Single-Cell Transcriptome Analysis Revealed Heterogeneity and Identified Novel Therapeutic Targets for Breast Cancer Subtypes

Abstract

Breast cancer (BC) is a heterogeneous disease, which is primarily classified according to hormone receptors and HER2 expression. Despite the many advances in BC diagnosis and management, the identification of novel actionable therapeutic targets expressed by cancerous cells has always been a daunting task due to the large heterogeneity of the disease and the presence of non-cancerous cells (i.e., immune cells and stromal cells) within the tumor microenvironment. In the current study, we employed computational algorithms to decipher the cellular composition of estrogen receptor-positive (ER+), HER2+, ER+HER2+, and triple-negative BC (TNBC) subtypes from a total of 49,899 single cells’ publicly available transcriptomic data derived from 26 BC patients. Restricting the analysis to EPCAM+Lin− tumor epithelial cells, we identified the enriched gene sets in each BC molecular subtype. Integration of single-cell transcriptomic with CRISPR-Cas9 functional screen data identified 13 potential therapeutic targets for ER+, 44 potential therapeutic targets for HER2+, and 29 potential therapeutic targets for TNBC. Interestingly, several of the identified therapeutic targets outperformed the current standard of care for each BC subtype. Given the aggressive nature and lack of targeted therapies for TNBC, elevated expression of ENO1, FDPS, CCT6A, TUBB2A, and PGK1 predicted worse relapse-free survival (RFS) in basal BC (n = 442), while elevated expression of ENO1, FDPS, CCT6A, and PGK1 was observed in the most aggressive BLIS TNBC subtype. Mechanistically, targeted depletion of ENO1 and FDPS halted TNBC cell proliferation, colony formation, and organoid tumor growth under 3-dimensional conditions and increased cell death, suggesting their potential use as novel therapeutic targets for TNBC. Differential expression and gene set enrichment analysis in TNBC revealed enrichment in the cycle and mitosis functional categories in FDPShigh, while ENO1high was associated with numerous functional categories, including cell cycle, glycolysis, and ATP metabolic processes. Taken together, our data are the first to unravel the unique gene signatures and to identify novel dependencies and therapeutic vulnerabilities for each BC molecular subtype, thus setting the foundation for the future development of more effective targeted therapies for BC.

ENO1 as a biomarker of breast cancer progression and metastasis – a bioinformatic approach

“…The poor prognostic effect is observed in advanced (Stage 3) disease, and is present in HER-2 positive and triple negative breast cancers. Our data agrees with previous recent studies showing an association of high ENO1 expression with the aggressive basal subtype and a favourable prognosis in patients with early stage breast cancer but not with advanced stage disease and/or basal breast cancer [16,26,38]. We also observed that methylation of the gene is associated with a good prognostic effect, consistent with the expected effect from reducing ENO1 gene expression.…”

“…In breast cancer, it’s been shown in vitro that silencing ENO1 inhibits the proliferation, migration and invasion of breast cancer cells [24] and in a xenograft mouse model, inhibition of ENO1 expression increased tolerance to hypoxia in tumour cells, showing also slow reduced tumour size, cell growth and increased apoptosis [25]. A recent, single-cell transcriptomic profiling of breast cancer patients, identified higher ENO1 expression in the aggressive basal subtype, compared to hormone-and/or HER2-positive subtypes and this overexpression was linked to worse relapse-free survival [26]. They also showed that depletion of ENO1 in triple-negative breast cancer cell lines halted cell proliferation, colony formation and tumour growths (3D-organoids) and increased cell death suggesting that ENO1 could be used as a therapeutic target in this aggressive subtype [26].…”

“…A recent, single-cell transcriptomic profiling of breast cancer patients, identified higher ENO1 expression in the aggressive basal subtype, compared to hormone-and/or HER2-positive subtypes and this overexpression was linked to worse relapse-free survival [26]. They also showed that depletion of ENO1 in triple-negative breast cancer cell lines halted cell proliferation, colony formation and tumour growths (3D-organoids) and increased cell death suggesting that ENO1 could be used as a therapeutic target in this aggressive subtype [26]. However, a more comprehensive data analysis will allow us to better characterise the role of ENO1 in breast cancer progression and its potential use as a targeted therapeutic agent.…”

Enhancer regulatory networks globally connect non-coding breast cancer loci to cancer genes

“…5a). Consistent with this subtype specificity of transcriptional programs, previous studies show that both transcriptome and epigenome landscapes are distinct between ER+ and ERpatients 89,90 , single cell RNA-seq reveals transcriptome diversity between different subtypes of breast cancer 91 , and risk loci identified in GWAS show specificity between ER+ and ERpatients 8 . To examine enhancer specificity in different breast cancer molecular subtypes, we repeated the single-cell CRISPRi screen targeting the same genomic regions in (ER-) MDA-MB-231 (Fig.…”

Transcriptome profiling and network enrichment analyses identify subtype-specific therapeutic gene targets for breast cancer and their microRNA regulatory networks

“…The 3D organoid cultures were conducted as we previously described [ 21 ]. Briefly, 250,000 transfected cells were mixed with overnight thawed Matrigel (Corning cat.…”