Targeted therapeutics are needed for triple-negative breast cancer (TNBC). In this study, we investigated the activation of Src family of cytoplasmic tyrosine kinases (SFKs) and two SFK substratesdCUB-domain containing protein 1 (CDCP1) and protein kinase C d (PKCd)din 56 formalin-fixed, paraffin-embedded (FFPE) TNBCs. Expression of SFK phosphorylated at Y416 (SFK_pY416 þ ) in tumor cells was strongly associated with phosphorylation of CDCP1 and PKCd (CDCP1_ pY743 þ and PKCd_pY311 þ ), as assessed by immunohistochemistry, indicating increased SFK activity in situ. To enable biochemical analysis, protein extraction from FFPE tissue was optimized. Cleaved CDCP1 isoform (70 kDa) was expressed to a varying degree in all samples but only phosphorylated in TNBC tumor cells that expressed SFK_pY416. Interestingly, active SFK was found to be biphosphorylated (SFK_pY416 þ /pY527 þ ). Biphosphorylated active SFK was observed more frequently in forkhead box protein A1 (FOXA1) À TNBCs. In addition, in SFK_pY416 À samples, FOXA1 þ TNBC tended to be SFK_pY527 þ (classic inactive SFK), and FOXA1 À TNBC tended to be SFK_pY527 À (SFK poised for activation). Strong SFK_pY416 staining was also observed in tumor-infiltrating lymphocytes in a subset of TNBCs with high tumor-infiltrating lymphocyte content. This report will facilitate protein biochemical analysis of FFPE tumor samples and justifies the development of therapies targeting the SFK/ CDCP1/PKCd pathway for TNBC treatment.
Clear cell renal cell carcinoma (CC-RCC) remains one of the most deadly forms of kidney cancer despite recent advancements in targeted therapeutics, including tyrosine kinase and immune checkpoint inhibitors. Unfortunately, these therapies have not been able to show better than a 16% complete response rate. In this study we evaluated a cyclin-dependent kinase inhibitor, Dinaciclib, as a potential new targeted therapeutic for CC-RCC. In vitro , Dinaciclib showed anti-proliferative and pro-apoptotic effects on CC-RCC cell lines in Cell Titer Glo, Crystal Violet, FACS-based cell cycle analysis, and TUNEL assays. Additionally, these responses were accompanied by a reduction in phospho-Rb and pro-survival MCL-1 cell signaling responses, as well as the induction of caspase 3 and PARP cleavage. In vivo , Dinaciclib efficiently inhibited primary tumor growth in an orthotopic, patient-derived xenograft-based CC-RCC mouse model. Importantly, Dinaciclib targeted both CD105 + cancer stem cells (CSCs) and CD105 − non-CSCs in vivo . Moreover, normal cell lines, as well as a CC-RCC cell line with re-expressed von-Hippel Lindau ( VHL ) tumor suppressor gene, were protected from Dinaciclib-induced cytotoxicity when not actively dividing, indicating an effective therapeutic window due to synthetic lethality of Dinaciclib treatment with VHL loss. Thus, Dinaciclib represents a novel potential therapeutic for CC-RCC.
Targeted therapeutics are needed for triple-negative breast cancer (TNBC). In this study, we investigated the activation of Src family of cytoplasmic tyrosine kinases (SFKs) and two SFK substrates: CUB-domain containing protein 1 (CDCP1) and protein kinase C delta (PKCd) in 56 formalin-fixed, paraffin-embedded (FFPE) TNBCs. Expression of SFK phosphorylated at Y416 (SFK_pY416+) in tumor cells was strongly associated with phosphorylation of CDCP1 and PKCd (CDCP1_ pY743+ and PKCd_pY311+), as assessed by immunohistochemistry, indicating increased SFK activity in situ. To enable biochemical analysis, protein extraction from FFPE tissue was optimized. Cleaved CDCP1 isoform (70 kDa) was expressed to a varying degree in all samples but only phosphorylated in TNBC tumor cells that expressed SFK_pY416. Interestingly, active SFK was found to be biphosphorylated (SFK_pY416+/pY527+). Biphosphorylated active SFK was observed more frequently in forkhead box protein A1 (FOXA1)-TNBCs. In addition, in SFK_pY416-samples, FOXA1+ TNBC tended to be SFK_pY527+ (classic inactive SFK), and FOXA1- TNBC tended to be SFK_pY527-(SFK poised for activation). Strong SFK_pY416 staining was also observed in tumor-infiltrating lymphocytes in a subset of TNBCs with high tumor-infiltrating lymphocyte content. This report will facilitate protein biochemical analysis of FFPE tumor samples and justifies the development of therapies targeting the SFK/CDCP1/PKCd pathway for TNBC treatment. Citation Format: Olga V. Razorenova, Luke Nelson, Heather Wright, Nguyen Dinh, Kevin Nguyen, Scott Heinemann. Biphosphorylated Src at Y416/527 is active in triple-negative breast cancer cell lines and a subset of patient samples [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 LB202.
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