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Supplementary Figures 1 to 9 from Distinct TP63 Isoform-Driven Transcriptional Signatures Predict Tumor Progression and Clinical Outcomes

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posted on 2023-03-31, 01:22 authored by Hussein A. Abbas, Ngoc Hoang Bao Bui, Kimal Rajapakshe, Justin Wong, Preethi Gunaratne, Kenneth Y. Tsai, Cristian Coarfa, Elsa R. Flores

Supplementary Figure 1. Pathways enriched in PCSSTAp63 and PCSSÎ"Np63 signatures. a-b, -Log10(p-value) of pathways enriched in (a) PCSSTAp63 and (b) PCSSÎ"Np63 signatures. Supplementary Figure 2. Differential expression of Î"Np63 in MCF10-CA1D, HCC95 and Caki-1 cells. a-b, (a) Western blot analysis for Î"Np63 expression in MCF10-CA1D, HCC95 and Caki-1 cells. NHEK-WT and NHEK-shÎ"Np63 were used as positive and negative control for Î"Np63 expression. Actin was used as internal loading control. (b) Western blot analysis for either Î"Np63 knockdown efficiency in MCF10-CA1D and HCC95 treated with either siC or siÎ"Np63, or Î"Np63 overexpression in Caki-1 cells treated with either empty vector (EV) or Î"Np63α-overexpressing plasmid (Î"Np63α). Supplementary Figure 3: Lef1 is a downstream target of Î"Np63 in MCF10-CA1D and HCC95. a-e, (a) Western blot analysis for Î"Np63 and Lef1 expression in MCF10-CA1D and HCC95 cells treated with siÎ"Np63 and Lef1-overexpressing plasmid (Lef1). Actin was used as internal loading control. (b-e) Quantification and fluorescence representative images of EdU (green) incorporation of indicated cell lines. Data are mean {plus minus} SD, n = 3. Asterisks indicate statistical significance, p < 0.05 versus siC or siÎ"Np63, two-tailed t test. Supplementary Figure 4. Lef1 is a downstream target of Î"Np63 in Caki-1. a-j, (a) qRT-PCR for Lef1 in Caki-1 cells expressing either empty vector (EV) or Î"Np63α-overexpressing plasmid (Î"Np63α). Asterisks indicate statistical significance, p < 0.005 two-tailed t test. (b) Western blot analysis for Lef1 in Caki-1 cells expressing either empty vector (EV) or Î"Np63α-overexpressing plasmid (Î"Np63α). Actin was used as internal loading control. (c) Western blot analysis for Lef1 overexpression efficiency in Caki-1 cells expressing either with empty vector (EV) or Î"Np63α-overexpressing plasmid (Î"Np63α). Actin was used as internal loading control. (d-e) Quantification and bright field representative images of anchorage-independent colony formation of indicated cell lines in soft agar assay (per 10X field). Data are mean {plus minus} SD, n = 3. (f) Western blot analysis for Î"Np63 and Lef1 expression in Caki-1 cells treated with Î"Np63α-overexpressing plasmid (Î"Np63α) and siLef1. Actin was used as internal loading control. (g-h) Quantification and fluorescence representative images of EdU (green) incorporation of indicated cell lines. Data are mean {plus minus} SD, n = 3. (i-j) Quantification and bright field representative images of anchorage-independent colony formation of indicated cell lines in soft agar assay (per 10X field). Data are mean {plus minus} SD, n = 3. Asterisks indicate statistical significance, p < 0.0005 versus EV, two-tailed t test. Supplementary Figure 5. Circos plots of the overlap and expression of TAp63 and Î"Np63 signatures with progression signatures of BLCA, KIRC, KIRP and LGG. a-b. (a) Expression profile of genes common to TAp63-/- signature and each cancer progression signature. (b) Expression profile of genes common to Î"Np63-/- signature and each cancer progression signature. Supplementary Figure 6. Validation of TAp63 and Î"Np63 activities in independent KIRC cohorts. a-b, (a) TAp63KIRC and (b) Î"Np63KIRC activities in independent cohort of KIRC across high and low stages. Supplementary Figure 7. Common pathways enriched in corresponding TAp63 and Î"Np63 signatures of BLCA, KIRC, KIRP and LGG. a-f. (a) -Log10(p-value) of pathways enriched in TAp63KIRC, TAp63KIRP and TAp63LGG. (b) -Log10(p-value) of pathways enriched in Î"Np63BLCA, Î"Np63KIRC, Î"Np63KIRP and Î"Np63LGG. ClueGo plots of overlap in corresponding TAp63 and Î"Np63 in (c) BLCA, (d) KIRC, (e) KIRP and (f) LGG progression signatures. Supplementary Figure 8. Î"Np63CTHRC1/COL5A1 in tumor progression of BLCA, KIRC, KIRP and LGG. a-b, (a) Activity and (b) Kaplan-Meier survival of BLCA, KIRC, KIRP and LGG with respect to Î"Np63CTHRC1/COL5A1. Supplementary Figure 9. Survival of BLCA, KIRC, KIRP and LGG cancers with respect to bottom and top quartile gene expression of (a) CTHRC1 and (b) COL5A1.

Funding

NCI

CPRIT

University of Texas M.D. Anderson Cancer Center

Leukemia and Lymphoma Society

Cancer Prevention & Research Institute grants

Texas Proteomics & Metabolomics Core Facility

University of Texas MD Anderson Cancer Center

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ARTICLE ABSTRACT

TP63 is required to maintain stem cell pluripotency and suppresses the metastatic potential of cancer cells through multiple mechanisms. These functions are differentially regulated by individual isoforms, necessitating a deeper understanding of how the distinct transcriptional programs controlled by these isoforms affect cancer progression and outcomes. In this study, we conducted a pan-cancer analysis of The Cancer Genome Atlas to identify transcriptional networks regulated by TAp63 and ΔNp63 using transcriptomes derived from epidermal cells of TAp63−/− and ΔNp63−/− mice. Analysis of 17 cancer developmental and 27 cancer progression signatures revealed a consistent tumor suppressive pattern for TAp63. In contrast, we identified pleiotropic roles for ΔNp63 in tumor development and found that its regulation of Lef1 was crucial for its oncogenic role. ΔNp63 performed a distinctive role as suppressor of tumor progression by cooperating with TAp63 to modulate key biological pathways, principally cell-cycle regulation, extracellular matrix remodeling, epithelial-to-mesenchymal transition, and the enrichment of pluripotent stem cells. Importantly, these TAp63 and ΔNp63 signatures prognosticated progression and survival, even within specific stages, in bladder and renal carcinomas as well as low-grade gliomas. These data describe a novel approach for understanding transcriptional activities of TP63 isoforms across a large number of cancer types, potentially enabling identification of patient subsets most likely to benefit from therapies predicated on manipulating specific TP63 isoforms.Significance: Transcriptomic analyses of patient samples and murine knockout models highlight the prognostic role of several critical mechanisms of tumor suppression that are regulated by TP63. Cancer Res; 78(2); 451–62. ©2017 AACR.

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    Cancer Research

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    Computational MethodsGene expression profilingMolecular modelingProgression, Invasion & MetastasisStem Cell BiologyTumor MicroenvironmentTumor-stromal cell interactions

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