RESUMEN
Activated Cdc42-associated Kinase, ACK1, is a non-receptor tyrosine kinase with numerous interacting partners, including Cdc42 and EGFR. Gene amplification and overexpression of ACK1 were found in many cancer types such as those of the lung and prostate. Previously, we identified both somatic- and germ line missense mutations in the ACK1 coding sequence, by surveying 261 cancer cell lines and 15 control tissues. Here, we verified and characterized the non-synonymous mutation, ACK-S985 N, located in the ubiquitin association domain of the protein. Both overexpression and silencing experiments in MCF7 and A498 cells, respectively, demonstrated a role of the ACK1 S985 N mutation in enhancing cell proliferation, migration and anchorage-independent growth as well as the epithelial-mesenchymal transition. Further, we showed that the ACK1 S985 N mutant is unable to bind ubiquitin, unlike the wild type kinase. This contributed to ACK1 protein stability and stabilized EGFR after EGF stimulation, thereby prolonging mitogenic signaling in cancer cells. In addition, the ACK1 S985 N-EGFR interaction is enhanced, but not the ubiquitination of the receptor. Intriguingly, silencing of ACK1 in A498 cells sensitized the renal carcinoma cells to gefitinib, against which they are otherwise resistant. The work demonstrates that other than gene amplification, a single somatic mutation in ACK1 can result in extended protein stability enabling the oncoprotein to exert its oncogenic function in tumor progression. It also provides a rationale to target ACK1 in combination with other chemotherapeutic drugs, such as EGFR inhibitors, to potentiate therapeutic action against resistant tumors.
Asunto(s)
Carcinoma de Células Renales/metabolismo , Receptores ErbB/metabolismo , Neoplasias Renales/metabolismo , Mutación , Proteínas Tirosina Quinasas/genética , Transducción de Señal , Ubiquitina/metabolismo , Antineoplásicos/metabolismo , Secuencia de Bases , Carcinoma de Células Renales/genética , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Receptores ErbB/genética , Gefitinib , Silenciador del Gen , Humanos , Neoplasias Renales/genética , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/metabolismo , Quinazolinas/metabolismo , Ubiquitina/genéticaRESUMEN
Protein tyrosine kinases (PTKs) play a critical role in the manifestation of cancer cell properties, and respective signaling mechanisms have been studied extensively on immortalized tumor cells. To characterize and analyze commonly used cancer cell lines with regard to variations in the primary structure of all expressed PTKs, we conducted a cDNA-based sequence analysis of the entire tyrosine kinase transcriptome of 254 established tumor cell lines. The profiles of cell line intrinsic PTK transcript alterations and the evaluation of 155 identified polymorphisms and 234 somatic mutations are made available in a database designated "Tykiva" (tyrosine kinome variant). Tissue distribution analysis and/or the localization within defined protein domains indicate functional relevance of several genetic alterations. The cysteine replacement of the highly conserved Y367 residue in fibroblast growth factor receptor 4 or the Q26X nonsense mutation in the tumor-suppressor kinase CSK are examples, and may contribute to cell line-specific signaling characteristics and tumor progression. Moreover, known variants, such as epidermal growth factor receptor G719S, that were shown to mediate anticancer drug sensitivity could be detected in other than the previously reported tumor types. Our data therefore provide extensive system information for the design and interpretation of cell line-based cancer research, and may stimulate further investigations into broader clinical applications of current cancer therapeutics.