Rendering to the carcinogenesis models, cancer cells can originate from any cell of the body and have potential to grow at a specific site or become malignant, presenting unlimited proliferation following multiple mutations. Evidence has suggested that the capability of cells to initiate a tumor is a unique characteristic of cells with stemness properties. These putative CSCs have been isolated from different cell types including CRC, a significant disease worldwide. Every year over a quarter of million people are observed to be affected by CRCs. The CRC is the second most common cause of cancer relating deaths worldwide with one million new cases diagnosed per year [1-3]. Evidence has shown that the lifetime risk of occurrence of CRC in industrialized nations is approximately 5%, and the lifetime risk of developing an adenoma is 20% [4-6]. When the cancer is only at its primary site, there is a 70% to 90 % chance for acure. However, mortality rate increases in the metastatic state. CRC is rated as one of the top three reasons for cancer-related death worldwide [4]. At ShaukatKhanum Memorial Cancer Hospital and Research Center (Pakistan), colorectal cancers are among the top five malignancies (4.8% of 16,000 cases registered from 1994-2014) (https://www.shaukatkhanum.org.pk/images/skm_img/downloads/pdf/ccrr-1994-2014.pdf).

CSCs have been observed to be resistant to chemotherapy [7] compared to non-CSC populations [8,9] leading towards the increased risk of cancer recurrence. Over the years many drugs have been developed for targeting the CSC population [10]. For this purpose, it is important to identify the regulatory mechanisms and signaling pathways which are involved in CSCs renewal. Hence, these investigations require testing the ability of drugs which kill CSCs to prevent the relapse of disease.

Key Words: Colorectal Cancer Stem Cells (CRCSC); Drugs causing cell cycle arrest; Drugs targeting WNT; EMT pathways; Plant derived drugs against colorectal cancer stem cells.

Abbreviations:

CSCs              :               Cancer Stem Cells (CSCs)

CRC               :               Colorectal Cancer

SCs                 :               Stem Cells

ESCs              :               Embryonic Stem Cells

NSCs              :               Neural Stem Cells

HSCs              :               Hematopoietic Stem Cells

ASCs              :               Adult Stem Cells

LTSCs            :               Long-Term Stem Cells

STSCs            :               Short-Term Stem Cells

AML              :               Acute Myeloid Leukemia

mCRC           :               Metastatic Colorectal Cancer

VEGF             :               Vascular Endothelium Growth Factor

Cmab             :               Cetuximab

Pmab             :               Panitumumab

EGF                :               Epidermal Growth Factor

EGFR             :               Epidermal Growth Factor Receptor

GF                   :               Griseofulvin

ND                  :               Nocodazole

FZ                   :               Frizzle

APC                :               Adenomatous Polyposis Coli

DC                  :               Destruction Complex

TCF4              :               Transcription Factor 4

ART               :               Artesunate

NSAID           :               Non Steroidal Anti-Inflammatory Drugs

COX               :               Cyclo Oxygenase

EMT               :               Epithelial to Mesenchymal Transition

SMA               :               Smooth Muscle Actin

FN                   :               Fibronectin

CKs                :               Cytokeratin’s

HAD               :               Histone Deacetylase

TSA                :               Trichostatin A

VPA                :               Valproic acid

SFN                :               Sulforaphane

TET                :               Tetrandrine

LUT                :               Luteolin

AOM              :               Azomethane

Stem cells

Normal Stem Cells

Undifferentiated cells possessing the ability to self-renew, generating one daughter cell identical to mother cell (containing self-renewal potential) and the second more specialized cell, are known as stem cells (SCs) [11]. SCs with the property of self-renewal and heterogeneity [12] have various categories with respect to their differentiation pattern. SCs are either totipotent (able to give rise to a new full organism), pluripotent (able to give rise to all tissues of the body except trophoblast of placenta) or multipotent (able to produce all cell types in a certain organ or location) [11]. Embryonic stem cells (ESCs), Neural Stem Cells (NSCs) and Hematopoietic Stem Cells (HSCs) are the best characterized types of SCs [13]. Between the developmental stages (change from totipotent to pluripotent), appoint comes when the cells preserve the state of undifferentiating by self-renewal, known as Adult Stem Cells (ASCs). However these are dedicated to the specific lineages of the organ to which they belong [14]. Studies have shown that ASCs having greater developmental potential, remain quiescent (non-dividing) for long periods of time, in G₀ phase of the cell cycle [14-17], however cellular differentiation asymmetrically takes place when they become activated by a normal need for more cells to maintain tissues, or in drastic conditions (differentiate in symmetric fashion for the maintenance of tissue homeostasis/tissue repair after injury) [18-22]. It has been shown that rapidly regenerating tissues have certain heterogeneity in cell cycle kinetics among SCs [15]. According to cellular division cycles there are two types of SCs present – long-term stem cells LTSCs, having unlimited self-renewal capacity, and short-term stem cells STSCs having limited potential to self-renew [20].

Cancer Stem Cells

CSCs may be the derivatives of normal self-renewing cells after abnormal differentiation pattern or from progenitor cells that might gain oncogenic activity and lose tumor suppressor function, thereby instigating de-differentiation [23-25]. These CSCs go into symmetric fashioned cell division pattern and this evidence was shown by Cicalese’s group who worked on an advanced assay with a fluorescent dye by working on breast CSCs [26]. They found that normal cells mostly divide asymmetrically by losing self-renewal potential in cultures as compared to tumor cells which divide in symmetrical way but are nearly immortal, increasing five-folds after every passage [26]. In a tumor, a heterogeneous population of cells is always present and contains mature cells which express differentiation markers reflecting their origin. Furthermore, the cells which don’t express surface markers have an immature morphology [27-29].The existence of CSCs was first reported by Bonnet and Dick [30]. They reported that only those cells which haveCD34⁺/CD38^–markers can produce acute myeloid leukemia (AML) in NOD/SCID mice. The first report about existence of CSCs in solid tumor came in 2003, by Al-Hajj et al. [31] who worked on breast cancer [31]. Thus far, CSCs have been found in numerous solid tumors, including lung cancer [32], colorectal cancer [33], prostate cancer [34], brain cancer [35], and melanoma [36]. It has been reported that CD133⁺CD44⁺CD166⁺EpCAM⁺CD24⁺ are the colon cancer markers [33,37-39]. Moreover, within CRC, CD133⁺ or CD44⁺ cell subpopulations have been recognized as CSCs, which motivate tumor progress and recurrence [33,38,40-42].

Drugs targeting CRC and colorectal cancer stem cell (CCSCs)

Almost all CRCs start from benign polyps and then slowly progress into malignant tumors [2].Colonoscopy can be used for screening these pre-cancerous polyps, facilitating the detachment before malignant transformation [43]. Evidence indicates that liver is the most common site of Metastatic Colorectal Cancer (mCRC) [44-47]. Typically conventional adjuvant and neo-adjuvant types of chemotherapy is effective [45]. For mCRCFOLFOX (a combination of 5-flourouracil, leucovorin, and oxaliplatin) and FOLFIRI (a combination of 5-flourouracil, leucovorin, and irinotecan) is commonly administered [48]. In neo-adjuvant chemotherapy, anti-angiogenic drugs, such as Bevacizumab (Avastin^®; targets the vascular endothelium growth factor, VEGF, pathway) are combined with Cetuximab (Cmab) or panitumumab (Pmab) (target epidermal growth factor (EGF) pathway by acting on its receptor (EGFR), [47,49,50]. Todaro et al. states that these type of combination therapies work for many patients survival but are not effective for mCRC patient soften resulting in relapse [51]. This may be due to intervention of chemotherapeutic drugs with rapidly growing tumor cells, but not with CSCs, leading to tumor recurrence.

Todaro et al. [52] studied the exposure of oxaliplatin on colon CSCs derived xenografts, and found a decrease in tumor size, however there was a considerable amplification in the proportion of CD133⁺ cells [52]. They also studied the inhibition of IL-4 signaling transduction pathway with an anti-IL-4 neutralizing antibody or anIL-4 receptor α-antagonist sensitized CSCs to chemotherapeutics through down-regulation of anti-apoptotic proteins, such as cFLIP, Bcl-xL, and PED [52]. Also a number of immune therapies and differentiation therapies have been studied against colon CSCs. Moreover, it has been detected using a tumor sphere assay that Salinomycin a polyether ionophore antibiotic isolated from Streptomyces albus, but not oxaliplatin or cisplatin, was able to decrease the CSCs population [53]. Salinomycin causes CCSCs apoptosis by selective target on CD133⁺ sub-population and decreases malignancy [53]. Although these drugs have been shown to be very effective against signaling pathways specific to CSCs. Particularly, the bio molecules, Salinomycin, parthenolide and biguanide metformin have been reported to produce tumor cell death in various cancers, and may contribute to eradicate cancer more efficiently than compounds which target CSCs or regular cancer cells [54-56].

Drugs targeting CCSC cycle checkpoints

The cell cycle machinery controls cell proliferation, and cancer is a disease of inappropriate cell proliferation [57]. Although many anti-cancer drugs have been discovered, causing inhibition of cell growth at some level, however, CSCs like NSCs undergo G₀, G, S, G₂ and M phase for division with their specific property of self-renewal [58]. Reports say that AEE788 (dual receptor TKI of both EGFR and VEGFR)causes cell cycle arrest in various human cancer models [59,60] including colon cancer cells [61]. In addition, celecoxib has been studied as a COX-2 inhibitor and causes cell cycle arrest on G₀/G₁ phase thus inhibiting the transition to S phase [62]. Valverde et al. [63] used the combination therapy of AEE788 and celecoxib and demonstrated not only enhanced efficacy to inhibit colon cancer cell proliferation, migration and angiogenesis but also reduced colon CSCs sub-population by targeting stemness-related pathways [63].They also showed that AEE788 caused EGFR down-stream signaling thus constraining the cell proliferation and cell cycle arrest at G1 phase. Nonetheless, the antitumor action of this combination therapy is reliant on wild type cell K-Ras status. Another oral antifungal drug, Griseofulvin (GF) at micro molar concentrations, caused cell cycle arrest at G₂/M phase by abnormal microtubule formation, elevation of cyclin B1/cdc2 kinase activity and down-regulation of myt-1 protein expression [64]. The combined drug therapy of Griseofulvin and Nocodazole (ND) was used in athymic mice bearing human colorectal cancer xenografts, which concluded that both drugs combine causes synergistic induction of apoptosis and G₂/M arrest [64]. It has been reported previously that the inhibition mechanism of Wnt/β-catenin signaling down-regulates the expression of cdc25c, cdc2, and cyclinB, resulting in G₂ arrest. Also, the target gene of β-catenin activity, Axin2 play role in mitosis. Axin2 is highly up-regulated in colorectal cancers [65]. Furthermore they reported that NC043 (diterpinoid derivative 15-oxospiramilactone) perhaps regulates the activity of Axin2, leading to cell cycle arrest at G₂/M phase [66].

Drugs targeting Wnt signaling pathway

The activities of the Wnt signaling pathway has been reported to be involved in the regulation of normal stem cells, tumor cells and cancer stem cells [67,68]. For the control of intestinal epithelial stem cell function , Wnt signaling pathway is regulated, in which Wnt binds to Frizzle (FZ), activating one of the two pathways [69]. In first, canonical, β-catenin becomes active which is being regulated by highly conserved proteins (controls cellular proliferation) and in second, non-canonical, Ca²⁺ is involved (pedals cellular movement and polarity) [70-72]. In the canonical/β-catenin pathway, when Wnt signals are absent, the tumor suppressor, adenomatous polyposis coli (APC) and Axin/Axin2 proteins in the destruction complex (DC) (composed of the tumor-suppressor protein APC, glycogen synthase kinase3β and axin), become activated for targeting β-catenin [73,74]. The outcome is serine phosphorylation of β-catenin, recognition by an E3 ubiquitin ligase, and its subsequent degradation [68]. If Wnt signal is present, the kinase activity of DC is blocked and β-catenin remain sun-phosphorylated, resulting in accumulation of β-catenin in the nucleus [68]. Hence, β-catenin remains bound to the transcription factor TCF4 that can activate downstream target genes such as the proto-oncogene MYC promoting entry of the cell into the S-phase of the cell cycle [75]. Evidence suggests that mutations in APC (tumor suppressor gene), serine/threonine residues, scaffolding protein Axin2 or the formation of nuclear TCF/β-catenin complexes results in uncontrolled TCF target gene transcription [76-79].

For colorectal cancers, associated with a hyperactive Wnt/β-catenin pathway, disruption of the signaling pathway provides a target for new anti-cancer drugs [80]. It has been reported that in the development of cancer, the mutation of Wnt/β-catenin components reduce normal ubiquitination and degradation of β-catenin protein [81,82]. Lin et al. [83] used Artesunate (ART) in vitro on CLY cell line. They reported that ART treatment caused β-catenin to translocate from the nucleus to adherent junctions of membrane which switched the function of β-catenin from promoting target genes expression to enhancing cell adherence. ART helps in decreasing mRNA level of Wnt/β-catenin target genes such as c-myc and surviving, thus promotes anti-tumor activity [83].

Aspirin (non-steroidal anti-inflammatory drug) constrains β-catenin/TCF4 signaling in colon cancer cells [84-86]. Coxibs (such as celecoxib and rofecoxib) also affect CCSCs independently of COX-2 expression [87]. Tuynman et al. [88] suggested that celecoxib limits c-Met-dependent signaling, resulting in down-regulation of oncogenic Wnt signaling in CRC [88]. Similarly, Salinomycin, has been reported to inhibit CSCs growth in different types of human cancers including CRC, almost certainly by snooping with ABC drug transporters, the Wnt/β-catenin signaling pathway, and other CSC pathways [89].

Nangia-Makker et al. [90] studied the effect of metformin in combination with 5-FU and oxaliplatin (FuOx) on CCSCc by down-regulation of Wnt/β-catenin signaling pathway [90]. An et al. [91] studied the effect of a LP SN (Lactobacillus plantarum (LP) supernatant (SN) with combination of 5-FU, and their results showed that LP SN can increase the therapeutic effect of 5-FU for colon cancer, and lessen CCSC by reversing the development of resistance to anti-cancer drugs. So, they concluded that probiotic (a microorganism introduced into the body for its beneficial qualities) substances may be a useful therapeutic alternatives as bio-therapeutics for chemo-resistant CRC [91].

Drugs targeting EMT pathway

The Epithelial-to-Mesenchymal Transition (EMT) program is a diverse series of events that outcomes in the epithelial cells’ losing their epithelial characters and obtaining numerous properties of mesenchymal cells (including increased expression of vimentin, Smooth Muscle Actin (SMA), FibroNectin (FN), matrix metalloproteinases and N-cadherin same as shift to fibroblastic morphology in monolayer culture [92-94]. Epithelial cells become more motile and lose their cell to cell polarity and cytoskeletal reorganization because they transit to mesenchymal cells by decreasing expression of epithelial Cytokeratin’s (CKs), like CK-8 and CK-18, in addition to reduced expression of cell-to-cell adhesion proteins (e.g., E-cadherin and plankoglobin (leading towards disassembling of adherens junctions and desmosomes respectively) [92-94]. Transcriptional repression of E-cadherin promotors, the proteolytic cleavage of E-cadherin protein and any somatic or chromosomal mutations can be the reason of inactivation of E-cadherin [95].

Throughout cancer development, various stimuli can trigger EMT by secreting many molecules, such as Hedgehog, EGF, hepatocyte growth factor, and members of the TGF-β, Wnt, fibroblast growth factor, and insulin-like growth factor families [96]. Recent studies have highlighted a link between EMT and CSC formation that it is sufficient to endow differentiated normal and cancer cells with stem cell properties. This relationship between EMT and CSCs might have many implications in tumor progression; hence explore the importance of these links in the development of improved antitumor therapies. EMT pathway causes drug resistance in CSCs and ultimately results in tumor relapse [96]. Salinomycin prompts expression of E-cadherin by RNA interference and down-regulates expression of vimentin in HT29 tumorous cell line [53]. Histone deacetylase (HDA) inhibitors like trichostatin A (TSA) and valproic acid (VPA) have been recently reported to induce mesenchymal features in CRC by decrease in E-cadherin and increase in vimentin expression at mRNA and protein levels [97]. A study reported that PS341 (Bortezomib) is first proteasome drug that inhibit mCRC by inhibition of cell proliferation, Epithelial-Mesenchymal Transition (EMT), the expression of stemness-related genes, cell migration and invasiveness [98].

Plant Derived drug therapies

Natural extracts causing CCSC cycle arrest

Resveratrol is a naturally occurring polyphenol (derived from grapes) [99,100] with cancer chemo-preventive properties [101]. The effect of this compound on human colonic adeno carcinoma cell line CACO-2 is an increase of apoptotic activity of cells after 24 and 48h of treatment with 200 µmol/L resveratrol, by measuring caspase-3 activity. A disrupted cell cycle progression from S to G₂ phase was observed up to 50 µmol/L concentration (cell cycle arrest), while higher concentrations led to reversal of S-phase arrest. Furthermore, down-regulation of cyclin D₁/CDK₄protein complex was also observed [102]. Another group studied the effect of Resveratrol on HCT116 colon cancer cell line, and they concluded that a dose dependent activity of this compound on CSC of CRC, and the cell cycle arrest at G0/G1 phase with promotion of cell apoptosis [103]. Sulforaphane (SFN) (naturally occurring Isothiocyanate, found in cruciferous vegetables) has also anti-tumoral properties causing cell cycle arrest at G₂/ M phase in HT29 human colon cancer cell line, correlated with greater than before expression of cyclin A and B1 [104-106]. A number of studies have also reported that sulforaphane may target CSCs in different types of cancer through modulation of NF-κB, SHH, epithelial-mesenchymal transition and Wnt/β-catenin pathways [107]. Flavonoids, present in many fruits and vegetables, have been comprehensively studied [108-112]. It has been reported that flavonoids (like Quercetin) inhibits cell cycle progression at G1/S phase by effect on tyrosine kinase as well as other kinase activities [113-115] and another Genistein arrest cell cycle at G2/M phase by specific inhibition of tyrosine kinase activity [116]. Quercetin has also been reported to obstruct G2/M phase of CSCs [117].

Plant derived drug therapies against Wnt pathway

A natural product bis benzyl isoquinoline alkaloid tetrandrine (TET) exhibits anti-cancer activity against colon cancer cell line HCT116. TET effectively targets Wnt/β-catenin signaling pathway in human colon cancer cells by inhibiting the activity of TCF/LEF reporter, TOP-Luc and Myc/Max-Luc of a well characterized downstream target c-Myc. TET’s synergizes with 5-FU in an anti-proliferation effect on human colon cancer cells [118]. Luteolin (LUT), a flavonoid, which inhibited colon carcinoma by reducing Azomethane (AOM)-induced cell proliferation by the involvement of key components of Wnt signaling pathway, β-catenin, GSK-3β enzyme and cyclin D1 [119]. Similarly, Fisetin (flavonoid) treatment resulted in down-regulation of COX2 protein expression and Wnt-signaling activity through down-regulation of β-catenin and TCF4 (T cell factor 4) and decreased the expression of target genes such as cyclin D1 and matrix metalloproteinase-7. Fisetin treatment suppresses the growth of colon cancer cells by inhibition of COX2 and Wnt/EGFR/NF-Ò¡B signaling pathways [120]. Curcumin and piperinehave also been described to inhibit Wnt/β-catenin pathway in CSCs [121].

Plant derived drug therapies against EMT pathway

It has been reported previously, that Curcumin (derivative of rhizomes of plant curcuma longa) and its analogues have been shown to be effective in dropping tumor relapse by targeting the CSC population on signaling pathways (Wnt/β-catenin, Notch and Hedgehog) and EMT at multiple levels [122]. Resveratrol has also been reported to suppress EMT trough TGF-β1/ SMADS signaling pathway mediated SNAIL/ E-cadherin expression [123].

Conclusion

Targeted therapy by identifying new targets in colorectal cancer stem cells followed by discovery of novel drugs to directly kill these resistant cell populations is required to eradicate the disease completely.

Acknowledgment

Authors thank the Shaukat Khanum Memorial Cancer Hospital and Research Center, Lahore, Pakistan.

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Citation: Saleem S, Tariq S (2016) Drugs in Discovery Pipeline Targeting Colorectal Cancer Stem Cells. Gavin J Stem Cell Res Ther 2016: 4-11.