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Oncogenic mutations in B-type Raf Kinase (BRAF) (particularly V600E) are present in 7-10% of metastatic colorectal cancer (mCRC), conferring resistance to conventional therapies and a poor prognosis. Src Family Kinases (SFKs) play a relevant role in CRC, where Src activation is associated with an aggressive phenotype and a worse prognosis; and could be involved in chemoresistance mechanisms. Dasatinib is a potent Src inhibitor with demonstrated anti-proliferative activity, although deeper knowledge is needed. Thus, we aimed to explore the role of Src in BRAF V600E CRC and to investigate this tumor subgroup in response to Dasatinib. SRC activation was evaluated by Western Blot. We generated Knock-Out (KO) Src cell lines by CRISPR/Cas9 system and stable Src overexpression (OE) by lentivirus. Cell viability was analysed by crystal violet and MTS assay. Phospho-kinase proteins screening was performed using the Proteome Profiler Human Phospho-Kinase Array kit. Expression levels of Src and phospho-Src (p-Src) displayed an inverse correlation with IC50 values for Dasatinib in a panel of 7 CRC cell lines including BRAF V600E (HT29, COLO201, RKO, LS411N) and BRAF WT (DIFI, HCA7 and LIM1215), and these doses were able to inhibit Src activation in all cell lines. Focusing on HT29 cells, inhibition of p-Src occurs in a dose-dependent manner and in shortened periods of treatment. Dasatinib showed a significant inhibition of proliferation and migration on HT29. Moreover, stable Src-OE HT29 cells showed higher clonogenicity, migration and proliferation capacities, as well as higher resistance to Dasatinib. Conversely, HT29 Src KO cells were more sensitive. In line with those results, silencing Src in other BRAF V600E cells (RKO and LS411N) also resulted in a remarkable reduction in clonogenicity and migration abilities. Xenograft mouse model of SRC depletion showed that the growth of SRC KO tumors was significantly slower than WT tumors. Phospho-kinase array quantification in basal and Src-OE HT29 cells showed up- and downregulated proteins related to classical tumorigenic signaling pathways such as MAPK (JNK1/2, MSK), PI3K/Akt (Akt, TOR, GSK3), and others. Further studies to explore resistance mechanisms mediated by Src/SFKs are required. To conclude, lower p-Src expression at baseline is related to a higher resistance to Dasatinib in all CRC cell lines. Dasatinib showed a significant anti-proliferative activity in a subset of CRC cell lines and regulates the activity of Src in a time and dose-dependent manner. Modified expression of Src (OE and KO) in BRAF V600E CRC cells significantly regulates clonogenicity, proliferation and migration abilities in vitro. Src overexpression is responsible of the acquired resistance to Dasatinib and regulates several pathways that could be involved in the tumorigenesis of BRAF V600E CRC, although further studies to explore resistance mechanisms mediated by Src/SFKs are required. Citation Format: Beatriz Rubio Cuesta, Beatriz Gil Calderón, Patricia LLamas Granda, Jacinto Sarmentero, Carlos Carretero Puche, María del Carmen Riesco, Rocío García Carbonero, Beatriz Soldevilla. Molecular characterization of BRAF V600E CRC in response to Src-targeted therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 355. |
