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Supplementary Figures 1 - 9 from Hedgehog Signaling Alters Reliance on EGF Receptor Signaling and Mediates Anti-EGFR Therapeutic Resistance in Head and Neck Cancer

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posted on 2023-03-30, 21:51 authored by Stephen B. Keysar, Phuong N. Le, Ryan T. Anderson, J. Jason Morton, Daniel W. Bowles, Jeramiah J. Paylor, Brian W. Vogler, Jackie Thorburn, Pamela Fernandez, Magdalena J. Glogowska, Sarah M. Takimoto, Daniel B. Sehrt, Gregory N. Gan, Justin R. Eagles-Soukup, Hilary Serracino, Fred R. Hirsch, M. Scott Lucia, Andrew Thorburn, John I. Song, Xiao-Jing Wang, Antonio Jimeno

PDF file - 1255K, Confirmation of GLI1 modulation by EGFR activation (S1); Epithelial gene expression profile and morphology predicts erlotinib sensitivity in HNSCC cell lines (S2); Zeb1 transcription factor is key driver of EGF induced EMT (S3); EGFR activation induces EMT in HNSCC cell lines characterized by expression of Vimentin (S4); Inter-pathway crosstalk as well as EGF induced EMT are blunted in erlotinib resistant cells (S5); Silencing of HhP transcription factor GLI1 increases EGFR driven EMT in Tu-167 cells (S6); MEK/ERK signaling is required for EMT-like state while both MEK/ERK and PI3K/AKT are required for invasion through Matrigel (S7); Proliferative inhibition of erltotinib (1μM and 2μM) plus IPI-926 (1μM) or SHH (250ng/ml) (S8); Cetuximab and IPI-926 modulate EGFR, HhP and EMT gene expression in vivo (S9).

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ARTICLE ABSTRACT

The EGF receptor (EGFR)-directed monoclonal antibody cetuximab is the only targeted therapy approved for the treatment of squamous cell carcinoma of the head and neck (HNSCC) but is only effective in a minority of patients. Epithelial-to-mesenchymal transition (EMT) has been implicated as a drug resistance mechanism in multiple cancers, and the EGFR and Hedgehog pathways (HhP) are relevant to this process, but the interplay between the two pathways has not been defined in HNSCC. Here, we show that HNSCC cells that were naturally sensitive to EGFR inhibition over time developed increased expression of the HhP transcription factor GLI1 as they became resistant after long-term EGFR inhibitor exposure. This robustly correlated with an increase in vimentin expression. Conversely, the HhP negatively regulated an EGFR-dependent, EMT-like state in HNSCC cells, and pharmacologic or genetic inhibition of HhP signaling pushed cells further into an EGFR-dependent phenotype, increasing expression of ZEB1 and VIM. In vivo treatment with cetuximab resulted in tumor shrinkage in four of six HNSCC patient-derived xenografts; however, they eventually regrew. Cetuximab in combination with the HhP inhibitor IPI-926 eliminated tumors in two cases and significantly delayed regrowth in the other two cases. Expression of EMT genes TWIST and ZEB2 was increased in sensitive xenografts, suggesting a possible resistant mesenchymal population. In summary, we report that EGFR-dependent HNSCC cells can undergo both EGFR-dependent and -independent EMT and HhP signaling is a regulator in both processes. Cetuximab plus IPI-926 forces tumor cells into an EGFR-dependent state, delaying or completely blocking tumor recurrence. Cancer Res; 73(11); 3381–92. ©2013 AACR.

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