Emerging agents that target signaling pathways to eradicate colorectal cancer stem cells

2.1.1.1 Phenethyl isothiocyanate

Phenethyl isothiocyanate (PEITC) is a natural product present in many cruciferous vegetables with anti-cancer and chemopreventive activities in clinical trials and has been associated with the induction of oxidative stress [60]. Interestingly, PEITC has also been reported to be able to eliminate CSCs of different types of cancer, including cervical [61, 62], lung [63], ovarian [64], and breast cancers [64, 65]. Importantly, PEITC inhibited the properties of CRC-SCs through downregulation of the Wnt/β-catenin pathway, inhibition of proliferation, and induction of apoptosis in CRC DLD-1 and SW480 cell lines. PEITC decreased the percentage of CD133⁺ cells by approximately 3–16-fold [66]. Activation of the Wnt/β-catenin pathway with lithium chloride, an agonist of canonical Wnt signaling, elevated CD133 expression and suppressed the effects of PEITC on CRC-SCs [66]. PEITC also suppressed the expression of pluripotency factors, including OCT4, SOX2, and NANOG, in NCCIT cells, a pluripotent stem cell line from mediastinal mixed germ cell tumors [67, 68]. In vitro self-renewal capacity and clonogenicity and in vivo tumor growth and expression of pluripotency factors were also observed to be reduced after treatment with PEITC in an EpCAM-expressing CSC model derived from the CRC HCT116 cell line [67, 68]. These results support that PEITC is a candidate to eradicate CRC-SCs targeting Wnt signaling.

2.1.1.2 Sulforaphane

Sulforaphane is an isothiocyanate obtained from cruciferous vegetables with anti-cancer properties. Some studies indicate that it can act on CSCs [69-72]. Interestingly, sulforaphane reduced pancreatic CSCs by blocking the sonic hedgehog-GLI family zinc finger (GLI) [70] and NF-κB pathways [71] and enhanced imatinib against CSCs of chronic myelogenous leukemia by inhibiting Wnt/β-catenin function [72]. In CRC, sulforaphane caused cell cycle arrest and apoptosis in CRC HT-29 and Caco-2 cell lines [73, 74]. Recently, Chen et al. [75] demonstrated that sulforaphane inhibited CRC-SC properties by suppressing the transactivating p63 isoform α (TAp63α)/LGR5/β-catenin axis in vitro and in vivo. In addition, activation of the Wnt/β-catenin pathway leads to the expression of LGR5, which is also a promoter of the Wnt/β-catenin pathway when binding to R-spondin (RSPO), suggesting that LGR5 may be used not only as a marker but also as a target in CRC-SCs. LGR5 acts through TAp63α to enhance Wnt signaling and the activation of β-catenin targets, promoting the maintenance of stem cells. Sulforaphane treatment led to downregulation of CRC-SC markers (CD133, CD44, NANOG, and OCT4), suppressing the formation of colonies in a spheroid cell model with CRC HCT116 and SW480 cell lines. Moreover, the upregulation of TAp63α blocked the inhibitory effect of sulforaphane. These data indicated that sulforaphane is a useful drug to eliminate CRC-SCs by suppressing Wnt signaling.

2.1.1.3 Salinomycin

Salinomycin, an anti-bacterial polyether isolated from Streptomyces albus, selectively eliminated CD133⁺ cells in CRC [76]. Salinomycin induced caspase activation, increased DNA damage and caused disruption of the Wnt/β-catenin/TCF complex and apoptosis of human CRC-SCs, and decreased tumor growth and expression of CSC-related Wnt genes, including LGR5 [77, 78]. Treatment with salinomycin alone or in combination with the FOLFOX regimen (5-FU, folic acid plus oxaliplatin) was also evaluated in primary CSCs of patients with hepatic metastases of CRC or primary CRC [79]. Salinomycin exhibited superior anti-CSC activity and tumor growth inhibition when compared to the FOLFOX protocol, and salinomycin plus FOLFOX resulted in improvement of these effects when compared to salinomycin alone. Salinomycin increased the expression of LGR5 and mitigated the expression of ALDH1. Furthermore, salinomycin also caused apoptosis related to decreased cellular ATP production and the accumulation of dysfunctional mitochondria and reactive oxygen species [79]. Likewise, Singh et al. [80] demonstrated that salinomycin improved cell death receptors in CRC-SCs by inhibiting enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a histone-lysine N-methyltransferase enzyme. Importantly, salinomycin has also been reported to target CSCs of various types of cancer, including melanoma [81], osteosarcoma [82], breast [83], liver [84], brain [85], ovarian [86], and oral cancers [87]. Salinomycin specifically eliminated epithelial CSCs [88].

Wang et al. [89] developed salinomycin nanocrystals to improve their oral bioavailability/solubility and reduce toxicity. Salinomycin nanocrystals exhibited greater cell uptake efficiency, cytotoxicity and Wnt-inhibiting effects three times better than regular salinomycin, indicating that salinomycin nanocrystals have great potential for the clinical treatment of CRC. The combination of SN38, the active form of irinotecan, with salinomycin in nanoformulations also diminished the adverse effects and enhanced the treatment efficacy against CRC-SCs [90]. These results suggest salinomycin as a drug capable of eliminating CRC-SCs by targeting Wnt signaling and improving conventional chemotherapeutic effects, including the FOLFOX protocol.

2.1.1.4 Epigallocatechin gallate

Epigallocatechin gallate (EGCG), also known as epigallocatechin-3-gallate, is one of the catechins found in green tea. It has been widely studied as an anti-CSC agent against different types of cancer, including lung [91], bladder [92], pancreas [93], and breast cancers [94]. In combination with cisplatin or oxaliplatin, EGCG increased the therapeutic effect on human CRC cells [95]. Furthermore, EGCG was also effective in suppressing CRC-SCs by downregulating CD133, CD44, NANOG, OCT4, ALDH1, and Notch1 expression, inhibiting the ability to form spheres of CRC cells, and upregulating the expression of GSK-3β, a negative regulator of Wnt/β-catenin [96, 97]. In addition, EGCG induced the sensitivity of CRC-SCs to 5-FU [97].

In a pilot study with 125 patients, green tea extract reduced the recurrence of colorectal adenoma in approximately 50% of patients [98]. Moreover, in a randomized controlled clinical trial, 10 volunteers received 800 mg EGCG, and 11 volunteers received the same amount of EGCG plus Brazil nuts for 6 weeks. DNA methyltransferase (DNMT1) (DNA methylation is a molecular marker in CRC) and NF-κB expression were decreased in rectal biopsies of both groups [99]. In a clinical trial with 223 healthy Japanese individuals, Escherichia coli containing polyketide synthase (tumorigenic bacteria for CRC) in the gut microbiota was downregulated by ingestion of green tea [100].

Taken together, these preclinical results support that EGCG reduces the population of CRC-SCs and enhances the effect of cisplatin and oxaliplatin. Furthermore, clinical data suggest EGCG and/or green tea extract as a beneficial strategy in CRC therapy/prevention.

2.1.1.5 Farnesyl dimethyl chromanol

Farnesyl dimethyl chromanol (FDMC), a chemical structure that is part of the tocopherol and tocotrienol molecules (vitamin E), exhibits antioxidant properties [101]. Recently, Husain et al. [102] demonstrated that FDMC decreased the viability and self-renewal of CRC-SCs by downregulating Wnt/β-catenin signaling in vitro and in vivo. FDMC reduced spheroid and organoid formation and downregulated the expression of the pluripotent transcription factors NANOG, OCT4 and SOX2 in CD24⁺/CD44⁺/LGR5⁺ CRC cells. Induction of DNA fragmentation, exposure to phosphatidylserine, cleaved caspase 3 and cleaved PARP and reduced migration, invasion, VEGF and NF-κB and Wnt signaling were also observed in CRC-SCs treated with FDMC in vitro. In an orthotopic xenograft metastasis model, FDMC inhibited tumor growth and liver metastasis and suppressed angiogenesis and NF-κB and β-catenin signaling. This indicates that FDMC is effective in eradicating CRC-SCs.

2.1.1.6 4-Acetyl-antroquinonol B

4-Acetyl-antroquinonol B (4-AAQB), a mycelial from Antrodia camphorate, has been reported as a cytotoxic agent [103, 104]. In CRC, 4-AAQB reduced migration, invasion and clonogenicity in CRC DLD1 and HCT116 cell lines, with less effect on the viability and proliferation of normal colon cells. Downregulation of N-cadherin, vimentin, MYC and BcL-xL and upregulation of E-cadherin and BAX proteins were found in 4-AAQB-treated CRC cells. 4-AAQB also reduced the superoxide dismutase 2 (SOD2)-mediated CSC phenotype by hsa-miR-324. In vivo, 4-AAQB reduced tumor growth and enhanced the FOLFOX protocol by inhibiting SOD2 and increasing hsa-miR-324-5p expression [103]. In addition, 4-AAQB also downregulated the LGR5/Wnt/β-catenin, JAK/STAT, and non-transmembrane receptor tyrosine kinase signaling pathways and stemness-related factors in CRC cell lines. A reduction in tumor size was also observed after 4-AAQB treatment in CRC animal models [104]. These results suggest that 4-AAQB is useful to reduce the stemness properties of CRC cells and may improve the effect of conventional chemotherapeutics such as the FOLFOX protocol.

2.1.1.7 Diallyl trisulfide

Diallyl trisulfide (DATS) is a garlic-derived organosulfur compound with anti-tumor properties. Zhang et al. [105] reported that DATS reduced the size and number of spheroid forms of the CRC SW480 and DLD-1 cell lines and decreased the expression of CD133, CD44, ALDH1, OCT-4 and NANOG. The characteristics of cell death by apoptosis were found to be increased, including decreased Bcl-2 levels and increased BAX, caspase-3, -8 and -9. This effect was associated with the suppression of the activity of the Wnt/β-catenin pathway, observed by the increased expression of GSK-3β and decreased expression of β-catenin, MYC and cyclin D1. Pretreatment of the culture with lithium chloride blocked the effects of DATS. These results indicate that DATS can inhibit proliferation and induce apoptosis of CRC-SCs by suppressing Wnt signaling.

2.1.1.8 Zerumbone

Zerumbone, a sesquiterpene from Zingiber zerumbone, has anti-tumor effects in various types of cancer. In the CRC HCT-116 and SW-48 cell lines, zerumbone suppressed epithelial-mesenchymal transition (EMT), as observed by the increased expression of E-cadherin and reduced expression of vimentin, ZEB1 and N-cadherin [106]. A reduction in cell invasion, migration and spheroid formation was also found, as well as in the expression of CSC markers, including CD133, CD44, BMI1 and ALDH1. Zerumbon also reduced β-catenin expression. Interestingly, silencing of miR-200c inhibited these zerumbone effects, indicating that the inhibition of the β-catenin pathway through miR-200c may be a target of the anti-CRC-SC properties of zerumbone [106].

2.1.2.1 Retinoids

Homeobox A5 (HOXA5), a regulator of gene expression, morphogenesis, and differentiation, is downregulated in CRC. It inhibits Wnt signaling to reinforce differentiation through antagonists of APC downregulated 1 (APCDD1), CXXC finger protein 4 (CXXC4), and NKD inhibitor of Wnt signaling pathway 1 (NKD1) pathways [58]. All-trans retinoic acid and 9-cis retinoic acid upregulated HOXA5 levels, leading to tumor regression in the ALDH1⁺ HT-29 cell line. In this context, tumor regression via HOXA5 induction by retinoids suppressed Wnt signaling, leading to the differentiation and elimination of CRC-SCs, preventing tumor development, and inhibiting metastases [58]. All-trans retinoic acid also eliminated CSCs of head and neck cancer by inhibiting the Wnt/β-catenin pathway [107].

2.1.2.2 JIB-04

JIB-04 is an epigenetic modulator that acts as a pan-selective inhibitor of histone demethylases [108]. JIB-04 decreased the viability of HCT116 cells and reduced CD133 mRNA expression, CD133 protein expression levels, and clonogenic potential, indicating that JIB-04 resulted in a reduction in the CRC-SC population and self-renewal to form colonies [109]. Beyond that, JIB-04 mitigated invasion, migration, and growth/recurrence in an in vitro model. The expression level of E-cadherin mRNA (an epithelial marker) was increased, while those of vimentin and N-cadherin (mesenchymal markers) were decreased in cells treated with JIB-04. It also reduced the tumorigenic activity of CRC-SCs in vivo. These data were associated with increases in the expression of AXIN1 (axin-1) and GSK3β, genes involved in the inhibition of β-catenin signaling, and decreases in the expression of β-catenin target genes, including ALDH1 and SRY-box transcription factor 4 (SOX4) [109]. Importantly, JIB-04 had less effect on the viability of normal cells [110]. These results suggest that JIB-04 can be used to specifically target CRC-SCs.

2.1.2.3 IC-2

IC-2, a derivative of ICG-001 (a Wnt modulator), efficiently represses Wnt/β-catenin signaling [111]. Interestingly, IC-2 reduced the transcriptional activity of Wnt/β-catenin in CRC cells more effectively than 5-FU. IC-2 reduced the expression levels of CSC marker proteins, such as CD44, CD133, OCT3/4, NANOG, and LGR5. Then, CD44^high and CD44^low DLD-1 cells were isolated by cell sorting, and CD44^high sphere numbers were selectively reduced by IC-2, indicating that IC-2 preferentially suppresses CRC-SCs. Nevertheless, IC-2 did not affect viability at concentrations below 10 μmol/L. IC-2 also improved the cytotoxic effect of 5-FU, suggesting that IC-2 sensitizes CRC cells to 5-FU through the suppression of CRC-SCs [111]. Interestingly, IC-2 has also been reported as an anti-CSC agent against liver [112] and oral cancers [113].

2.1.2.4 NCB-0846

NCB-0846 is the first TRAF2- and NCK-interacting kinase (TNIK) inhibitor available orally with high activity against Wnt signaling by binding to TNIK, an essential activator of Wnt target genes. The anti-tumor and anti-CRC-SC activities of NCB-0846 were demonstrated [57, 114]. Pharmaceutical companies have developed other TNIK inhibitors that are in clinical trials, including ON108600 (OnconovaTherapeutics, Newtown, PA, USA), which was efficient in acting on CSCs [115]. Mebendazole, an anthelmintic agent, has also been identified as a selective TNIK inhibitor [116]. The combination of mebendazole and sulindac, a non-steroidal anti-inflammatory agent, reduced tumor initiation in CRC in a preclinical model [117].

2.1.2.5 36-077

36-077, an inhibitor of phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), was synthetized by Kumar et al. [118] as a potent autophagy inhibitor. In particular, 36-077 improved the efficacy of 5-FU in CRC Caco-2 and HCT116 cell lines grown in monolayers. Treatment of Caco-2 cells growing in spheroids or colon tumoroids derived from Apc^Min/+ mice with 5-FU plus 36-077 also reduced spheroid growth more than 5-FU or 36-077 alone. This effect was associated with the inhibition of the CSC population through GSK-3β/Wnt/β-catenin signaling. These data indicate that 36-077 can inhibit CRC-SCs by suppressing Wnt/β-catenin signaling and enhances 5-FU treatment.

2.1.2.6 CBB1003

CBB1003, a lysine-specific demethylase 1 (LSD1) inhibitor, has anti-tumor properties. Treatment with CBB1003 in HCT116 cells inhibited proliferation and colony formation and decreased the levels of the CSC marker LGR5 [119]. Moreover, the overexpression of LGR5 reduced cell death caused by CBB1003. Inactivation of β-catenin/TCF signaling was observed after treatment with CBB1003. The expression of LSD1, LGR5, β-catenin and MYC was found to be lower in adjacent normal tissues than in CRC tissues as well as in CRC cell lines [119]. These data indicate LSD1 as a target to affect CRC-SCs, inhibiting Wnt signaling. CBB1003 may be a useful anti-CRC-SC drug, acting by inhibiting LSD1.

2.1.2.7 Rimonabant

Rimonabant is an antagonist/inverse agonist of cannabinoid receptor 1 that can induce β-catenin degradation in CRC cell lines. The target genes Wnt/β-catenin, cyclin D1, MYC and cyclooxygenase-2 were reduced after treatment with rimonabant [120]. Non-canonical Wnt signaling was activated in CRC cells treated with rimonabant, as observed by the induction of Wnt5A and activation of calcium/calmodulin-dependent protein kinase II. Rimonabant also inhibited tumor growth and β-catenin transfer to cell nuclei in mice xenografted with HCT116 cells [120]. Fiore et al. [121] reported that rimonabant reduced primary CRC-SCs in culture through downregulation of β-catenin without affecting healthy colon epithelial cells. These results suggest rimonabant as an effective drug to eliminate CRC-SCs.

2.1.2.8 FH535

FH535 is a dual inhibitor of β-catenin/TCF/LEF and PPARs with anti-tumor action [122]. It reduced the migration and invasion of SW480 cells and downregulated the expression of matrix metalloproteinases 7 and 9, vimentin, and snail. FH535 also inhibited the expression of the CSC markers CD24, CD44 and CD133 in HT29 cells by inhibiting the Wnt/β-catenin pathway. FH535 also reduced tumor growth in mice xenografted with HT-29 [123]. This suggests that FH535 is able to eradicate CRC-SCs by targeting Wnt signaling.

2.1.3.1 HZ8CV2

Progastrin is overexpressed and detectable in the supernatant of CRC cell lines and patient-derived CRC cells and stimulates the survival of CSCs [124]. Downregulation of progastrin decreases Wnt/β-catenin activity and Wnt-induced tumorigenesis [125]. Prieur et al. [126] created a humanized anti-progastrin monoclonal antibody (HZ8CV2) that was effective against CRC with a KRAS mutation. Treatment of SW480 cells with HZ8CV2 for 96 h decreased the expression of survivin, a key component of the Wnt/β-catenin signaling pathway, by 40.7%. Beyond that, combination chemotherapy with the HZ8CV2 antibody increased chemosensitivity and delayed recurrence. HZ8CV2 in combination with 5-FU decreased 57.9% of the CSC population in KRAS-mutated T84 cells. In the xenograft model, the combination of HZ8CV2 with irinotecan decreased the frequency of CSCs by 55.9% and 70.1% in T84 and SW620 tumors, respectively, inhibiting the migratory and invasive properties of these CRC cells. These data corroborate that progastrin is a target to eliminate CRC-SCs and that HZ8CV2 is an effective anti-progastrin monoclonal antibody that inhibits CRC-SCs and improves 5-FU and irinotecan treatments.

These data indicate that the Wnt/β-catenin signaling pathway is a critical pathway to eradicate CRC-SCs. On the other hand, crosstalk among pathways can lead to resistance to single-pathway inhibitors and maintenance of the CSC phenotype [127, 128]. Therefore, multiple cellular pathways must be targeted to effectively eradicate CRC-SCs.

2.2.1.1 Honokiol

Honokiol, a naturally active biphenolic compound from Magnolia species used in traditional Chinese medicine, has anti-tumor properties against various types of cancer [134]. Honokiol is a potent inhibitor of melanoma stem cells that suppresses Notch signaling [135]. It also inhibits tumor progression and stem cells in glioma [136], breast cancer [137], and oral carcinoma cells [138]. In CRC, the combination of honokiol with ionizing radiation (IR) inhibited the levels of expression of CSC proteins, such as doublecortin-like kinase-1 (DCLK1), SRY-box transcription factor 9 (SOX9), CD133, CD44, Notch signaling-related proteins, and proteins of the γ-secretase complex in vitro, using HCT116 and SW480 cell lines, and in xenograft tissues using the HCT116 cell line [139]. Honokiol also slowed the growth of the tumor xenograft, inhibited proliferation, and promoted apoptosis. Furthermore, ectopic expression of the Notch intracellular domain partially rescues the honokiol effect, indicating that this compound is an anti-CRC-SC agent targeting Notch signaling [139].

Honokiol also suppressed the Hippo signaling pathway in CRC cells [140]. In this pathway, YAP1 is translocated to the nucleus to induce gene expression, which is inhibited by phosphorylation at Ser127. Honokiol inhibited YAP1 phosphorylation and caused apoptosis in HCT116 and SW480 cells. Inhibition of the expression of the CSC marker DCLK1 was also observed. In addition, honokiol prevented colitis-associated cancer growth in a mouse model using azoxymethane and dextran sulfate sodium [140].

2.2.1.2 Cucurbitacins

Cucurbitacin-B (C-B) and cucurbitacin-I (C-I) are phytochemicals present in various fruits and vegetables, such as bitter melon [141]. C-B and C-I inhibited cell proliferation and induced cell cycle arrest and apoptosis in CRC HCT116 and SW480 cell lines by downregulating ADAM9, which participates in the cleavage of the Notch receptor at the extracellular domain [142]. In addition, C-B and C-I reduced the spheroid and colony formation of CRC cells and inhibited the expression of the CRC-SC markers CD44, DCLK1, and LGR5. C-B and C-I also reduced tumor growth in mice xenografted with HCT116, as such reduced the expression of CSC markers and Notch signaling components in tumor tissues [142]. Together, these data suggest that cucurbitacins inhibit CRC, including CRC-SCs, by downregulating the Notch signaling pathway. Additionally, C-B was also able to eliminate liver [143] and gastric CSCs [144]. C-I also eliminated CSCs of the lung [145], brain [146], and head and neck [147] by blocking the JAK2/STAT3 signaling pathway.

2.2.1.3 Evodiamine

Evodiamine, a derivative of the traditional herbal medicine Evodia rutaecarpa, has an antiproliferative effect in the CRC LoVo cell line, leading to the induction of caspase-dependent apoptosis and S-phase arrest [148, 149]. The surviving cells after treatment with evodiamine were plated using serial dilutions and under conditions for the enrichment of CSCs by spheroids or implanted subcutaneously in immunodeficient SCID mice [150]. The results showed that evodiamine decreased the self-renewal of surviving cells in vitro and in vivo. Beyond that, the expression of the genes related to Wnt/β-catenin and Notch signaling was suppressed by treatment with evodiamine, indicating the elimination of CRC-SCs [150]. This indicates that evodiamine is an effective drug to eradicate CRC-SCs, suppressing Wnt and Notch signaling.

2.2.1.4 Quercetin

Quercetin, a natural flavonoid, is a compound that enhances radiosensitivity [151]. Quercetin plus IR eliminated CRC-SCs by inhibiting Notch1 signaling. Reductions in CSC markers (DCLK1, CD24, LGR5, CD29, CD44, and CD133) were observed in in vitro and in vivo studies. Quercetin plus IR suppressed the growth of both primary and secondary colonospheres and inhibited the Notch signaling pathway by downregulating γ-secretase complex proteins, Jagged-1, and Notch target genes hairy and enhancer of split-1 (HES1) and Hes-related with YRPW motif-1 (HEY1)[151]. This indicates that quercetin can induce radiosensitivity and eliminate CRC-SCs.

2.2.1.5 Portulaca oleracea extract

Portulaca oleracea is a grassy plant with succulent leaves and presents anti-tumor activities [152]. Portulaca oleracea extract inhibited the proliferation of CSCs (CD133⁺ and CD44⁺) by inducing apoptosis and downregulating the expression of Notch1 and β-catenin in CRC-SCs [153]. This indicates that Portulaca oleracea extract is a source of anti-CRC-SC agents.

2.2.1.6 α-Mangostine

α-Mangostine is a natural xanthone from Garcinia mangostana. It has antioxidant, anti-microbial, anti-cancer and anti-inflammatory properties [154]. α-Mangostine encapsulated in poly(D,L-lactic-coglycolic acid) (PLGA) nanoparticles inhibited self-renewal capacity, the expression of CSC markers, including CD133, CD44, Musashi, and LGR5, and pluripotency maintenance factors, such as OCT4, SOX2, KLF4, MYC, and NANOG. These effects were associated with inhibition of Notch signaling, suppression of the expression of Notch receptors (Notch1 and Notch2), their ligands (Jagged 1 and DLL4), protein of the γ-secretase complex (Nicastrin), and downstream target (Hes-1) in CRC HT-29 and HCT116 cell lines [155]. These results indicate α-mangostine as a drug with anti-CRC-SC properties.

2.2.1.7 Pien Tze Huang formula

Pien Tze Huang is a traditional Chinese herbal formula with anti-tumor properties. It inhibited CRC-CSs in the SW480 cell line [156]. In addition, Pien Tze Huang formula also decreased Notch1 and Hes1 expression in CRC-SCs [156]. This indicates that Pien Tze Huang formula is able to eliminate CRC-SCs by suppressing the Notch signaling pathway.

2.2.2.1 Anti-DLL4 antibodies

Anti-human and anti-mouse DLL4 antibodies have been developed as anti-tumor agents [157]. Moreover, anti-DLL4 antibodies were effective against both KRAS wild-type and mutant CRC cells. In combination with irinotecan, anti-DLL4 antibodies also increased CRC growth inhibition in a patient-derived xenograft model. They were also found to reduce the CRC-SC population [158]. These data indicate that these antibodies against the Notch DDL4 ligand are effective in eradicating CRC-SCs and in potentiating the effects of irinotecan. Notch signaling can interact and influence a large number of cancer-relevant pathways, including Wnt. Furthermore, crosstalk between the Wnt and Hedgehog pathways can also determine the general effect of Notch signaling [159, 160]. In this context, all cross-signaling networks in CSCs must be considered in a context-dependent manner. Clinical trials with Notch pathway inhibitors are still limited in the treatment of CRC. As observed above, these targeted agents have already been shown to be effective in preclinical studies and should also be tested in combination with classical chemotherapy drugs or other inhibitors of the signaling pathway in clinical trials to better understand their role in CRC therapy.

2.3.1.1 Cyclopamine

Cyclopamine, a naturally occurring alkaloid, is a classic inhibitor of the HH signaling pathway through the inhibition of SMO [172]. HCT-116 cells grown in serum-free non-adherent spheroids were treated with cyclopamine and showed reduced mRNA levels of CSC markers and genes related to HH signaling, including PTCH1, SMO, and GLI1 [173].

Zhou et al. [174], using bioinformatics analysis, selected a series of non-coding RNAs related to stemness in CRC-SCs and identified long non-coding RNAs (lncRNA-cCSC1) that are highly expressed in CRC and CRC-SCs and indicated a poor prognosis. After characterization, they observed that the depletion of lncRNA-cCSC1 levels was proportional to the increased sensitivity of CRC cells to 5-FU and to the inhibition of CRC-SCs’ self-renewal capacity via the HH pathway. The overexpression of lncRNA-cCSC1 increased the protein levels of factors related to stemness, such as CD133, CD44, and NANOG, and effector proteins of the HH signaling pathway, SMO and GLI1. In contrast, the use of cyclopamine caused a reduction in the levels of biomarkers related to stemness. These data indicate the potential action of the anti-SMO drug cyclopamine in eliminating CRC-SCs.

2.3.2.1 Vismodegib

Vismodegib (also known as GDC-0449) is a potent, orally bioavailable, specific SMO receptor antagonist. It is used to treat basal cell skin cancer in clinical practice approved by the United States-Food and Drug Administration [175]. Although still controversial, vismodegib has been reported as a drug able to inhibit CRC-SCs, since Wu et al. [176] showed that vismodegib inhibited cell proliferation and triggered apoptotic cell death with downregulation of Bcl-2 in CRC cells. In addition, it reduced the expression of the CRC-SC markers CD44 and ALDH by approximately 70%.

On the other hand, Chen et al. [177] demonstrated that vismodegib did not activate apoptosis in human CRC cells. Moreover, in a clinical trial of 199 patients with CRC treated with the FOLFOX or FOLFIRI (5-FU, leucovorin plus irinotecan) protocol combined with vismodegib, no benefit associated with the combination was observed [178].

Although some negative results have been found with HH signaling inhibitors, new inhibitors must be evaluated in CRC-SCs, especially compounds capable of inhibiting non-canonical HH signaling. These new data can help understand the potential of HH signaling inhibitors in CRC therapy.

2.5.1.1 Curcumin and GO-Y030

Curcumin is a polyphenol from Curcuma longa, and GO-Y030 is a novel curcumin analog. ALDH⁺/CD133⁺ subpopulations from CRC express high levels of phosphorylated STAT3, which enhances the proliferation and survival of CRC-SC lines [195]. Interestingly, curcumin and GO-Y030 inhibited STAT3 phosphorylation and induced apoptosis, as observed by increased expression of cleaved PARP and cleaved caspase-3 in CRC-SCs. In addition, curcumin induced cell cycle arrest, inhibited cell viability, decreased the ability to form spheroids, and suppressed tumor growth in a xenograft model. The effects of GO-Y030 were more potent than those of curcumin [195]. These data indicate curcumin and its analog GO-Y030 as drug candidates to eliminate CRC-SCs by suppressing STAT3 activity.

2.5.2.1 Napabucasin

Napabucasin (also known as BBI608) is an orally administered STAT3 inhibitor with anti-cancer activity against various types of cancer. It killed CRC-SC SW480 cells isolated by Hoechst-33342 dye exclusion sorting [196]. In a phase I clinical trial with four patients with metastatic CRC, napabucasin (240 mg twice daily) plus FOLFIRI and bevacizumab showed a safety profile in Japanese patients with metastatic CRC [197]. All four patients had diarrhea, two patients reported decreased appetite, and decreased neutrophil counts were observed in three patients. Deaths or serious adverse effects were not reported. A phase III clinical trial with patients with advanced p-STAT3⁺ CRC treated with napabucasin showed a significant survival benefit compared to placebo (median overall survival of 5.1 vs. 3 months, hazard ratio = 0.41, P = 0.0025) [198].

These preclinical and clinical data indicate that napabucasin is a promising anti-CSC drug in CRC therapy. It is the most advanced drug in development that targets cell signaling pathways to eradicate CRC-SCs and the only one with published results from phase III clinical trials. Napabucasin may be the first anti-CRC drug approved for clinical use targeting CSCs.

2.5.2.2 SC-43 and SC-78

SC-43 and SC-78, STAT3 inhibitors that stimulate Src homology 2 domain-containing protein tyrosine phosphatase 1 (SHP-1) to inactivate STAT3, suppressed the activation of STAT3 in CRC HCT-116 and HT-29 cell lines [199]. They also reduced the ability to form spheroids in an assay with cells grown on non-adherent spheroids without serum and supplemented with growth factors and to form colonies in a soft agar colony formation assay. A decrease in the CD133⁺/CD44⁺ subpopulation was also observed. Moreover, they synergize with oxaliplatin and/or irinotecan to inhibit sphere formation of these cells.

These data corroborate that STAT3 inhibitors may be useful to improve anti-CRC therapy targeting CRC-SCs.

2.6.1.1 GW6471

GW6471, a PPARα antagonist, interacts directly with PPARα activation function 2 (AF2) and prevents it from assuming an active conformation [204]. A CRC study using SW620, HT-29, WiDr, and SW480 cell lines showed that the PPARα pathway is active in CRC-SCs. Interestingly, GW6471 reduced the expression of CSC markers (SOX2, NANOG, and OCT4) and the ability of CRC cells to form spheroids [204]. These results suggest that GW6471 is a drug able to eliminate CRC-SCs. The relationship between the PPAR pathway and CRC-SCs is consistent, although more robust studies are needed to confirm the importance of this pathway in CRC-SCs.

2.7.1.1 Piplartine/piperlongumine

Piplartine, also known as piperlongumine, is an alkaloid amide isolated from peppers (the Piperaceae family). It presents multiple pharmacological activities, including cytotoxicity and in vivo anti-tumor effects against different types of cancers. The mechanism of action of piplartine has been associated with its capacity to induce oxidative stress by inhibiting the antioxidant system, including depletion of glutathione [213-217]. In particular, piplartine inhibited the Ras/PI3K/Akt/mTOR pathway and reduced tumor cell growth in a dimethylhydrazine/dextran sulfate sodium-induced colon carcinogenesis animal model [218].

Auranofin is a clinically approved synthetic drug for the treatment of rheumatoid arthritis. Anticancer properties have been reported for this gold molecule, where its main mechanism of action is inhibition of cellular antioxidant enzymes, such as thioredoxin reductase, causing oxidative stress and cell death in cancer cells [219]. Auranofin demonstrated selective capacity to improve the inhibition of CT26 colon tumor growth in a mouse model of abdominal irradiation without enhancing radiation toxicity in the normal intestine [220]. Auranofin also sensitized HCT116 and SW620 cells to 5Z-7-oxozeaenol, an inhibitor of TGF-β-activated kinase 1 [221].

Moreover, Tanaka et al. [222] demonstrated that piplartine plus auranofin decreased the expression level of the CD44v9 surface marker and reduced CRC growth in a patient-derived xenograft model. Piplartine also showed lower cytotoxicity to fibroblast cells than cancer cells growing in spheroids. This indicates that piplartine plus auranofin is able to eliminate CRC-SCs. Interestingly, piplartine also inhibited stemness properties in leukemia [223] and oral cancer [224] and had less effect on the viability of normal cells [213, 214].

2.7.1.2 Atractylenolide I

Atractylenolide I (ATL-1) is a plant-based compound found in the rhizome of Atractylodes macrocephala used in traditional Chinese medicine. In a study using CRC COLO205 and HCT116 cell lines, ATL-1 treatment induced apoptosis, inhibited invasion, downregulated the Akt/mTOR pathway by reducing the phosphorylation of pathway-related proteins, and reduced the stem cell phenotype [225]. In an in vivo experiment, ATL-1 reduced tumor weight and reduced CRC-SCs. This suggests that ATL-1 can inhibit CRC-SCs in vitro and in vivo.

2.7.1.3 Rapamycin

Rapamycin, a macrolide from Streptomyces hygroscopicus, is clinically used due to its immunosuppressant functions. It is a known mTOR inhibitor. Interestingly, rapamycin treatment decreased the spheroid-forming ability and ALDH1 activity in CRC Caco-2 and SW480 cell lines [211]. These effects were also observed in co-treatment with 5-FU and oxaliplatin. Importantly, 5-FU and oxaliplatin generally increased the CRC-SC subpopulation, but co-treatment with rapamycin reduced this cell subpopulation. These data indicate that rapamycin can inhibit CRC-SCs and potentiate the effect of 5-FU and oxaliplatin.

2.7.2.1 Dactolisib

Dactolisib (also known as BEZ235), a synthetic imidazoquinoline derivative, is a dual PI3K/mTOR inhibitor that selectively inhibits PI3K class I (p110α, β, δ, and γ), mTORC1, and mTORC2, inhibiting their catalytic activities and cell signaling [226]. Dactolisib suppressed the proliferation of CRC-SCs and reduced the expression of CD133 and LGR5 in HCT116 cells. In particular, insulin, a positive regulator of the PI3K/Akt/mTOR pathway, reduced the effect of treatment with dactolisib. This result suggests that dactolisib is a drug with the ability to eliminate CRC-SCs.

2.7.2.2 LY294002

LY294002, a synthetic compound developed based on the flavonoid quercetin, acts as a PI3K inhibitor. Treatment with LY294002 resulted in decreased proliferation, spheroid formation, and self-renewal properties, together with reduced Akt phosphorylation and cyclin D1 expression in CRC-SCs in vitro [209]. In an in vivo experiment, LY294002 reduced tumorigenicity, increased the detection of cleaved caspase 3, and increased the expression of the inflammatory chemokine IL-8. These data indicate that LY294002 is capable of eliminating CRC-SCs in preclinical models.

2.7.2.3 Taselisib, miransertib, and buparlisib

Taselisib, an inhibitor of PI3K class I, selectively inhibits phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA) and its mutant forms in the PI3K/Akt/mTOR pathway. Treatment of CRC-SCs from 60 samples from CRC patients with trastuzumab (an ERBB2 inhibitor), dabrafenib (a MEK inhibitor) or taselisib induced in vitro cell death and regression of tumor xenografts, including those with KRAS and PIK3CA mutations [210]. Furthermore, taselisib alone was also able to kill CRC-SCs and inhibited the formation of liver metastases in immunocompromised mice after injection of CRC sphere cells. Buparlisib (also known as BKM120), another PI3K inhibitor, and miransertib, an Akt inhibitor, also reduced the viability of CD44v6⁺ cells in vitro [210].

In a phase Ib clinical trial in patients with KRAS wild-type advanced CRC, buparlisib (60 mg given 5 out of 7 days per week) plus panitumumab (6 mg/kg injected intravenously every 2 weeks) was recommended for a phase II clinical trial [227].

2.7.2.4 Metformin

Metformin, an oral anti-diabetic drug, has also been reported as a cytotoxic agent with the ability to reduce the CSC population in different types of cancer [228-230]. In both flow cytometric analysis (CD44⁺/CD133⁺) and tumor spheroid growth assays, CSCs decreased after treatment with metformin in CRC HT-29 and DLD-1 cell lines [231]. Augmentation of the phosphorylation of adenosine monophosphate-activated protein kinase (AMPK) and reduction of mTOR (p-S6) were also observed after treatment with metformin. In a xenograft mouse model using HT-29 cells, tumor growth and CSC populations were decreased by metformin administration. Metformin effects were prevented by mevalonate treatment [231]. Interestingly, metformin also reduced CSCs in patients with colorectal and other gastrointestinal cancers in a pilot clinical trial [232].

These preclinical and clinical results indicate metformin as a promising anti-CRC-SC drug, but more clinical data are needed to use it in CRC therapy.

2.7.2.5 Torin 1, Torkinib and MK-2206

Torin 1, a selective inhibitor of mTOR, has anti-tumor properties. Torin 1 was able to induce cell death in CRC-SCs in vitro and in vivo without affecting the proliferation of normal colon stem cells in vivo [233]. Torkinib (PP242) is an ATP-competitive second-generation inhibitor of mTOR. It reduced CRC-SCs in in vitro models when it was used alone or in combination with 5-FU and oxaliplatin [211]. MK-2206 is an allosteric Akt inhibitor. It inhibited CRC-SCs, as observed by the reduction of the capacity to form spheres in vitro and to initiate tumor formation in vivo using the CRC SW480 cell line [212]. MK-2206 was also effective in reducing patient-derived spheroid formation.

These data indicate the importance of the PI3K/Akt/mTOR pathway in the control of CRC-SCs, showing that this pathway may be a target for therapeutic agents and can improve CRC therapy in the future.

2.8.1.1 Baicalin

Baicalin is a monomeric flavonoid from the root of Scutellaria baicalensis, a traditional Chinese herb. Baicalin has several pharmacological effects, including anti-cancer, anti-inflammatory, antioxidant and neuroprotective effects [243]. In CRC RKO and HCT116 cell lines, baicalin caused cell cycle arrest in the G₁ phase, p53-independent cell apoptosis, and EMT inhibition by inhibiting the TGF-β/Smad pathway [244]. Baicalin also inhibited the CRC-SC subpopulation in in vitro and in vivo models using the CRC HCT116 cell line.

Altogether, the TGF-β/Smad signaling pathway also plays an important role in CRC-SC maintenance, and novel selective inhibitors must be screened for CRC-targeting stemness properties.