CREB5 promotes tumorigenicity and upregulates druggable cell surface modalities in basal-like breast cancer

Abstract

Basal-like breast cancers (BLBC) have limited targeted therapies and poor outcomes. We found that CREB5 is a transcription factor overexpressed in 15% of BLBCs and was upregulated in breast cancers that metastasize to the brain. In cell lines, CREB5 overexpression regulated cell phenotypes and transcriptional changes, including IL13RA2, a cell surface receptor that is currently druggable and represents a novel target in BLBC.

Fig. 1. Clinical characterization of CREB5 in BC subtypes.

Box plots showing CREB5 median expression across A TCGA and B METABRIC datasets with the boxes representing the ends of the first and third quartiles in breast cancer molecular subtypes. Red dotted line denotes the global median while blue dotted lines are global standard deviations. C Percent of tumors with CREB5 amplifications or overexpression in BLBC as compared to all other BC subtypes in the same two datasets. Overexpression is defined by a z-score >2 and amplifications are defined by cBioPortal parameters. D Kaplan–Meier plot comparing overall survival of CREB5 high (red line) versus low (black line) BLBC patients (n = 309) from KMPlot.com. E BCBMs acquired by Cosgrove et al. are stratified by BC subtypes including basal (n = 16), HER2 (n = 9), luminal A (n = 11), and luminal B (n = 8). Median CREB5 expression across subtypes are shown in a box plot with whiskers extending to minimum and maximum values. *p < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001, ns not significant. F Waterfall plot shows the percent change of CREB5 expression in each patient from the primary BC biopsy to the BCBM biopsy in the same dataset.

Fig. 2. Phenotypic and transcriptomic effects of CREB5 in BLBC cell models.

Proliferation assay showing median live cell count over 10 days for A MDA-MB-231 and B HCC1806 cell lines with either CREB5 or luciferase (LUC) overexpressed. C Tumorsphere formation assay results for MDA-MB-231 (top row) and HCC1806 (bottom row) cell lines with CREB5 (left panels) or luciferase overexpressed. Scale bar shown is 200 μm. Quantitative analysis of median D tumorsphere formation efficiency and E tumorsphere size (μm²). F Volcano plots illustrating changes in gene expression from RNA sequencing in MDA-MB-231 (left panels) and HCC1806 (right panels) cell lines, in 2D (top row) and 3D (bottom row) culture. Each dot represents a gene that either passes the significant thresholds for both the p-value and log-fold changes (red), for only the p-value (blue), or neither (gray). G Net enrichment scores (NES) from GSEA based on C6 oncogenic signatures depicted across cell lines in 3D culture. Red circles: significantly upregulated both cell lines. Orange circles: only significantly upregulated in the MDA-MB-231 cells. Purple circles: only significantly upregulated in the HCC1806 cells. Significance is based on FDR < 0.05.

Fig. 3. Evaluation of CREB5’s promotion of IL13RA2.

A Bar chart depicts the counts of unique cell surface proteins increased by CREB5 overexpression in MDA-MB-231 and HCC1806 cell lines, as categorized by set ranges of abundance ratios. B Venn diagram compares cell surface proteins with greater than 10- fold changes in MDA-MB-231 and HCC1806 cell lines. The overlapping proteins (n = 3) are detailed in the table beneath. IL13RA2 mRNA expression in C MDA-MB-231 and D HCC1806 cell lines with CREB5 or luciferase overexpressed in 2D and 3D cultures. Box plots display median expression levels with whiskers extending to minimum and maximum values. p ** < 0.01, **** < 0.0001. E Log2 fold changes in IL13RA2 expression with respect to primary BC biopsies from patients with BCBMs, as organized by BC subtypes from Cosgrove et al.. Each circle represents the change in expression of a paired sample (n = 44). The dotted line at 0 indicates no change in expression. F Percentage of BCBM samples with IL13RA2 expression increased from the primary BC sample by 0–100%, ≥100%, or ≤0%. Change in IL13RA2 expression is shown across BC subtypes including basal (n = 16), HER2 (n = 9), luminal A (n = 11), and luminal B (n = 8). G Bar graph displays the frequency of IL13RA2 genetic alterations including high mRNA expression, amplification, and homozygous deletions, across multiple cancer types from TCGA.