A Strategic Approach to Identification of Selective Inhibitors of Cancer Stem Cells.

Cancer stem-like cells (CSC) have been implicated in resistance to conventional chemotherapy as well as invasion and metastasis resulting in tumor relapse in majority of epithelial cancers including colorectal cancer. Hence, targeting CSC by small molecules is likely to improve therapeutic outcomes. Glycosaminoglycans (GAGs) are long linear polysaccharide molecules with varying degrees of sulfation that allows specific GAG-protein interaction which plays a key role in regulating cancer hallmarks such as cellular growth, angiogenesis, and immune modulation. However, identifying selective CSC-targeting GAG mimetic has been marred by difficulties associated with isolating and enriching CSC in vitro. Herein, we discuss two distinct methods, spheroid growth and EMT-transformed cells, to enrich CSC and set up medium- and high-throughput screen to identify selective CSC-targeting agents.

A Unique Nonsaccharide Mimetic of Heparin Hexasaccharide Inhibits Colon Cancer Stem Cells via p38 MAP Kinase Activation.

Targeting of cancer stem cells (CSC) is expected to be a paradigm-shifting approach for the treatment of cancers. Cell surface proteoglycans bearing sulfated glycosaminoglycan (GAG) chains are known to play a critical role in the regulation of stem cell fate. Here, we show for the first time that G2.2, a sulfated nonsaccharide GAG mimetic (NSGM) of heparin hexasaccharide, selectively inhibits colonic CSCs in vivo G2.2-reduced CSCs (CD133+/CXCR4+, Dual hi) induced HT-29 and HCT 116 colon xenografts' growth in a dose-dependent fashion. G2.2 also significantly delayed the growth of colon xenograft further enriched in CSCs following oxaliplatin and 5-fluorouracil treatment compared with vehicle-treated xenograft controls. In fact, G2.2 robustly inhibited CSCs' abundance (measured by levels of CSC markers, e.g., CD133, DCMLK1, LGR5, and LRIG1) and self-renewal (quaternary spheroids) in colon cancer xenografts. Intriguingly, G2.2 selectively induced apoptosis in the Dual hi CSCs in vivo eluding to its CSC targeting effects. More importantly, G2.2 displayed none to minimal toxicity as observed through morphologic and biochemical studies of vital organ functions, blood coagulation profile, and ex vivo analyses of normal intestinal (and bone marrow) progenitor cell growth. Through extensive in vitro, in vivo, and ex vivo mechanistic studies, we showed that G2.2's inhibition of CSC self-renewal was mediated through activation of p38α, uncovering important signaling that can be targeted to deplete CSCs selectively while minimizing host toxicity. Hence, G2.2 represents a first-in-class (NSGM) anticancer agent to reduce colorectal CSCs.

Heparan sulfate hexasaccharide selectively inhibits cancer stem cells self-renewal by activating p38 MAP kinase.

Heparan sulfate (HS) plays a role in the majority of essential hallmarks of cancer, yet its ability to modulate self-renewal, especially of cancer stem cells (CSCs), remains unknown. We have discovered that a non-anticoagulant HS hexasaccharide (HS06) sequence, but not other shorter or longer sequences, selectively inhibited CSC self-renewal and induced apoptosis in colorectal, pancreatic, and breast CSCs suggesting a very general phenomenon. HS06 inhibition of CSCs relied upon early and sustained activation of p38α/ß mitogen activated protein kinase (MAPK) but not other MAPKs family members i.e. ERK and JNK. In contrast, polymeric HS induced exactly opposite changes in MAPK activation and failed to inhibit CSCs. In fact, TCF4 signaling, a critical regulator of CSC self-renewal, was inhibited by HS06 in a p38 activation dependent fashion. In conclusion, HS06 selectively inhibits CSCs self-renewal by causing isoform specific activation of p38MAPK to inhibit TCF4 signaling. These observations on chain length-induced specificity carry major mechanistic implications with regard to HS in cancer biology, while also presenting a novel paradigm for developing novel anti-CSC hexasaccharides that prevent cancer relapse.Heparan sulfate (HS) of specific length, i.e., hexasaccharide (HS06), but not longer or shorter sequences, selectively inhibit cancer stem cells (CSCs) through isoform specific activation of p38 mitogen-activated protein kinase. These findings will have major implication for developing chemical probes to decipher complex signaling events that govern cancer stem cells. Additionally, there are direct implications for designing glycosaminoglycan based cancer therapies to selectively target CSCs that escape killing by traditional chemotherapy threatening cancer relapse.

Inhibition of insulin-like growth factor receptor/AKT/mammalian target of rapamycin axis targets colorectal cancer stem cells by attenuating mevalonate-isoprenoid pathway in vitro and in vivo.

We observed a co-upregulation of the insulin-like growth factor receptor (IGF-1R)/AKT/mammalian target of rapamycin (mTOR) [InAT] axis and the mevalonate-isoprenoid biosynthesis (MIB) pathways in colorectal cancer stem cells (CSCs) in an unbiased approach. Hence, we hypothesized that the InAT axis might regulate the MIB pathway to govern colorectal CSCs growth. Stimulation (IGF-1) or inhibition (IGF-1R depletion and pharmacological inhibition of IGF-1R/mTOR) of the InAT axis produced induction or attenuation of CSC growth as well as expression of CSC markers and self-renewal factors respectively. Intriguingly, activation of the InAT axis (IGF-1) caused significant upregulation of the MIB pathway genes (both mRNA and protein); while its inhibition produced the opposite effects in colonospheres. More importantly, supplementation with dimethylallyl- and farnesyl-PP, MIB metabolites downstream of isopentenyl-diphosphate delta isomerase (IDI), but not mevalonate and isopentenyl-pp that are upstream of IDI, resulted in a near-complete reversal of the suppressive effect of the InAT axis inhibitors on CSCs growth. The latter findings suggest a specific regulation of the MIB pathway by the InAT axis distal to the target of statins that inhibit 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Effects of IGF-1R inhibition on colonic CSCs proliferation and the MIB pathway were confirmed in an 'in vivo' HCT-116 xenograft model. These observations establish a novel mechanistic link between the InAT axis that is commonly deregulated in colorectal cancer and the MIB pathway in regulation of colonic CSCs growth. Hence, the InAT-MIB corridor is a novel target for developing paradigm shifting optimum anti-CSCs therapies for colorectal cancer.

A strategic approach to identification of selective inhibitors of cancer stem cells.

Cancer stem-like cells (CSC) have been implicated in resistance to conventional chemotherapy as well as invasion and metastasis resulting in tumor relapse in majority of epithelial cancers including colorectal cancer. Hence, targeting CSC by small molecules is likely to improve therapeutic outcomes. Glycosaminoglycans (GAGs) are long linear polysaccharide molecules with varying degrees of sulfation that allows specific GAG-protein interaction which plays a key role in regulating cancer hallmarks such as cellular growth, angiogenesis, and immune modulation. However, identifying selective CSC-targeting GAG mimetic has been marred by difficulties associated with isolating and enriching CSC in vitro. Herein, we discuss two distinct methods, spheroid growth and EMT-transformed cells, to enrich CSC and set up medium- and high-throughput screen to identify selective CSC-targeting agents.

Inhibition of C-terminal binding protein attenuates transcription factor 4 signaling to selectively target colon cancer stem cells.

Selective targeting of cancer stem cells (CSCs), implicated in tumor relapse, holds great promise in the treatment of colorectal cancer. Overexpression of C-terminal binding protein (CtBP), an NADH dependent transcriptional regulator, is often observed in colon cancer. Of note, TCF-4 signaling is also up-regulated in colonic CSCs. We hypothesized that CtBP, whose dehydrogenase activity is amenable to pharmacological inhibition by 4-methylthio-2-oxobutyric acid (MTOB), positively regulates TCF-4 signaling, leading to CSC growth and self-renewal. CSCs demonstrated significant upregulation of CtBP1 and CtBP2 levels (mRNA and protein) and activity partly due to increased NADH/NAD ratio, as well as increased TCF/LEF transcriptional activity, compared to respective controls. Depletion of CtBP2 inhibited, while its overexpression enhanced, CSC growth (1° spheroids) and self-renewal (2°/3° spheroids). Similarly, MTOB caused a robust inhibition of spheroid growth and self-renewal in a dose dependent manner. MTOB displayed significantly greater selectivity for growth inhibition in the spheroids, at least in part through induction of apoptosis, compared to monolayer controls. Moreover, MTOB inhibited basal as well as induced (by GSK-3ß inhibitor) TCF/LEF activity while suppressing mRNA and protein levels of several ß-catenin target genes (CD44, Snail, C-MYC and LGR5). Lastly, CtBP physically interacted with TCF-4, and this interaction was significantly inhibited in the presence of MTOB. The above findings point to a novel role of CtBPs in the promotion of CSC growth and self-renewal through direct regulation of TCF/LEF transcription. Moreover, small molecular inhibition of its function can selectively target CSCs, presenting a novel approach for treatment of colorectal cancer focused on targeting of CSCs.

Synthetic, non-saccharide, glycosaminoglycan mimetics selectively target colon cancer stem cells.

Selective targeting of cancer stem-like cells (CSCs) is a paradigm-shifting approach. We hypothesized that CSCs can be targeted by interfering with functions of sulfated glycosaminoglycans, which play key roles in cancer cell growth, invasion and metastasis. We developed a tandem, dual screen strategy involving (1) assessing inhibition of monolayer versus spheroid growth and (2) assessing inhibition of primary versus secondary spheroid growth to identify G2.2, a unique sulfated nonsaccharide GAG mimetic (NSGM) from a focused library of 53 molecules, as a selective inhibitor of colon CSCs. The NSGM down-regulated several CSC markers through regulation of gene transcription, while closely related, inactive NSGMs G1.4 and G4.1 demonstrated no such changes. G2.2's effects on CSCs were mediated, in part, through induction of apoptosis and inhibition of self-renewal factors. Overall, this work presents the proof-of-principle that CSCs can be selectively targeted through novel NSGMs, which are likely to advance fundamental understanding on CSCs while also aiding development of novel therapeutic agents.