Figure S1. Specificity of PBX3 protein detection. Figure S2. Effects of WNT repression on PBX3 in colon cancer cells. Figure S3. Lack of transcriptional regulation of PBX3 expression by WNT. Figure S4. Effects of EMT induction with a conditional ZEB1 allele in LS174T and DLD-1 colon cancer cells. Figure S5. Effects of ZEB1 depletion on PBX3 expression. Figure S6. Effects of PBX3 depletion on VIM expression upon EMT induction by SNAIL. Figure S7. PBX3, ZEB1 and SNAIL mRNA expression and disease free survival in n=786 colon cancer cases. Table S1. Antibodies used. Table S2. qRT-PCR primers. Table S3. Differentially expressed transcription factors. Table S4. PBX3 in UICC II colorectal cancer. Table S5. Multivariate analysis of cancer specific survival in UICC stage II colorectal cancer. Table S6. Multivariate analysis of disease free survival in UICC stage II colorectal cancer. Table S7. PBX3 expression in colon cancers with and without distant metastasis. Table S8. Multivariate analysis for PBX3 mRNA expression. Table S9. Multivariate analysis for ZEB1 mRNA expression. Table S10. Multivariate analysis for SNAIL mRNA expression.
ARTICLE ABSTRACT
Purpose: Colorectal cancers are composed of phenotypically different tumor cell subpopulations within the same core genetic background. Here, we identify high expression of the TALE transcription factor PBX3 in tumor cells undergoing epithelial–mesenchymal transition (EMT), analyze PBX3 regulation, and determine clinical associations in colorectal cancer.Experimental design: We used transcriptomic and in situ analyses to identify PBX3 expression in colorectal cancer and cell biology approaches to determine its regulation and function. Clinical associations were analyzed in independent tissue collections and gene expression datasets of colorectal cancers with recorded follow-up data.Results: PBX3 was expressed in tumor cells with high WNT activity undergoing EMT at the leading tumor edge of colorectal cancers, whereas stromal cells were PBX3 negative. PBX3 expression was induced by WNT activation and by the EMT transcription factors SNAIL and ZEB1, whereas these effects were mediated indirectly through microRNA miR-200. PBX3 was required for a full EMT phenotype in colon cancer cells. On the protein level, PBX3 expression indicated poor cancer-specific and disease-free survival in a cohort of 244 UICC stage II colorectal cancers, and was associated with metastasis in a case–control collection consisting of 90 cases with or without distant metastasis. On the mRNA level, high PBX3 expression was strongly linked to poor disease-free survival.Conclusions: PBX3 is a novel indicator of EMT in colorectal cancer, part of an EMT regulatory network, and a promising prognostic predictor that may aid in therapeutic decision making for patients with colorectal cancer. Clin Cancer Res; 24(8); 1974–86. ©2018 AACR.
