Abstract
Cellular phenotype plasticity between the epithelial and mesenchymal states has been linked to metastasis and heterogeneous responses to cancer therapy, and remains a challenge for the treatment of triple‐negative breast cancer (TNBC). Here, we used isogenic human breast epithelial cell lines, D492 and D492M, representing the epithelial and mesenchymal phenotypes, respectively. We employed a CRISPR‐Cas9 loss‐of‐function screen targeting a 2240‐gene ‘druggable genome’ to identify phenotype‐specific vulnerabilities. Cells with the epithelial phenotype were more vulnerable to the loss of genes related to EGFR‐RAS‐MAPK signaling, while the mesenchymal‐like cells had increased sensitivity to knockout of G2‐M cell cycle regulators. Furthermore, we discovered knockouts that sensitize to the mTOR inhibitor everolimus and the chemotherapeutic drug fluorouracil in a phenotype‐specific manner. Specifically, loss of EGFR and fatty acid synthase (FASN) increased the effectiveness of the drugs in the epithelial and mesenchymal phenotypes, respectively. These phenotype‐associated genetic vulnerabilities were confirmed using targeted inhibitors of EGFR (gefitinib), G2‐M transition (STLC), and FASN (Fasnall). In conclusion, a CRISPR‐Cas9 loss‐of‐function screen enables the identification of phenotype‐specific genetic vulnerabilities that can pinpoint actionable targets and promising therapeutic combinations.
Highlights
Despite the substantial improvements in therapy over the last few decades, high-risk and metastatic breast cancer (BC) continues to be a demanding clinical challenge, causing the death of over 600 000 women globally each year [1]
In agreement with previous studies [10,11,12], we determined that D492 cells express high levels of epithelial markers such as E-cadherin and cytokeratins, while the D492M cells lost the expression of these proteins and gained expression of mesenchymal markers, including N-cadherin, vimentin, and several others (Fig. 1A,B)
When testing cell proliferation in 2D cultures, we observed faster growth of D492 cells compared with D492M (Fig. 1C), which is a common difference for their respective phenotypes
Summary
Despite the substantial improvements in therapy over the last few decades, high-risk and metastatic breast cancer (BC) continues to be a demanding clinical challenge, causing the death of over 600 000 women globally each year [1]. The failure of BC management is caused, in part, by the phenotypic plasticity of the cancer cells that contributes to metastasis and heterogeneous responses to therapy facilitating treatment resistance [2,3]. Phenotype plasticity, encompassing both epithelial–mesenchymal transition (EMT) and the reverse process, describes the cells’ ability to interconvert between phenotypic states along the EMT spectrum [4]. The transition to a mesenchymal phenotype includes the loss of polarity, the shift to a vimentin-based cytoskeleton, reduced cell–cell adhesion associated with an E-cadherin to N-cadherin switch, and increased motility and invasiveness [5]. It is important to identify genes or pathways that represent phenotype-associated vulnerabilities and could be exploited as actionable targets in a phenotypespecific manner
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