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

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-ß 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.

Cancer stem cells and self-renewal.

The cancer stem cell (CSC) or cancer-initiating cancer (C-IC) model has garnered considerable attention over the past several years since Dick and colleagues published a seminal report showing that a hierarchy exists among leukemic cells. In more recent years, a similar hierarchical organization, at the apex of which exists the CSC, has been identified in a variety of solid tumors. Human CSCs are defined by their ability to: (i) generate a xenograft that histologically resembles the parent tumor from which it was derived, (ii) be serially transplanted in a xenograft assay thereby showing the ability to self-renew (regenerate), and (iii) generate daughter cells that possess some proliferative capacity but are unable to initiate or maintain the cancer because they lack intrinsic regenerative potential. The emerging complexity of the CSC phenotype and function is at times daunting and has led to some confusion in the field. However, at its core, the CSC model is about identifying and characterizing the cancer cells that possess the greatest capacity to regenerate all aspects of the tumor. It is becoming clear that cancer cells evolve as a result of their ability to hijack normal self-renewal pathways, a process that can drive malignant transformation. Studying self-renewal in the context of cancer and CSC maintenance will lead to a better understanding of the mechanisms driving tumor growth. This review will address some of the main controversies in the CSC field and emphasize the importance of focusing first and foremost on the defining feature of CSCs: dysregulated self-renewal capacity.

Cancer stem cells in solid tumors: an overview.

It has long been appreciated that significant functional and morphologic heterogeneity can exist within the individual cells that comprise a tumor. Increasing evidence indicates that many solid tumors are organized in a hierarchical manner in which tumor growth is driven by a small subset of cancer stem cells (CSCs) or tumor-initiating cells. Although these cells represent a small percentage of the overall tumor population, they are the only cells capable of initiating and driving tumor growth. Emerging evidence indicates that these cells are also resistant to chemotherapy and radiation therapy, which has led to much speculation and interest surrounding the potential clinical applicability of CSCs.

Colon cancer stem cells.

This unit describes protocols for working with colon cancer stem cells. To work with these cells one must start by generating single-cell suspensions from human colon cancer tissue. These cell suspensions are sorted using flow cytometry-assisted cell sorting to fractionate the cells into tumor-initiating and nontumor-initiating subsets. Once the cells have been fractionated, they must be functionally tested to determine tumor-forming capacity, the gold standard being the in vivo xenograft assay. Methods have also been developed to grow these cells in vitro in a sphere-forming assay. This unit will describe how to isolate and functionally test colon cancer stem cells, as well as provide advice on the potential challenges of the research.