|Year : 2014 | Volume
| Issue : 8 | Page : 233-239
Updates in colorectal cancer stem cell research
Chun-Jie Li1, Xueqian Zhang2, Guan-Wei Fan3
1 Institute of Traditional Chinese Medicine Research, Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193; Department of Emergency Medicine, Tianjin Chest Hospital, Tianjin 300060, China
2 Department of Editorial Office, Tianjin Huanhu Hospital, Tianjin 300060, China
3 Institute of Traditional Chinese Medicine Research, Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China
|Date of Web Publication||17-Feb-2015|
Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193
Source of Support: None, Conflict of Interest: None
Colorectal cancer (CRC) is one of the world most common malignant tumors, also is the main disease, which cause tumor-associated death. Surgery and chemotherapy are the most used treatment of CRC. Recent research reported that, cancer stem cells (CSCs) are considered as the origin of tumor genesis, development, metastasis and recurrence in theory. At present, it has been proved that, CSCs existed in many tumors including CRC. In this review, we summary the identification of CSCs according to the cell surface markers, and the development of drugs that target colorectal cancer stem cells.
Keywords: Biomarker, cancer stem cell, colorectal cancer, target therapy
|How to cite this article:|
Li CJ, Zhang X, Fan GW. Updates in colorectal cancer stem cell research. J Can Res Ther 2014;10, Suppl S4:233-9
Xueqian Zhang - #Co-first author
| > Introduction|| |
Even though the tumor therapy methods are improving, colorectal cancer (CRC) is still the main cause of tumor-related death.  Although most CRC patients are treated with surgery to remove the tumor tissue, some of the CRC patients recurred because of the undiscovered minimal residual tumor focus.  Other treatment methods such as radiation and chemotherapy are used as adjuvant therapy to treat the residual tumor cells. Whereas these treatments are useless in tumor cells with drug-resistance. ,, Therefore, developing new therapeutic strategies to eliminate tumor is ultimately in need.
Not only in pathogenesis aspects but molecular events aspects, the development of CRC is a multi-step process. , The adenoma-carcinoma model of CRC genesis is shown in [Figure 1]. In molecular level, the signal transduction pathways are considered as having important regulation roles. In the research of family CRC syndrome, changes of different key genes were found; later research also revealed that these genes had obvious changes in sporadic cancer. In the tumorigenesis of CRC, Wnt signaling pathway is the cause of hereditary cancer predisposing syndrome and familial adenomatous polyposis (FAP), which plays an important role in the abnormal gene mutation of adenomatous polyposis coli (APC). ,,
Except FAP-related malignant tumor, APC gene mutation is found in 80% sporadic CRC. In these cases, apart from APC gene, genes in Wnt signaling pathway cascade are also found, including β-catenin mutation gene. , Meanwhile, in bone morphogenesis protein 4 (BMP4) signal pathway, the mutation of BMP receptor 1A (BMPR1A) and SMAD4 also found in hereditary cancer predisposing syndrome and juvenile polyposis. ,, Then, SMAD4 gene somatic mutation was found in 30% sporadic CRC. ,, In other sporadic tumor, hereditary nonpolyposis CRC mismatch gene mutation repair was found, including MLH1, MSH2, PMS1, PMS2, and MSH6. , This kind of genetic heterogeneity also reflects on the phenotypic heterogeneity of CRC, which is characterized as the difference of tumor sits, progress and treatment response.
In molecular level, human cancer can be composed of cells with phenotypic heterogeneity.  Otherwise, tumor cell can present functional heterogeneity, such as, the growth characteristics difference of proliferation during in vitro determination,  or the difference of tumorigenicity in vivo and maintenance potential of tumor growth.  These differences to some extent demonstrate the ongoing gene mutation. This conclusion is the basic of the cancer stem cell (CSC) model.  In CSCs theory, only the subset of CSCs can proliferate widely and drive tumor growth that induced morphological and functional diversity daughter cells, including nontumorigenicity cells group. Therefore, CSCs are a small group of cells in tumor tissue with the character of stem cell, such as the potential of self-renew, multi-lineage differentiation and infinite multiplication, , they also show characteristics of tumor cell, such as the abnormal of proto-oncogene and tumor suppressor gene, their daughter cells are also incomplete differentiation and with functional defects. When transplant CSCs to animals, they can differentiate into different phenotype cell subpopulation, which is similar to the original tumor but without tumorigenicity. ,,,,,, Recent research showed that, CSCs could cause the recovery of CRC. ,,, This review summarizes the development of colorectal cancer stem cell (CR-CSC).
|Figure 1: The adenoma-carcinoma model of human colorectal carcinogenesis. Adenomatous polyposis coli (APC) or β-catenin mutations initiate the neoplastic process. Tumor progression results from mutations in other genes (e.g., K-ras, Smad 4 and p53) and genomic instability. Patients with familial adenomatous polyposis inherit APC mutations and develop numerous aberrant crypt foci, some of which progress as they acquire other genetic mutations|
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| > The identification of colorectal cancer stem cells|| |
A large amount of evidence proved that in human CRC tissues, there are CR-CSCs. ,,, Meanwhile, CR-CSCs are the main reason of chemotherapy resistance and treatment failure, which prevent the development of clinical oncology.
Recent researchers including Dalerba P, Papailiou J, Zhu L,Vermeulen L,Yeung TM,Takahashi H, et al select or enrich CR-CSCs according to possible stem cell markers, CD133, CD44, CD24, CD166, LGR-5 and aldehyde dehydrogenase-1 (ALDH-1) ,,,,,,, are current discovered CR-CSCs markers. [Table 1]. In an in vivo research, different amount of cancer cell subpopulations, which were classified according to whether CD133 expression or not, were respectively injected into immune deficiency mice, only CD133 + colon cancer cell subpopulation have tumorigenic ability. CD133 + cells account for 2-25%  or 1-6%  in CRC tissue, 1 × 10 2 or 1.5 × 10 3 CD133 + cells can cause tumor, while 1 × l0 5 CD133− cell can't cause tumor. Meanwhile, in other research, 2.5 × 10 5 CD133− cancer cells were injected into 9 mice, only one of them acquired tumor.  In addition, research also showed that, unlike CD133− cells, CD133 + colon cancer cell could be alive for at least 1-year as CR-CSC spheres in vivo. However, CD133 as CR-CSCs marker was doubted in the following research of Shmelkov et al.  Because they found that CD133− CR-CSC also can cause primary tumor and metastasis tumor, and can be serial cultured in vivo. Conversely, Li et al.  agreed to consider CD133 as CR-CSCs marker, in their research, clone population of CD133 + single cell isolated from CRC cell line SW480 has more tumorigenic ability than CD133− cell. Meanwhile, CD133 overexpression cell population have more invasion ability than CD133 down expression cell population. In metastasis tumor, CD133 can be considered as a marker of increased tumorigenic ability. Furthermore, in derived culture of clinical tumor only CD133 + signal cell showed the potential of multi-lineage differentiation. 
Dalerba et al.  also found another cell population of human CRC with epithelial cell adhesion molecule (EpCAM) (epithelial specific antigen [ESA]) high /CD44 + phenotype had tumorigenic ability. 0.03-38% of the total alive tumor cell expressed both of these markers. Injection nonobese diabetic/severe combined immunodeficient mice with minimal 2 × 10 2 this kind of phenotype cells can cause implant tumor whereas 1 × 10 5 EpCAM low /CD44− cells can't cause tumor. And only one mouse occurred tumor originated from injection of 2 × 10 5 EpCAM low /CD44− cells. However, in this research, long-term cell self-renew ability was not assessed, and tumor cell hierarchy organ xenografts were either not conducted.  Interestingly, cells with phenotype of EpCAM high /CD44 + also expressed mesenchymal stem cell marker CD166, which was also found related to the progress of CRC  and human malignant melanoma stem cell-like cancer cell  in previous research. CD166 also can be considered as a candidate marker of CR-CSCs enrichment. The primary selecting experiment results demonstrate that the tumorigenic cell can be restricted in the ESA high /CD44 + /CD166 + CRC cell population.  Moreover, it is proved that two kinds of human colon cancer cell line (HT-29 and CaCO 2 ) express both CD133 and CD44, meanwhile, the tumorigeneic ability of CD133 + /CD44 + cell is higher than cells with one positive marker.  In one following research, ALDH-1 is overexpressed in the two hyperplasia colon crypts and colon adenoma of FAP patients.  ALDH-1 + cell population showed effective ability to induce xenograft tumor, also can passage in-line in vivo, while ALDH-1− cells don't have this ability. ALDH-1 + cells also represent the subpopulation of CD133 + or CD44 + cells, which means ALDH-1 can be considered as another CR-CSCs marker.
Recent research using lineage-tracing experiments in mice cancer model revealed that CR-CSCs could come from stem cell in physiological tissue. For example, using the latest intestinal stem cell marker LGR5,  Barker et al. proved that the regulation of Wnt signal on APC was lost in LGR5 + cell instead of LGR5− cells, which induced the progress of intestinal adenoma.  It is also demonstrated that in CD133 + cells, internal Wnt signaling pathway was activated though mutated β-catenin, which induced the damage of colon crypts structure and the disproportionate expansion of CD133 + cell in intestinal crypt stem cell. Therefore, some potential marker or marker group exist in the enriched CR-CSCs cells.
Especially when considering future therapy design, identification of the function of CSC surface marker is an important problem to solve. Du et al.  proved that CD133 knock-out have no effect on the formation of in vitro clone or in vivo xenotransplanted tumor, indicating that CD133 can be considered as the passive marker of CR-CSCs. However, knockout CD44 can induce limited colony formation, and significantly reduce the formation of xenotransplanted tumor, which strongly implied that CD44 plays a role in CRC tumorigenesis. These data support a single genetic model independently; knocking out CD44 in APC min/+ mice can limit the progress of intestinal adenoma.  CD44 transmembrane glycoprotein is a cell adhesion molecular, which transmit extracellular matrix signal from hyaluronic acid into the cell.  Wnt signaling pathway can regulate the expression of CD44, and the modulation of Wnt signaling pathway may also regulate CR-CSCs. ,,
| > Drug-Resistance of Colorectal Cancer Stem Cells|| |
Traditional cancer therapies, including chemotherapy and radiotherapy, depend on the rapid cycling cell cycle and aim at specific cell division phase, such as 5-fluoroucil (5-FU), the inhibitor of thymidylate synthase in cell mitotic S-stage, and oxaliplatin, a platinum compounds agent.  Nevertheless, there is evidence that showed drug-resistance exists in CR-CSCs, which may be associated with the slow growth of CR-CSCs in G0 stage. A large sample of clinical research conducted in 501 human CRC patients demonstrated that, tumors with CD133 overexpression showed more resistance in 5-FU based chemotherapy and the expression of CD133 related to poor prognosis.  In oxaliplatin treated SW620 and LoVo CRC cells, overexpression of CD133 was also observed.  Recently, Dallas et al. found that the high concentration of 5-FU or oxaliplatin treated human HT-29 CRC cells showed enrichment of CD133 + and CD44 + CR-CSCs and decreased in vitro proliferation. Insulin-like growth factor receptor I (IGF-1R) is overexpressed in chemoresistant HT-29 cell line, treatment with IGF-1R inhibitor AVE-1642 can inhibit the growth of in vivo xenograft model.  Todaro et al.  reported that human CD133 + CR-CSCs can regulate death receptor and inhibit chemotherapy-induced cell apoptosis through specifically expressed interleukin-4 (IL-4). Treated CD133 + CR-CSCs with anti-IL-4 neutralizing antibody can increase the sensibility of 5-FU or oxaliplatin-based therapy.  Cammareri et al.  reported other mechanism of CD133 + CR-CSCs 5-FU and oxaliplatin resistance: Aurora-A kinase, a regulator of mitosis affecting the process of cell cycle, is overexpressed in CR-CSCs; and Aurora-A silence can result in the growth inhibition, down-regulation of the apoptosis protein expression and increase of chemotherapy sensitivity, which then induce the death of CR-CSCs. MicroRNAs (miRNA) also play a role in the regulation of cell proliferation and can regulate CR-CSCs specific signal pathway to increase CR-CSCs drug-resistance. For example, miRNA-140 inhibits the proliferation of CD133 +high /CD44 +high human CRC cells through regulating histone deacetylase 4, leading to resistance to methotrexate (MTX) and 5-FU.  Though inhibiting cell proliferate and regulating nuclear and centrosomal protein DTL to block cell cycle and induce cell G2 stage retardation, miRNA-215 can increase the resistance of CD133 + CD44 + CRC to MTX and tomudex.  It is meaningful to study this invasion cancer cell subpopulation with specific targets and their drug-resistance mechanism, and to improve the effect of traditional cancer therapy and treatment method. 
In the treatment of metastasis cancer, chemotherapy effect also impacted by different mechanism induced primary or secondary tumors, this phenomenon is called multi-drug-resistance (MDR).  Reduce the enriched drug in the tumor is the most studied in tumor MDR mechanism. It is found that part of the tumor cells express adenosine triphosphate (ATP) depended on drug efflux transporter p-glycoprotein (P-gp, MDR1, ABCB1). , In clinical, the overexpression of ABCB1 is found in CRC, kidney, adrenal hepatocellular tumor, and acute myeloid leukemia.  However, till now, the relation between ABC transduction and CR-CSCs has not been identified. It is reported that human MDR family-ABCB5 expressed in human skin  and human malignant melanoma. , Meanwhile, the analysis of ABCB5-mRNA revealed that its expression also exist in normal colonic tissue and other CRC tissue.  Notably, in CD133 + stem cell phenotype-expressing tumor cells in malignant melanoma cultures and clinical primary and metastatic melanomas, the specific expression of ABCB5 was found.  In a multi-center drug selecting research (NCI-60) supported by National Cancer Institute, it is found that lots of cancer cell lines express ABCB5, which is related to the chemotherapy drug-resistance. , In CD133 + stem cell phenotype-expressing melanoma cells, ABCB5 can mediate doxorubicin resistance through drug efflux transport.  Follow-up research have identified that ABCB5 mediated doxorubicin resistance in melanoma and liver cancer cells, , furthermore, in melanoma cells, ABCB5 resisted to camptothecin and 5-FU, which are two commonly used anti-CRC drugs.  Because the expression of ABCB5 means drug-resistance, ,,,, it is evidenced that in solid malignant tumor, there are novel, potential and key relations among CSCs, tumor development and chemotherapy resistance. ABCB5 + CSCs may be the main factor of malignant disease development and chemotherapy resistance. Therefore, target CSCs therapy is a new treatment strategy in metastasis tumor.  All these results indicated that, ABCB5 expression is closely related to the CSCs-driven cancer, ,, ABCB5 expressing or not in CRC may represent another drug-resistant mechanism.  Therapies target CR-CSCs subpopulation with tumorigenicity and chemotherapy resistance can improve the therapy efficacy in these malignant tumors. 
Some research suggested that CSC is resistant to radiotherapy, such as, compared with CD133− tumor cell, the chemotherapy resistance increased in CD133 + human brain glioma stem cell, meanwhile, CD133 + tumor cells presented preferentially activation of DNA damage checkpoint in response to radiotherapy-induced DNA damage and repair.  The exact relationship between CR-CSCs and chemotherapy in still unclear, several research demonstrated that, in CRC, CD133 + CR-CSCs survived after adjuvant chemoradiotherapy.  As the radiation dose increasing, the expression of CD133 also increased in vitro. 
| > Colorectal cancer stem cells target therapy|| |
Recent researches provide new target therapy strategy for advanced colon cancer [Table 2]. The purpose of this therapy is to damage the necessary way of tumor growth, survival and metastasis, and specific cytotoxic. , Currently, there are two targets of this therapy, epidermal growth factor receptor (EGFR) and vascular endothelial growth factor (VEGF). EGFR is a member of ErbB family, which is abnormally activated in many tumors.  VEGF promotes angiogenesis through inducing epithelial cell growth and differentiation, VEGF expresses in 40-60% colon cancer, the activation process is related to tumor development and metastasis. , Some clinical trials indicated that, in addition to 5-FU therapy, target therapy including anti-EGFR (such as cetuximab) and anti-VEGF monoclonal antibody (bevacizumab) could increase the survival of advance colon cancer patients for over 20 months. , Therefore, treatment targeting to specific cell population after chemotherapy is feasible to eradicate tumor successfully.
|Table 2: Novel targeting approaches against colorectal cancer stem cells|
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Recent discoveries of specific molecular signaling pathway in chemotherapy resistance, self-renew and tumorigenicity provide potential targets for the future treatment of CR-CSCs. The role of Wnt/β-catenin signaling pathway in maintaining the phenotype of intestinal crypt stem cell has been evidenced. APC function loss or β-catenin mutation induced Wnt signaling disorder are the leading causes of most CRC. , Recent findings revealed that LGR5, a marker of intestinal stem cell, could regulate CRC cell proliferation and survival through targeting the Wnt/β-catenin signaling pathway. , CD44v6 is found expressed in all CR-CSCs, inhibition of phosphatidylinositol 3-kinase selectively killed CD44v6 CR-CSCs and decreased metastatic growth.  Meanwhile, Notch signaling pathway is overexpressed in CR-CSCs, and plays a role in CR-CSCs tumorigenicity and self-renew by inhibiting cell cycle kinase inhibitor p27 and transcription factor ATOH1.  In APC min CRC model, γ-secretase inhibition mediated Notch signaling dysfunction induced the APC min proliferating cells in intestinal adenoma turning into goblet cells, then leading to the growth stagnation of tumor cells.  BMP4 plays a key role in the development of physiological intestinal crypts.  Mutation of BMP4 signaling pathway (SMAD4 and BMPRIA) or blocking the BMP4 signaling pathway by expression of BMP4 antistatic agent, Noggin, induced the development of intestinal crypts, cancer susceptibility syndrome, and juvenile polyposis syndrome.  The expression of BMPR2 and SMAD4 were also found in an immunohistochemical analysis of 72 sporadic CRC patients, revealing that blocking BMP4 signaling pathway can potentially contribute to the formation of colorectal tumor.  In this aspect, Lombardo et al. demonstrated that, BMP4 could induce the differentiation of CD133 + CR-CSCs, improving the sensitivity of oxaliplatin and 5-FU in the treatment of xenografted tumor.  It is reported that, actin-binding peptide thymosin β4 (Tβ4) is overexpressed in CD133 + CR-CSCs. Targeting Tβ4 can damage the growth and migration of CR-CSCs in vitro, also, though regulating integrin-linked kinase/Akt transduction cascades in vivo can reduce the tumor size of CD133 + CR-CSCs based xenografts in mice. 
In a word, these data demonstrated the efficacy of CSCs therapy, providing new approaches to discover new mechanism of cancer treatment resistance by detection of more invasive cancer and cancer cell subpopulation proliferation. If researches limited to unclassified tumor cells, the discovery of new therapy method is impossible.
| > Future prospective|| |
Despite the observed proliferation of CR-CSCs is very slow, the knowledge of the metabolism character of CR-CSCs remains limited. Metabolomics that is an emerging research field, points out the way. Warburg effect, which believes that cancer cells utilize glycolysis as ATP energy source in priority instead of fatty acid oxidation, also can be applied as broad-spectrum anticancer drugs. , A recent research of Akao et al. found the primary evidence of the metabolomics changes in drug-resistance cells. SIRT1, the key regulator, is overexpressed in 5-FU resistance DLD-1 cells.  In addition, there are many approaches for high throughput screening CR-CSCs, such as RNA interference library, drug screening libraries, mass spectrometry analysis, differentiation therapy, microarray chip and gene therapy, paved the way for future identifying different characteristics of CR-CSCs related molecular signal pathways and the effective methods to eradicate tumors.
| > Conclusion|| |
Since the first report of CR-CSCs in 2007, there are great advancements in the identification CR-CSCs and targeting CR-CSCs treatments. At present, although a large amount of research have been done, more novel molecular characters of CR-CSCs are still to be explored in the future. Patient oriented new therapy strategy is under development. Once the efficacy of targeting CR-CSCs and tumor side population cells therapy were identified and verified, the survival of patients can be obviously improved.
| > Acknowledgements|| |
We thank the financial supports from the National Natural Science Foundation of China (81001659, 81273891), and Tianjin Innovative teams in Colleges and Universities (TD12-50-31)
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[Table 1], [Table 2]