Sam68 offers selectively aimed modulation of transcription in cancer stem cells

SUMMARY

In this issue of Cell Chemical Biology, Benoit et al. (2017) report the selective targeting of cancer stem cells (CSCs) by the ICG-001/CWP family of molecules. Their findings reveal that Sam68 is a novel transcriptional modulator uniquely required for the dysregulated Wnt/β-catenin signaling in CSCs over healthy stem cells.

MAIN TEXT

Ever expanding evidence suggests that cancer stem cells (CSCs), a rare cell population with enhanced survival and self-renewal capacity, are pivotal for tumorigenesis. Since their original identification in transformed hematopoietic tissue of adult myeloid leukemia (AML) patients (Bonnet and Dick, 1997), human CSCs have been found in a spectrum of cancers including blood and solid tumors (O’Brien et al., 2009). As a conserved feature among a variety of cancers, CSCs have long served as a biomarker for cancers and have emerged as a promising therapeutic target, particularly for targeting the crucial and unique signaling pathways that regulate survival and self-renewal in CSCs (Kahn, 2014; Takahashi-Yanaga and Kahn, 2010).

The Wnt/β-catenin pathway is an evolutionarily conserved signaling pathway that is crucial for normal healthy stem cells (SCs) as it helps maintain the SCs pluripotent state and regulates SC proliferation, differentiation and apoptosis (Clevers and Nusse, 2012). The canonical Wnt/β-catenin signaling pathway is activated through the stabilization and nuclear translocation of β-catenin. β-catenin then binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) transcription factors. This recruits the CREB-binding protein (CBP) and E1A-binding protein p300 (p300) transcriptional coactivators, which initiates transcription of Wnt genomic targets (Clevers and Nusse, 2012; Kahn, 2014). Interestingly, CBP/p300-dependent dysregulation of Wnt/β-catenin signaling and transcription has been recognized as a characteristic feature shared among a substantial number of human CSCs, regardless of tissue type (Kahn, 2014). Unfortunately, targeting the Wnt/β-catenin signaling pathway in CSCs has remained a challenge since healthy SCs also rely upon the pathway for survival and thus the normal SCs are also targeted. Indeed, various synthetic and natural small molecules exhibit Wnt/β-catenin inhibitory effects by antagonizing the interactions between β-catenin and TCF/LEF or β-catenin and CBP/p300; however, none of these inhibitors have successfully been transformed into cancer therapeutic agents. The apparent requirement of the Wnt/β-catenin signaling pathway in both CSCs and healthy resident SCs substantially reduces the selectivity of CSCs over SCs when targeting shared pathway component interactions, such as β-catenin-TCF/LEF and β-catenin-CBP/p300. It is therefore imperative that differences between the “dysregulated” Wnt/β-catenin signaling in CSCs versus the “regular” Wnt/β-catenin signaling in healthy SCs be identified. Finding these differences will ideally aid in the identification and exploitation of novel vulnerabilities unique to CSCs that can be targeted specifically with cancer therapeutics.

In this issue of Cell Chemical Biology, Benoit et al. ((Benoit et al., 2017) utilized the AML patient CSC xenograft model, a benchmark model of human CSC biology. The authors conducted rigorous in vivo assays with serial transplantation xenografts in mice to assess the impact of CWP232228, an analog of the well-known Wnt/β-catenin inhibitor ICG-001, on specifically targeting CSCs versus healthy hematopoietic SCs. Their findings demonstrate that CWP232228 selectively abolishes the self-renewal potential of CSCs derived from AML patients in vivo; whereas the small molecule does not affect normal SCs. Genome-wide transcriptome profiling data highlight that in vivo CWP232228 treatment substantially shifts the global gene expression pattern towards that of healthy hematopoietic SCs, particularly for Wnt genomic targets in AML patient-derived CSCs. Moreover, CWP232228 treatment significantly attenuates the recruitment of β-catenin and TCF to the promoters of a subset of Wnt target genes in CSCs, without impacting the β-catenin and TCF protein levels. These findings strongly suggest that CWP232228 treatment specifically alters the transcription of Wnt genomic targets in CSCs, but not in healthy SCs, thus selectively dampening the self-renewal potential of CSCs over normal SCs.

Mechanistically, Benoit et al. (2017) discovered that Sam68 (Src associated substrate during mitosis of 68 kDa) is overexpressed specifically in CSCs and functions as a novel transcriptional modulator uniquely required for CSC-specific Wnt gene transcription. Specifically, the CBP-β-catenin-TCF complex is responsible for the dysregulated transcription of Wnt genomic targets that favors CSC survival and self-renewal. Interestingly, CWP232228 treatment induces the functional SUMOylation of Sam68, which leads to the formation of a unique complex of Sam68 and CBP. The SUMOylated Sam68-CBP complex disrupts the CBP-β-catenin interaction, thus altering the transcriptional selectivity of Wnt target genes within CSCs and inducing apoptosis and differentiation. The SUMOylation mediated Sam68-CBP interaction within CSCs provides an opportunity to selectivity disrupt Wnt/β-catenin signaling and transcription in CSCs.

Sam68 is a versatile nucleic acid-binding protein that has emerged as a key modulator of promoter selectivity and transcriptional specificity under various pathophysiological conditions. Besides altering the transcription of Wnt genomic targets in CSCs, Sam68 has been reported to interact with other key transcription factors, including nuclear factor kappa B (NF-κB) and p53, and to be indispensable for NF-κB- and p53-conferred selective gene transcription (Fu et al., 2013; Li and Richard, 2016). The unique Sam68-CBP complex shifts the availability of transcriptional cofactors, hence switching Wnt transcriptional specificity; whereas the complex formed by Sam68 and p65 directly targets the binding of NF-κB to a select subset of promoters of NF-κB-dependent genes. These results underscore a pivotal role of Sam68 in directing the binding of transcription factors and cofactors to selective promoters and functionally modulating transcriptional specificity. The apparent distinct functions of Sam68 in switching the transcriptional specificity of Wnt/β-catenin and NF-κB could result from different post-translational modifications on Sam68. For example, SUMOylation of Sam68 is required for the formation of the Sam68-CBP complex that alters Wnt/β-catenin transcriptional selectivity. Additionally, phosphorylation of Sam68 on serines 113 and 117 has been proposed to be a prerequisite for Sam68 interactions with p65, which directs NF-κB to selective promoters (Fu et al., 2013). Dysregulated Wnt/β-catenin, NF-κB, and p53 signaling pathways and gene transcription are all associated with a wide variety of cancers and are critical in CSCs (Vazquez-Santillan et al., 2015). As Sam68 is involved in these pathways, it could be serving as an essential “switch” in CSCs that simultaneously modulates promoter selectivity and transcriptional specificity of Wnt/β-catenin, NF-κB, p53, and other critical transcription factors via unique and temporal post-translational modifications.

Overall, Benoit et al. (2017) provides important insights into the unique features of dysregulated Wnt/β-catenin signaling and transcription in CSCs as compared to the canonical Wnt/β-catenin signal transduction and gene expression required in normal SCs. These insights provide additional support for specifically targeting the unique vulnerabilities of CSCs as compared to healthy SCs and could provide a promising strategy for cancer therapeutics. For instance, the SUMOylated Sam68-CBP complex uniquely formed in CSCs offers CSC-targeting selectivity. The evidence that genetic ablation of Sam68 substantially dampens colon tumor development in genetically susceptible Min^Apc716/+ mice, without affecting normal colonic development (Fu et al., 2016), further supports that Sam68 could be a feasible CSC-unique vulnerability for therapeutics specifically targeting CSCs. Importantly, this study establishes a foundation for understanding the selective targeting of Sam68 in CSCs by small molecules in the ICG-001/CWP family. Since Sam68 is specifically overexpressed in CSCs and is involved in Wnt/β-catenin-, NF-κB-, and p53-conferred gene transcription in CSCs, targeting Sam68 selectively in the CSC microenvironment could be a safe and promising therapeutic option for cancer treatment. A comprehensive understanding of the Sam68 biology unique to CSCs as compared to normal SCs, and an increased understanding of the ICG-001/CWP family of molecules will greatly facilitate this encouraging endeavor.