CRISPR screens identify cholesterol biosynthesis as a therapeutic target on stemness and drug resistance of colon cancer

Shanshan Gao,Huarong Chen,Shiyan Wang,Chi Chun Wong,Catherine Adell O’Brien,Yong Zeng,Musaddeque Ahmed,Housheng Hansen He,Zhenjie Yang,Fraser Soares,Weixin Liu,Minnie Y Y Go,Joseph J Y Sung,Jun Yu

doi:10.1038/s41388-021-01882-7

Abstract

Cancer stem cells (CSCs) are responsible for tumor progression, recurrence, and drug resistance. To identify genetic vulnerabilities of colon cancer, we performed targeted CRISPR dropout screens comprising 657 Drugbank targets and 317 epigenetic regulators on two patient-derived colon CSC-enriched spheroids. Next-generation sequencing of pooled genomic DNAs isolated from surviving cells yielded therapeutic candidates. We unraveled 44 essential genes for colon CSC-enriched spheroids propagation, including key cholesterol biosynthetic genes (HMGCR, FDPS, and GGPS1). Cholesterol biosynthesis was induced in colon cancer tissues, especially CSC-enriched spheroids. The genetic and pharmacological inhibition of HMGCR/FDPS impaired self-renewal capacity and tumorigenic potential of the spheroid models in vitro and in vivo. Mechanistically, HMGCR or FDPS depletion impaired cancer stemness characteristics by activating TGF-β signaling, which in turn downregulated expression of inhibitors of differentiation (ID) proteins, key regulators of cancer stemness. Cholesterol and geranylgeranyl diphosphate (GGPP) rescued the growth inhibitory and signaling effect of HMGCR/FDPS blockade, implying a direct role of these metabolites in modulating stemness. Finally, cholesterol biosynthesis inhibitors and 5-FU demonstrated antitumor synergy in colon CSC-enriched spheroids, tumor organoids, and xenografts. Taken together, our study unravels novel genetic vulnerabilities of colon CSC-enriched spheroids and suggests cholesterol biosynthesis as a potential target in conjunction with traditional chemotherapy for colon cancer treatment.

Highlights

Colon cancer is one of the most commonly diagnosed malignancy worldwide
To unravel druggable addition, marked and coordinated upregulation of most cholesessential genes in these characterized Cancer stem cells (CSCs)-enriched spheroids, we terol biosynthesis enzymes were validated in 3D CSC-enriched performed CRISPR/Cas9 dropout screening using our in-house Epi- spheroids by real-time PCR (Fig. 3E)
Cholesterol biosynthesis genes are overexpressed in human addition, we found that loss of HMGCR or FDPS significantly colon cancer and CSC-enriched spheroids In light of our findings, we investigated the potential role of cholesterol biosynthesis pathway in human colon cancer

Summary

Introduction

Colon cancer is one of the most commonly diagnosed malignancy worldwide. The current standard of care for patients with colon cancer is maximal surgical removal of tumor, followed by adjuvant chemotherapy or targeted therapy [1]. CSCs from primary colon tumors can be enriched and maintained by the well-established three-dimensional (3D) spheroid culture system, which preserves the capacity to re-establish cellular hierarchy recapitulating that of parental tumor and patient-specific genotypic and epigenetic signatures [2, 9, 11, 12]. These in vitro CSC-enriched spheroid models are clinically relevant in terms of their intrinsic resistance to conventional drugs [13]. We aim to uncover genetic vulnerabilities of colon CSC-enriched spheroids with a novel high-throughput CRISPR dropout screen

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Conclusion