Additional file 1 of Yap governs a lineage-specific neuregulin1 pathway-driven adaptive resistance to RAF kinase inhibitors
Creators: name: Garcia-Rendueles, Maria E. R.
name: Krishnamoorthy, Gnana
name: Saqcena, Mahesh
name: Acuña-Ruiz, Adrian
name: Revilla, Giovanna
name: de Stanchina, Elisa
name: Knauf, Jeffrey A.
name: Lester, Rona
name: Xu, Bin
name: Ghossein, Ronald A.
name: Fagin, James A.
Publisher: name: figshare
Year: 2024
Subjects: subject: Genetics
subject: FOS: Biological sciences

description: Additional file 1: Fig Sup 1. A) Top: HIPPO pathway alterations in thyroid cancer cell lines derived from papillary thyroid carcinomas (PTC) or poorly differentiated or anaplastic thyroid cancers (PDTC/ATC). Bottom: Oncoprint of HIPPO pathway alterations in thyroid cancer cell lines, and in PTC, PDTC or ATC tissues. MUT: mutations; AMP: amplification; HOM del: homozygous deletion. B) Schematic design of transgenic lines used to investigate the role of Yap in transformation by HrasG12V in the context of Nf2 loss. C) IHC of Yap1 in thyroid sections of the indicated genotypes. D) Thyroid tumor volume by ultrasound at 10 and 20 weeks showing effects of Yap inactivation on tumor development in HrasG12V/Nf2flox mice. **p &lt; 0.01; ***p &lt; 0.001. Fig Sup 2. YAP Western blots of nuclear (N) and cytoplasmic (C) fractions of confluent cells. TATA-BS: nuclear fraction control. HK1: cytoplasmic fraction control. All cell lines were grown to &gt; 90% confluency in 10% FBS. Two top panels: NU-YAP cell lines; Two lower panels: CYT-YAP cell lines. Fig Sup 3. A) Immunofluorescence for YAP, FLAG and DAPI in NUC-YAP and CYT-YAP cell lines in 1�S following 3 days of dox-induced expression of shYAP or YAPS127A, respectively. B) Western blots probed against the indicated antibodies. Cells treated with dox for 3 days in 1�S. C) Cell viability after expression of FLAG-YAPS94A in NUC-YAP and CYT-YAP cell lines. D) Representative images of mechanical scratch assays at baseline and 24 h after the lesion in NUC-YAP (red) and CYT-YAP (blue) cell lines. E) Top: Representative images of 24 h mechanical scratch assays in NUC-YAP and CYT-YAP with or without dox-inducible expression of YAP shRNA. Bottom: Quantification of effects of YAP shRNA on wound healing in NU-YAP and CYT-YAP lines. F) Top: Wound healing in CYT-YAP cell lines with or without dox-induced expression of YAPS127A. Bottom: quantification of effects of expression of YAPS127A on wound healing in CYT-YAP lines. Data in panels C, E and F represent 3 independent experiments with each line and condition. *p &lt; 0.05 **p &lt; 0.01 ***p &lt; 0.001. Fig Sup 4. A) GSEA of RNA-seq of tumor cells sorted from YAPS127A-driven mouse thyroid tumors revealed a significant increase in the Cordenonsi_YAP_conserved gene expression signature compared with thyroid cells from WT mice. B) NES and Nom p-values for each YAP signature from the GSEA of RNA-seq of the indicated human thyroid cancer cell comparators (see Fig. 3G). C) Volcano plots from DEseq2 analysis of RNAseq of 3 NU-YAP (8505C, SW1736, HTH104), 4 CYT-YAP with or without expression of YAPS127A (BHT101, KTC2, MDAT32, MDAT41) comparing NU-YAP vs CYT-YAP and CYT-YAP vs CYT-YAPS127A. Statistically significant (p &lt; 0.05) up and downregulated genes are represented in light red and light blue, respectively, and YAP-Cluster 2 signature genes common to both groups are highlighted in the respective dark colors. There is an inverse relationship in expression of YAP-Cluster 2 genes in the two comparators: upregulated in NU-YAP vs CYT-YAP and downregulated in CYT-YAP vs CYT-YAPS127A. Fig Sup 5. Time course of vemurafenib (1000 nM) on expression and phosphorylation of the indicated proteins in NU-YAP cell lines (Hth104 and SW1736) after YAP silencing and CYT-YAP cell lines (MDAT32 and BHT101) after expression of YAPS127A in 10% FBS. Fig Sup 6. A) Baseline mRNA expression by real-time qRT-PCR of ERBB3 and ERBB2 in NU-YAP cell lines compared with CYT-YAP cell lines. B) Effect of dox-withdrawal on Erbb3 and Erbb2 mRNAs in Braf-YAPS127A mouse cell line (BY96s). C) ERBB3 and ERBB2 mRNA fold-change after vemurafenib relative to vehicle in NU-YAP vs CYT-YAP cell lines. D) Western blot showing the expression of the indicated proteins after a 48 h treatment with 1 μM vemurafenib and after silencing of ERBB2 or ERBB3 in a representative NU-YAP (8505C) cell line. The vertical lines represent the sites of a deleted lane. **p &lt; 0.01 ***p &lt; 0.001. Fig Sup 7. A) YAP immunofluorescence in Braf.pV600E-mutant melanoma (SK-MEL28 and MM96L) and colorectal (HT29, LS411N, LIM2405, RKO and Colo-205) cancer cell lines in sparse and confluent conditions in the presence of 10% FBS. B) Cell viability after dox-induced expression of shYAP or FLAG-YAPS127A in CYT-YAP melanoma and colorectal lines for 6d. C) Dose-dependent effects of a 6-day incubation with vemurafenib on cell viability of CYT-YAP cells with or without dox-induced expression of YAPS127A. D) Effect of YAPS127A on ERBB3 and ERBB2 mRNA after 24 h of vemurafenib treatment in melanoma cell lines. E) Time course of vemurafenib (1000 nM) on expression and phosphorylation of HER2 and HER3 in SK-MEL28 cells in the presence or absence of YAPS127A in 10% FBS. F) Effect of dox-induced expression of YAPS127A in SK-MEL-28 cells by vemurafenib, lapatinib or their combination. Growth was measured at day 6 after addition of the indicated concentrations of the drugs in 1% FBS. Sup Table 1. Thyroid histology of mouse GEMM models and frequency of metastasis. Sup Table 2. Classification of thyroid cancer cell lines based on YAP localization. Supplementary Methods. Sup Methods Table 1. Primers used for genotyping. Sup Methods Table 2. Primers for qRT-PCR.