Cell Biology International

REVIEW CD133: An emerging prognostic factor and therapeutic target in colorectal cancer

Morteza Akbari1,2,3, Navid Shomali2,4,5, Afsaneh Faraji2, Dariush Shanehbandi2, Milad Asadi2, Ahad Mokhtarzadeh2, Aliakbar Shabani3* and Behzad Baradaran2*


Colorectal cancer (CRC) is the third most common cancer worldwide affecting all races and both sexes almost equally. However, its incidence increases with age (Siegel et al., 2017). It is a multifactorial process affected by various genetic and environmental factors (Aran et al., 2016). Despite recent advances in its treatment, CRC remains an important healthcare issue, and in later stages, it bears a poor prognosis (Ren et al., 2013; Shirafkan et al., 2018). It has recently been suggested that, a small subgroup of cancer cells called cancer stem cells (CSC) has a self‐ renewal potential and induce tumor growth (Kawamoto et al., 2010). Evidences show that many malignancies such as CRC possess CSCs hidden in their tissues. This fact explains why many cancers recur after initial successful treatment. It also justifies tumor dormancy and metastasis (Batlle and Clevers, 2017). A great deal of effort has been put to identify CSCs in CRC using the expression of specific surface biomarkers. CD133, a five‐transmembrane glycoprotein, is one of these biomarkers whose role is the organization of the topology of the plasma membrane (Corbeil et al., 2001). Evidence suggests that CD133‐positive CRC cells have a potential of tumorigenesis. On the contrary, the CD133 negative cells are not able to induce tumor growth even after repeated transplantation in mice (Dalerba et al., 2007b). The identification of CD133‐positive cells is also of therapeutic and prognostic values. On the contrary, none of the proposed therapeutic measures for CRC is curative. The failure is believed to be the direct result of resistance acquisition in several tumoral cells due to mutations and natural selection (Ricci‐Vitiani et al., 2009).


5‐FU, 5‐fluorouracil; biAbs, bispecific antibodies; CCRT, concurrent chemoradiotherapy; CR, chemoradiation; CRC, colorectal cancer; CRT, chemoradiotherapy; CSC, cancer stem cell; EGFR, epidermal growth factor receptor; NSAIDs, non‐steroidal anti‐inflammatory drugs; OS, overall survival; PCI, photochemical internalization; PROM‐1, prominin 1; RFI, relapse‐free interval; TNM, tumor node metastasis; TSA, tumor‐specific antigens; VEGF, vascular endothelial growth factor (5‐FU) through the production of interleukin‐4 (Todaro et al., 2007). It has also been found that the Wnt signaling pathway may play a role in the CD133‐induced chemore- sistance in CRC (Deng et al., 2010; Karimi et al., 2017). A study also showed that the expression of CD133 in CRC cell bears a significantly poor prognosis and poorer clinical response to chemotherapy (Ong et al., 2010). Another study proposed that CD133 expression is accompanied by a higher rate of distant metastasis and metastatic relapse in CRC (Saigusa et al., 2009). It has been shown that, CD133‐targeted therapy of gastric and hepatocellular carcinoma has remarkably reduced cell proliferation and tumor growth both in vivo and in vitro in cells expressing CD133 marker (Smith et al., 2008). CD133 knockdown has also found to prevent glioma cell proliferation (Yao et al., 2009). Therefore, it can be a novel and promising target in the treatment of various malignancies including CRC (Zhao et al., 2015). This review aims to gather the old and most recent data on the prognostic and therapeutic values of CD133 and CD133‐ targeted therapies in CRC. This will then enlighten our view of how emerging chemotherapies may change the lives of patients suffering from end‐stage CRC (Montazami et al., 2015; Sadreddini et al., 2017). CD133 function and related pathways in CSCs Human CD133 (prominin‐1) is widely expressed by several progenitor and stem cells (Karbanova et al., 2008).

It is a five‐transmembrane glycoprotein that is localized to the membrane protrusions. CD133 possesses two isomeric subtypes CD133‐1 and CD133‐2 and has a molecular weight of 97–120 kD. Although its primary function remains unclear, it has been postulated that CD133 is involved in signal transduction. The marker is also widely used in CSC identification and isolation (Ren et al., 2013). Recent evidence suggests that CD133 is a CSC surface marker, see Figure 1. CD133 has been found to be expressed in ovarian, kidney, pancreas, lung, and brain CSCs (Hermann et al., 2007; Tirino et al., 2008; Yanagisawa et al., 2009; Salnikov et al., 2010; Wu et al., 2012a). It has been found that CD133 is co‐expressed with Akt in cancer cells that show a higher level of chemoresistance. The pathway involving Akt is also responsible for cell proliferation and growth, angiogenesis, and halts apop- tosis (Manning and Cantley, 2007). The pathway has also been found to play a role in increasing cell motility, decreasing cellular adhesions, and tumor progression (Bellacosa et al., 2005). A study showed that colon cancer cells that showed a higher resistance to chemoradiation Figure 1 A pictorial representation of CD133 structure. Schematic of the CD133 topology and putative epitopes of commercially available CD133 antibodies. The five‐transmembrane glycoproteins contain two large extracellular loops (EC2 and EC3), which comprise a total of nine N‐linked glycan residues. The commonly used CD133/1 and CD133/2 epitopes are located on the EC3 region of CD133 and have the potential for epitope masking or antibody inaccessibility due to changes in glycosylation patterns. had both higher levels of CD133 and Akt expression. The same study proved that Akt knockout reduced the expression of CD133 (Sahlberg et al., 2014).

The evidence suggested the role of Akt in CD133‐induced tumor growth and progression. The role of EGFR‐AKT and PI3K‐AKT is illustrated in Figures 2 and 3. Another study showed that the Ras/ERK signaling pathway is involved in CD133 expression and its blockage reduced side population in cancer cells. This study concluded that the pathway, at least partially, contributed to the CD133‐rendered CSC hallmarks (Tabu et al., 2010). As the Ras signaling pathway is regulated by Src in CRC, how Src phosphorylates FAK and FAK activates Src is shown in Figures 2 and 3. In another study, it was found that the Wnt signaling pathway is also linked to CD133. Some studies have confirmed the activation of the Wnt pathway in CD133(+) cells (Corbo et al., 2012). The Wnt pathway modulates cell renewal and proliferation, and its activation induces dysregulated CD133 expression in colonic crypts. The study argued that the Wnt pathway plays a role in CD133‐ mediated colon (Co)‐CSCs (Deng et al., 2009). This pathway is regulated at the level of β‐catenin, which is degraded by adenomatous polyposis coli (APC). Mutations in the APC gene are found in most colorectal tumors. As a result, β‐catenin accumulates in the nucleus and activates target genes that play an important role in CRC develop- ment. (de Sousa et al., 2011). Other pathways have also revealed to play a role in CD133 signaling in CSCs. For instance, Notch pathway has been found to be linked with CD133, and its inhibition resulted in apoptosis. Notch inhibition also resulted in a massive loss of CD133 positive cancer cells (Georgia et al., 2006). Also, the Hedgehog pathway is involved in CD133 tumorigenesis and modulates CD133 positive cells renewal. Evidence reveals that, Hedgehog pathway inhibition reduced these Figure 2 The diverse functional role of CD133 as CSC regulator.

CD133 controls the acquisition of cancer stem cell (CSC) properties by regulation of signal molecules and change of cancer metabolism. (1) The cholesterol‐binding protein, CD133 may changes lipid metabolism by induction of lipid raft through the cholesterol recomposition of cellular membrane. (2) CD133 upregulates EGFR‐AKT signaling through estimated glomerular filtration rate (EGFR) stabilization. The stabilization of EGFR may be due to direct binding to CD133. (3) PI3K‐AKT signaling is activated by the interaction between CD133 and P85 subunit. (4) Src kinase is phosphorylated by physical interaction to CD133. Activated FAK by Src induces cell migration and survival. (5) CD133 induces autophagosome formation through release from the membrane to the cytoplasm. (6) Lipid droplet induced by CD133 upregulates Wnt/β‐catenin tumor‐promoting signaling. Figure 3 Functional roles of CD133. A diagram that shows the different roles of CD133 in colorectal cancer (CRC). cells’ self‐renewal capacity (Clement et al., 2007). In addition, the CD133(+) CSCs may be relevant to the Ras‐ Raf (Kemper et al., 2012), STAT3 (Lin et al., 2011), Akt, mitogen‐activated protein kinase (Wang et al., 2010), hypoxia‐inducible factor‐1α (Saigusa et al., 2011), and microRNAs (Fang et al., 2012). Although CD133 was observed to be associated with active proliferating cells, few studies have investigated the role of CD133 in the cell cycle. However, these studies could not explain the function of CD133 directly (Ren et al., 2013). CD133 and epithelial–mesenchymal transition (EMT) Overall, the observations imply that tumor cells are highly plastic and may even transit from a differentiated to a CSC status regulated by environmental conditions. Further plasticity is apparent when epithelial cells undergo EMT (Kong et al., 2011; Scheel and Weinberg, 2011).

An association of CD133 expression and features of EMT was seen in a highly migratory subclone of the pancreatic cancer cell line Capan‐1. In this subclone, EMT‐related transcrip- tion factors (Slug, Snail) and mesenchymal molecules (fibronectin, N‐cadherin) were upregulated along with CD133 expression; knockdown of CD133 resulted in reduced migration potential and downregulation of Slug (Ding et al., 2012). CD133 detected along with EMT markers in pancreatic cancer cells treated with gemcitabine and radiotherapy, and in head and neck squamous cell carcinoma (HNSCC) which its upregulation led to a shift to the mesenchymal state while its silencing led to a shift to an epithelial phenotype. (Chen et al., 2011). Here, the phosphorylation status and activity of Src, a non‐receptor tyrosine kinase, correlated with the CD133 status. Activation of this kinase was also seen in breast cancer cells undergoing EMT, indicating a functional relationship between CD133 expression and EMT status (Nagaharu et al., 2011). Co‐CSCs and CD133 Separate studies have shown that colon cancer possesses CD133 positive cells (Co‐CSCs), which have the ability of tumor growth and self‐renewal (O’Brien et al., 2007; Ricci‐Vitiani et al., 2007). These cells were shown to have tumorigenic potential in the long run (Ricci‐Vitiani et al., 2009). A previous experiment showed that all colon‐ cancer‐initiating cells were CD133 positive, and the CD133 negative ones were devoid of self‐renewal properties (O’Brien et al., 2007). Sahlberg et al. (2014) found that colon cancer cell, which had a higher expression of CD133 (CD133high) showed more resistance to radiation.

Another study asserted that CD133 upregulation is associated with poorer prognosis and higher synchronous metastasis to the liver (Horst et al., 2009b). Ong et al. found that CD133‐positive CRC cell showed resistance to all regimens that included 5‐FU (Horst et al., 2009b; Ong et al., 2010). The conclusion was drawn from the fact that no patients in the stage 3 group benefited from chemotherapy. Also, patients in the end‐ stage (4) group had a less favorable response to chemotherapy than those with CD133 negative cells (Ong et al., 2010). Saigusa et al. revealed that post‐chemoradia- tion (CR) expression of CD133 with a bunch of other markers is linked to a poorer prognosis and a higher rate of distant metastasis. They also stated that the CD133 messenger RNA (mRNA) expression level could be a valuable marker for estimation of prognosis in patients undergoing preoperative CR and the following surgery (Saigusa et al., 2009). However, controversy exists over the role of CD133 in CSCs and whether it is a valid marker for their identification. Shmelkov et al. (2008b) stated that CD133 was neither a CSC‐specific marker nor played a crucial role in distant metastasis. Similarly, LaBarge and Bissell (2008) suggested that CD133 expression is not limited to CSCs and both CD133‐positive and ‐negative metastatic cells have the capacity of tumor production in immunodeficient mice. Another study argued that chemoresistance might be due to the varying levels of anti‐apoptotic proteins in CSCs and not CD133 (Yi et al., 2008). Dalerba et al. (2007a) proposed other CSC‐specific antigens that may play a role in tumor growth and its self‐renewal ability. In a study by Kawamoto et al. it was found that both CD133‐positive and ‐negative cells can initiate tumor growth. However, CD133 positive cells formed larger bulks of the tumor. They also demonstrated that tumors derived from injections of CD133‐negative cells had no CD133‐ positive cells whereas tumors derived from injections of CD133‐positive cells had CD133‐positive cells. This showed the self‐renewal ability of CD133‐positive cells (Kawamoto et al., 2010).

CD133 expression level in CSCs may also play a role in this regard and justify the discrepancies observed in the studies. In a study by Liao et al. (2010) it was revealed that CSCs with the higher expression level of CD133 (CD133high) showed a higher growth and renewal rate than others. The discrepancy may also arise from the fact that some of these studies have evaluated CD133 function in the primary tumors and others in metastatic sites (Ferrand et al., 2009). The discrepancy mandates the need for more studies in this regard though. Clinical significance of CD133 CD‐133 as an important tumor marker may be considered as a prognostic and diagnostic markers and also as a therapeutic approach by its blocking in patients who are diagnosed with CRC. Despite the lack of knowledge regarding the molecular underpinnings of CD133 in cancer, a majority of the current studies do suggest that CD133 exhibits a significant prognostic and predictive value to the overall survival, disease‐free survival, and progression‐free survival in many different solid cancers (Grosse‐Gehling et al., 2013). In two comparative gene expression profiling studies, CD133 expression correlated with predicting glioblastoma patient outcomes and re- sponse to therapy (Colman et al., 2010). Given that both CSCs and CD133pos cell fractions have been shown to exhibit chemoresistance and radioresistance, the ability to predict how patients will respond to therapy could fulfill a significant unmet clinical need in many cancers where CD133 is overexpressed (Chen et al., 2010) The prognostic value of CD133 in CRC Several studies have tried to assess the clinicopathological relevance of CD133 in CRC.

Increasing data indicate that CD133 is of prognostic value in CRC. Further studies have shown that high expression of CD133 is associated with 5‐year and lower survival rates, lymph node metastasis and TNM stage, and poorer tumor differentiation (Ma et al., 2008; Arne et al., 2011; Zhao et al., 2011; Wu et al., 2012b). In a study, Horst et al. (2009a) demonstrated that the survival rate was less than 5–10 years in patients who were CD133‐positive than patients who were negative for this marker. Similarly, Kemper et al. (2012) found that CD133 expression is an indicator of poor prognosis in the relapse‐free survival of the patients. However, the authors stated that mutation of K‐Ras could be a better prognosis‐predictor than CD133 in patients with CRC. Saigusa et al. (2009, 2011) showed that CD133 expression in patients with rectal cancer is linked to distant metastasis, metastatic growth of the tumor, relapse, and poorer prognosis after CRT. In another study, Jao et al. investigated the prognostic value of CD133 in the prediction of tumor regression in CRC patients under- going neoadjuvant concurrent chemoradiotherapy (CCRT). The authors found that cytoplasmic CD133 expression is linked to local tumor recurrence and survival of patients. It may also help to label‐retaining cancer cells after CCRT. Although CD133 is transferred to the cytoplasm, the mechanism of cytoplasmic CD133 function currently remains unclear, but studies showed that it can play an important role in autophagosome in the cytoplasm of CRC cells (Jao et al., 2012). In line with the study of Yasuda et al. (2009), it is revealed that elevated levels of CD133 expression, as opposed to vascular endothelial growth factor (VEGF), and epi- dermal growth factor receptor (EGFR) levels, predicts poorer survival and distant metastasis in patients with rectal cancer; in line with this study and also the significant role of EGFR which participated in CD133 signaling through alteration lipid metabolism and its downstream pathways, and also the role of EGFR‐AKT and its association with CD133 which is conducted by Li et al. showed that higher CD133 expression (higher than or equal to 55%) is a strong predictor of poor survival in patients with locally advanced CRC.

This indicated that CD133 might closely involve in tumor progression. The authors then argued that CD133 overexpression might indicate the overpopulation of CSCs (Li et al., 2009). In a direct relevance to the previous evidence, Artells et al. (2010) found that CD133 overexpression in patients with CRC is linked to a shorter relapse‐free interval (RFI) and poorer overall survival (OS) and thus can be a prognostic marker in this regard. In another study by Kojima et al. it was revealed that higher expression of CD133 in patients with moderate to well‐differentiated adenocarcinoma of the colon is a predictor of poorer survival (Kojima et al., 2008). Kashihara et al. (2014) obtained the same results in CRC patients and showed that CD133 overexpression is an independent prognostic factor in CRC. On the contrary, several other studies could not synthesize the mentioned results in their studies. Choi et al. showed that CD133 overexpression is associated with local invasiveness of tumor and predicts the differentiation in patients with CRC. However, it has no association with patient’s survival (Choi et al., 2009). Also, Kojima et al. (2008) showed that CD133 over- expression predicts the overall poorer survival but not recurrence‐free survival in CRC patients. Yamamoto et al. showed that lack of CD133 expression in liver metastasis of patients with CRC was a predictor of poorer survival. It was argued that increased invasiveness and proliferation in these cells could be a reason for the observed results (Yamamoto et al., 2014). The discrepancies may arise from the fact that these studies have been performed on a heterogeneous set of patients. Different study designs and use of commercial antibodies may also justify the differences seen in these studies (Ren et al., 2013). However, as emerges from the evidence presented above, CD133 overexpression may be a useful marker for the prediction of poorer overall survival, chemoresistance, relapse, and distant metastasis in CRC patients. These lines of evidence was proved in two separate meta‐analyses performed in 2012 and 2015, which linked CD133 overexpression with poorer patient survival (Wang et al., 2012; Zhao et al., 2016). CD133 as a therapeutic target in CRC patients Immunotherapy is an emerging strategy in the treat- ment of malignancies. The strategies such as targeting tumor‐specific antigens (TSA), T lymphocytes, and BiAbs have revolutionized the treatment of cancer patients (Yasuda et al., 2009; Ghasabi et al., 2019).

Recently, significant progress has been achieved in the production of BiAbs owing to the improvements in the protein engineering techniques. Evidence suggests that CD133 may be a novel valuable option for the treatment of CRC (Van Orden et al., 2007). Several studies have successfully tried anti‐CD133 Ab in various solid tumors such as glioblastoma and head and neck squamous cell carcinomas (Damek‐Poprawa et al., 2011; Wang et al., 2011). In a study by Zhao et al., the effects of mouse anti‐ human CD133 monoclonal antibody (MS133) was evaluated both in vivo and in vitro. It was found that anti‐CD133 BiAb comprised of a monomer of chimeric AC133 and humanized OKT3 showed considerable cytotoxicity against CD133high cells and could success- fully prevent tumor growth and increase lysis of CD133 high tumor cells in CRC. It was concluded that MS133 could be a novel promising treatment for CRC. It was also stated that due to the much lower expression of CD133 in normal cells the toxicity of the Ab to normal cells is limited (Zhao et al., 2015). In another study by Ning et al., anti‐CD133 antibody‐ conjugated SN‐38 nanoparticles were used to abolish the CSC population in CRC. The authors found that the treatment with mentioned Ab could effectively and selectively reduce CD133 CSCs in CRC model. It was also proposed that anti‐CD133 antibody‐conjugated SN‐ 38‐loaded nanoparticles could be a promising target therapy in patients with CRC (Ning et al., 2016). Bostad et al. applied a photochemical internalization (PCI)‐based method of targeting CD133 expressing CSCs in CRC cells using immunoconjugate AC133–saporin. Evidence emerging from this study proved that PCI‐based CD133‐targeting using AC133–saporin considerably in- hibited tumor growth both in the primary and metastatic sites. It was argued that this method protected normal cells against the adverse effects of the treatment (Bostad et al., 2015).

Other CD133‐based therapeutics have also been tried in CRC. Shen et al. investigated the effects of miR‐142‐ 3p, a crucial regulator of colon cancer cells via pathways involving CD133, on CRC. The results of this study demonstrated that miR‐142‐3p successfully inhibited colon cancer cell growth and reduced tumor bulk in cell line models of CRC. The mentioned microRNA also increased malignant cells sensitivity to chemotherapy in chemoresistant cells. Accordingly, it was argued that miR‐142‐3p is a potential novel treatment for CRC (Shen et al., 2013). Lonroth et al. argued that CD133 expression in CSC is linked to chemoresistance in CRC patients. Accordingly, they found that short‐term preoperative treatment with non‐steroidal anti‐inflammatory drugs (NSAIDs) namely indomethacin and Celebrex effec- tively reduced CD133 expression at both mRNA and protein levels and increased survival in CRC patients (Lonnroth et al., 2012). Similarly, in another study by Deng et al. revealed that celecoxib reduced CD133 expression at both protein and mRNA levels in a time‐ and dose‐dependent manner. When combined with other chemotherapeutic agents it also reduced CSCs bulk as well as chemoresistance and increased survival of CRC patients (Deng et al., 2009). Huang et al. applied anti‐notch pathway‐targeted therapy against Co‐CSCs. It was shown that colon spheres exhibiting higher chemoresistance against 5‐FU and oxaliplatin are replete with CD133. On the contrary, anti‐ notch‐targeted therapy with N‐[N‐(3,5‑difluoroph ena- cetyl)‐l‐alanyl]‐S‐phenylglycine t‐butyl ester (DAPT) was found to reduce the number of chemoresistant CSCs significantly. As previously discussed Notch pathway is involved in CD133 signaling in CSCs and the application of anti‐notch therapies is an indirect method of targeting CD133 in CRC (Huang et al., 2015). Deng et al. used Dickkopf‐1 (Dkk‐1) which is a Wnt signaling pathway inhibitor and involved in the CD133 signaling pathway in CSCs and in Co‐CSCs pre‐ treated with 5‐FU. This study showed that the treatment with 5‐FU increased the proliferation of CD133‐ positive cells. However, the addition of Dick‐kopf‐1 to the therapeutic regimen increased the sensitivity of CD133high cells to 5‐FU and their apoptosis rate. It also significantly decreased CD133 expression in these cells. This study signified the role of the Wnt pathway in CD133‐induced chemoresistance in CRC (Deng et al., 2010) (Table 1).

The role of the WNT/β‐ catenin signaling pathway is illustrated completely in Figures 2 and 3. Current challenges and future perspectives CD133 mRNAs are found in many human tissues including the pancreas, testis, and digestive tract, with the most prominent expression in the kidney, mammary gland, trachea, salivary gland, and placenta. Its complex gene transcription is controlled in a tissue‐specific manner by five alternative promoters (P1‐P5) generating at least 16 alternative splicing patterns of the 5′‐UTR of CD133 transcripts (Shmelkov et al., 2004; Tabu et al., 2008) There are two major targets for advanced CRC therapy: epidermal growth factor receptor and VEGF. The Wnt pathway is an additional potential target. Data have shown that Wnt pathway activity could be responsible for the chemoresistance of CD133(+) cells in CRC cells. Deng et al. (2010) demonstrated that 5‐FU upregulated Wnt activity in CD133(+) colon cancer stem‐like cells. Dickkopf‐1, an inhibitor of Wnt sig- naling, decreased the expression of CD133 and Lgr5. It also reduced the proliferation, migration, and invasion of colon cancer cells (Qi et al., 2012). This indicates that blocking the Wnt pathway may be one possible solution to the problem of chemoresistance. Furthermore, other pathways such as the Notch and Hedgehog signaling pathways involved in maintaining CSC identity and other regulators such as STAT3 and microRNAs could be conceivable targets (Lin et al., 2011). Conclusion CD133 has been found to be a surface marker for CSCs namely in CRC. It has been widely accepted that CD133 plays prognostic and therapeutic roles in CRC. Several studies have indicated that higher expression of CD133 in Co‐CSCs is associated with poorer response to CRT, poorer OS, and shorter RFI. On the contrary, increasing data suggest that anti‐CD133‐based therapies could be a novel promising option in the treatment of CRC patients, especially those with the chemoresistant profile. Targeting CD133 via BiAbs and its related pathways such as Wnt and notch via other chemicals are promising options.


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