the EGF receptor (EGFR)-directed therapeutic antibody cetuximab
We previously showed that de novo mode of cetuximab resistance in SC cells could be overcome by addition of the multi-RTK inhibitor crizotinib [21]
Summary
Receptor tyrosine kinase (RTK) signaling is one of the major dysregulated pathways in cancer that contributes to transformation and is a major therapeutic target [1,2,3]. In colorectal cancer (CRC), the RTK EGFR is overexpressed in more than 50% of cases and is linked to poor prognosis and metastasis [4]. EGFR-targeting monoclonal antibodies, cetuximab and panitumumab, are approved by the U.S FDA for the treatment of individuals with advanced wild-type KRAS CRC [5,6,7,8]. Cetuximab use is contraindicated with KRAS mutations, which lead to constitutive activation of downstream signaling, rendering EGFR-directed therapies ineffective [8, 9]. In advanced CRC in particular, there is a need to enhance the effectiveness of clinically approved targeted therapies (cetuximab, panitumumab, bevacizumab, and ramucirumab; Supplementary Table 1) and prevent or overcome emergence of resistance to these therapies
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