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Supplementary Figures S1-S15 from Targeting the c-Met/FZD8 Signaling Axis Eliminates Patient-Derived Cancer Stem–like Cells in Head and Neck Squamous Carcinomas

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posted on 2023-03-30, 22:49 authored by Shuyang Sun, Suling Liu, Sheng Zhong Duan, Lei Zhang, Henghua Zhou, Yongjie Hu, Xianghui Zhou, Chaoji Shi, Rong Zhou, Zhiyuan Zhang

Supplementary Figures S1-S15. Identification and enrichment of CSCs directly from clinical SCCHN samples ; qRT-PCR measurement of the c-Met mRNA level and western blot measurement of c-Met protein in HN-CSC#1 spheres, HN-CSC#2 spheres, and HN-CSC#3 spheres infected with lentiviral nonspecific shRNA (scramble) or c-Met-specific shRNAs ; Knockdown of c-Met inhibited the properties of HN-CSCs ; Dose-dependent effect of conventional chemotherapeutic agents (cisplatin, 5-Fu, and docetaxel) on the viability of tumor cells derived from patient HN-0-22, HN-0-29, or HN-0-46 ; Treatment with c-Met inhibitor PF-2341066 inhibited the formation of HN-CSC#2 spheres and HN-CSC#3 spheres in a dose-dependent manner ; Treatment with c-Met inhibitor SU11274 inhibited the formation of HN-CSC#1 spheres and HN-CSC#2 spheres in a dose-dependent manner ; The combination of c-Met inhibition and docetaxel treatment eliminates CSCs in patient-derived tumor xenografts ; Pharmacologic inhibition of c-Met enhances the therapeutic efficacy of conventional anti-cancer agent 5-Fu ; Western blot analysis shows that no anaplastic lymphoma kinase (ALK) expression was detected in seven SCCHN PDXs including HN-0-22, HN-0-29, and HN-0-46-derived xenografts ; Histologic confirmation of metastasis by H&E staining ; RT2 Profiler PCR Array and further qPCR validation ; Downregulation of FZD8-mediated Wnt/β-catenin signaling is a crucial step for the elimination of HN-CSCs caused by c-Met inhibition ; Quantification of the normalized photon flux, measured at weekly intervals following cells inoculation ; Western blot analyses ; C-Met functionally regulates FZD8 through an ERK/c-Fos cascade in HN-CSCs .

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ARTICLE ABSTRACT

Cancer stem–like cells (CSC) thought to contribute to head and neck squamous carcinomas (HNSCC) may offer attractive therapeutic targets if a tractable approach can be developed. In this study, we report that silencing c-Met is sufficient to suppress sphere formation, tumor initiation, and metastatic properties of HN-CSC. Pharmacologic inhibition of c-Met with the selective inhibitor PF-2341066 preferentially targeted CSC and synergized with conventional chemotherapy to improve efficacy in a mouse xenograft model of HNSCC, impeding tumor growth and reducing metastasis. Mechanistic investigations showed that CSC elimination was due to downregulation of Wnt/β-catenin signaling in HN-CSC and that the Wnt pathway receptor FZD8 was essential for interactions of c-Met and Wnt/β-catenin signaling in HN-CSC. Notably, ectopic expression of FZD8 rescued the impaired phenotype of HN-CSC where c-Met was inhibited. Furthermore, c-Met upregulated FZD8 through the ERK/c-Fos cascade in HN-CSC. Taken together, our results offer a preclinical proof-of-concept for targeting the c-Met/FZD8 signaling axis as a CSC-directed therapy to improve HNSCC treatment. Cancer Res; 74(24); 7546–59. ©2014 AACR.

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