Reporters of Cancer Stem Cells as a Tool for Drug Discovery

Abstract

In view of the importance of cancer stem cells (CSCs) in chemoresistance, metastasis and recurrence, the biology of CSCs were explored in detail. Based on that, several modalities were proposed to target them. In spite of the several clinical trials, a successful CSC-targeting drug is yet to be identified. The number of molecules screened and entered for clinical trial for CSC-targeting is comparatively low, compared to other drugs. The bottle neck is the lack of a high-throughput adaptable screening strategy for CSCs. This review is aimed to identify suitable reporters for CSCs that can be used to identify the heterogeneous CSC populations, including quiescent CSCs, proliferative CSCs, drug resistant CSCs and metastatic CSCs. Analysis of the tumor microenvironment regulating CSCs revealed that the factors in CSC-niche activates effector molecules that function as CSC markers, including pluripotency markers, CD133, ABCG2 and ALDH1A1. Among these factors OCT4, SOX2, NANOG, ABCG2 and ALDH1A1 are ideal for making reporters for CSCs. The pluripotency molecules, like OCT4, SOX2 and NANOG, regulate self-renewal, chemoresistance and metastasis. ABCG2 is a known regulator of drug resistance while ALDH1A1 modulates self-renewal, chemoresistance and metastasis. Considering the heterogeneity of CSCs, including a quiescent population and a proliferative population with metastatic ability, we propose the use of a combination of reporters. A dual reporter consisting of a pluripotency marker and a marker like ALDH1A1 will be useful in screening drugs that target CSCs.

Keywords: cancer stem cells; drug resistant CSCs; drug screening and discovery; fluorescent reporters; metastasis initiating cells.

Figure 1

The role of “CSC niche” in regulating cancer stem cell properties. The Tumor associated macrophages (TAM) secretes cytokines initiating JAK/STAT and TGF-β signaling pathways. The regions where blood supply reduces creates hypoxia, which activates Hedge Hog (HH) WNT, Notch and Hippo signaling pathways. The regions where there is no oxygen and nutrient supply undergo necrosis, which creates an acidic pH that activates Notch and Hippo. Cancer associated fibroblasts (CAFs) also secret factors that initiate different signaling pathways. As a result of the pathway activation, several CSC-associated molecules are activated that results in the induction of CSCs.

Figure 2

The regulation of drug resistant CSCs. The niche factors activates several self-renewal pathways like HIF1α, WNT, Hedgehog or TGF-β that regulate chemoresistance in a β-catenin dependent way. Other self-renewal markers like CD44 and CD133 can also regulate chemoresistance. CD133 regulates β-catenin, while CD44 regulates Hippo signaling (YAP/TAZ) to induce chemoresistance. The transcription complexes indicated in the figure results in the up-regulation of molecules like ALDH and MDR proteins including ABCG2.

Figure 3

The regulation of Metastatic CSCs. Different signaling pathways activate ALDH1A1, OCT4 and SOX2, which activates β-catenin to induce EMT. At the same time OCT4/SOX2 regulates EMT in a β-catenin independent way also. NANOG inhibits β-catenin, but activates EMT through other pathway. When there is induction of EMT, epithelial cells might acquire stemness and metastatic ability, and gradually lose the stemness. The hybrid cells showing stemness property and metastatic property together are the metastasis initiating cells (MICs).

Figure 4

Endogenous markers regulating properties of CSCs. Different signaling pathways regulate the expression of OCT4, SOX2 and NANOG which in turn up-regulates different molecules required for self-renewal, metastasis and chemoresistance.

Figure 5

ALDH1A1 in the regulation of CSC properties Both classical and non-classical pathways initiated by Retinol regulate stemness. Retinoic acid (RA) binds to its nuclear receptor RARα and activates their target genes for differentiation. When RA binds to PPARβ/δ/RXR or RARα/ERα, genes related to survival and stemness are up-regulated. The balance of these pathways maintain CSC self-renewal and differentiation. The enzyme activity of ALDH1A1, results in the detoxification of chemotherapeutic drugs to impart chemoresistance.