Colon Cancer Stem Cells Research Articles

IL-8/CXCR2 mediates tropism of human bone marrow-derived mesenchymal stem cells toward CD133+ /CD44+ Colon cancer stem cells.

In cancer treatment, the most attractive feature of mesenchymal stem cells (MSCs) is it's homing to tumor tissues. MSC is an important part of the "colon cancer stem cell niche", but little research has been done on the tropism of human MSCs toward colon cancer stem cells (CCSCs). In this study, we first compared the effects of three tissue-derived MSCs (bone marrow, adipose tissue, and placenta) in vivo on colon tumor xenograft growth. Then, we analyzed the tropism of bone marrow-derived MSCs (BMSCs) toward normal intestinal epithelial cells (NCM460), parental colon cancer cells, CD133- /CD44-, and CD133+ /CD44+ colon cancer cells in vitro. Microarray analysis and in vitro experiments explored the mechanism of mediating the homing of BMSCs toward CCSCs. Compared with the parental and CD133- /CD44- colon cancer cells, CD133+ /CD44+ cells have a stronger ability to recruit BMSCs. In addition, BMSCs were significantly transformed into cancer-associated fibroblasts after being recruited by CCSCs. After coculture of BMSCs and CCSCs, the expression of interleukin (IL)-6, IL-8, IL-32, and CCL20 was significantly increased. Compared with parental strains, CD133- /CD44- cells, and NCM460, BMSC secreted significantly more IL-8 after coculture with CD133+ /CD44+ cells. Low concentration of IL-8 peptide inhibitors (100 ng/ml) and CXC receptor 2 (CXCR2) inhibitors have little effect on the migration of BMSCs, but can effectively weaken CCSC stemness and promote dormant CSCs in the coculture system to re-enter into the cell cycle. The endogenous IL-8 knockout in BMSCs or BMSCs loaded with IL-8 and/or CXCR2 inhibitors will make the therapy of BMSC targeting CCSCs function at its best.

Diacerein treatment prevents colitis-associated cancer in mice.

BACKGROUNDInflammation is a well-established enabling factor for cancer development and provides a framework for the high prevalence of colon cancer in inflammatory bowel disease. In accordance, chronic inflammation has recently been implicated in the development of cancer stem cells (CSCs). However, the mechanism whereby anti-inflammatory drugs act in the prevention of colitis-associated cancer (CAC) is only partially understood.AIMTo evaluate the role of diacerein (DAR), an anti-inflammatory drug that mainly acts through the inhibition of interleukin (IL)-1β expression in the development of CSCs and CAC.METHODSThe effects of DAR on colon inflammation in mice with CAC were evaluated by inflammatory index, reverse real-time transcription polymerase chain reaction and western blot. Cytokine levels were measured by enzyme-linked immunosorbent assay. Cells assays evaluated the effects of DAR on CSCs. Immunohistochemistry and apoptosis assays were also used to evaluate the effects of DAR on tumorigenesis associated with inflammation.RESULTSDAR treatment reduced colon inflammation as well as the number and size of tumors in azoxymethane plus dextran sulphate sodium-treated animals. Accordingly, DAR treatment was associated with reduced intracellular signals of inflammation (inhibitor of nuclear factor kappa B kinase and c-Jun N-terminal kinase phosphorylation) in the colon. In addition, DAR treatment was associated with a decrease in colon CSC formation, suggesting that besides reducing colonic inflammation, DAR has a direct effect on the inhibition of colon carcinogenesis.CONCLUSIONTogether, these data indicate that DAR-mediated IL-1β suppression attenuates inflammation-induced colon cancer and CSC formation, highlighting DAR as a potential candidate for the chemoprevention of CAC.

Curcumin may be a potential adjuvant treatment drug for colon cancer by targeting CD44

Despite the considerable advances in treatment method development, the mortality rate related to colon cancer still ranks the fifth in all tumor-related diseases. Recently, there has been growing evidences supporting the existence of colon cancer stem cells (CSCs) might be one of the main causes for initiation, progression and recurrence of colon cancer. Curcumin has been shown to possess anticancer activities. It has also been suggested that curcumin was effective against colon CSCs by coupling with CD44, a robust marker and functional important molecule for colorectal CSC. In the present study, we confirmed that curcumin can inhibit the proliferation, colony formation, migration and tumor sphere formation of colon cancer cells. Results from real-time PCR and western blotting had suggested that curcumin could down-regulate the expression of CD44. Moreover, results from flow cytometry had further revealed that curcumin could decrease the proportion of CD44+ colon cancer cells. After the expression of CD44 had been knocked down by using siRNA, the inhibition effects of curcumin against CD44+ colon cancer cells were observed to be reduced significantly. Moreover, it had been observed that the cellular uptake of curcumin was significantly higher in CD44+ colon cancer cells. Results from flow cytometry had shown that curcumin could induce apoptosis in CD44+ colon cancer cells. Altogether, our results suggested that curcumin might be an adjuvant drug for the treatment of colorectal cancer by targeting CD44.

Targeting poor proteasomal function with radioiodine eliminates CT26 colon cancer stem cells resistant to bortezomib therapy

We tested the hypothesis that tumor response to conventional bortezomib (BTZ) treatment is enhanced by targeted radiotherapy of resistant cancer stem cells (CSCs) that have characteristically poor proteasome function. This was accomplished by augmenting 131I uptake through expression of a sodium-iodide symporter (NIS) fusion protein that accumulates in cells with low proteasome activity. The NIS gene fused with the C-terminal of ornithine decarboxylase degron (NIS-cODC) was cloned. Stably expressing CT26/NIS-cODC cells and tumorsphere-derived CSCs were evaluated for NIS expression and radioiodine uptake. CT26/NIS-cODC cells implanted into mice underwent PET imaging, and tumor-bearing mice were treated with BTZ alone or with BTZ plus 131I. CT26/NIS-cODC cells accumulated NIS protein, which led to high radioiodine uptake when proteasome activity was inhibited or after enrichment for stemness. The cell population that survived BTZ treatment was enriched with CSCs that were susceptible to 131I treatment, which suppressed stemness features. Positron emission tomography and uptake measurements confirmed high 124I and 131I uptake of CT26/NIS-cODC CSCs implanted in living mice. In CT26/NIS-cODC tumor-bearing mice, whereas BTZ treatment modestly retarded tumor growth and increased stemness markers, combining 131I therapy suppressed stemness features and achieved greater antitumor effects. The NIS-cODC system offer radioiodine-targeted elimination of CSCs that are tolerant to proteasome inhibition therapy.

Correlation of clinicopathological features and LGR5 expression in colon adenocarcinoma

Colon cancer stem cells (CSCs) are closely related to tumorigenesis and treatment response, and LGR5 is currently the most robust and reliable CSC marker in colorectal cancer (CRC). However, LGR5 expression in CRC tumor budding (TB) is not well understood. We examined the clinicopathological and prognostic significance of LGR5 in CRC TB. LGR5 expression was evaluated by RNAscope, a newly developed RNA in situ hybridization technique, using a tissue microarray consisting of 55 patient samples of TB in colon adenocarcinoma (CA) selected from the medical archives at our hospital. Patients were stratified into negative and positive LGR5 expression groups. Tumor-infiltrating lymphocytes (TILs) and histological grade were lower in the LGR5-positive group compared with the LGR5-negative group (P = .0407 and P = .0436, respectively). There was no significant difference in overall survival between the LGR5-positive group and the LGR5-negative group (log-rank test, P = .6931). LGR5 expression did not remain a predictor of prognosis in univariate analysis (OR = 0.84, 95% CI: 0.33–2.02, P = .6928). LGR5 expression may be affected by TILs, which have been demonstrated to be associated with worse prognosis in the budding area of CA and is an important potential marker of prognosis.

Salinomycin inhibits epigenetic modulator EZH2 to enhance death receptors in colon cancer stem cells

ABSTRACT Drug resistance is one of the trademark features of Cancer Stem Cells (CSCs). We and others have recently shown that paucity of functional death receptors (DR4/5) on the cell surface of tumour cells is one of the major reasons for drug resistance, but their involvement in the context of in CSCs is poorly understood. By harnessing CSC specific cytotoxic function of salinomycin, we discovered a critical role of epigenetic modulator EZH2 in regulating the expression of DRs in colon CSCs. Our unbiased proteome profiler array approach followed by ChIP analysis of salinomycin treated cells indicated that the expression of DRs, especially DR4 is epigenetically repressed in colon CSCs. Concurrently, EZH2 knockdown demonstrated increased expression of DR4/DR5, significant reduction of CSC phenotypes such as spheroid formation in-vitro and tumorigenic potential in-vivo in colon cancer. TCGA data analysis of human colon cancer clinical samples shows strong inverse correlation between EZH2 and DR4. Taken together, this study provides an insight about epigenetic regulation of DR4 in colon CSCs and advocates that drug-resistant colon cancer can be therapeutically targeted by combining TRAIL and small molecule EZH2 inhibitors.

Inhibition of MicroRNA-302c on Stemness of Colon Cancer Stem Cells via the CARF/Wnt/β-Catenin Axis.

Even though the relevance of microRNA (miR)-302c has been studied, little is known about its involvement in colon cancer (CC). Our aim here was to investigate the role of miR-302c in the cancer stem cells (CSCs) of CC. Firstly, the CSCs were screened out from cultured SW1116 and SW480 cells by flow cytometry, and the differentially expressed miRNAs in cell were obtained by microarray analysis. The expression of miR-302c, collaborator of ARF (CARF), and Wnt/β-catenin-related genes in CSCs was determined by means of RT-qPCR and Western blot. A dual-luciferase reporter assay was conducted to authenticate the binding relationship between miR-302c and CARF. Proliferation, migration, invasion, sphere formation as well as apoptosis of CSCs were assessed by cell counting kit-8, Transwell assay, sphere formation assay as well as flow cytometric analysis, respectively. The roles of miR-302c and CARF in tumor growth were determined in vivo. The expression of miR-302c in CC cells was reduced versus that in normal cells. The overexpression of miR-302c weakened the stemness, proliferation, invasion, and migration abilities while induced apoptosis of CSCs in CC. Also, miR-302c reduced tumor size and weight in mice, accompanied with lowered CARF expression. The mechanistic analysis manifested that miR-302c bound to CARF and suppressed its expression and disrupted the Wnt/β-catenin signaling. This study offers a novel characterization of miR-302c function in CSCs in CC, which may be beneficial to the development of capable therapeutic options for CC.

Compound Kushen Injection Promoted the Killing Effect of Cytokine-Induced Killer Cells Which Was Activated by Dendritic-Colon Cancer Stem Cell Fusion Cells on Colon Cancer Stem Cells

Colon cancer stem cells (cCSCs) are highly tumorigenic and resistant to traditional chemotherapeutic drugs. Therefore, they are an essential factor in colorectal cancer (CRC) metastasis and recurrence. Dendritic cells (DCs) could bind to the tumor cells and form the fusion cells (FCs). And these FCs could inhibit the development of malignant tumors. Furthermore, the cytokine induced killer (CIK) cells (CD3+ /CD56+ T lymphocytes) could also apply for the immunotherapy of cancer. And compound Kushen injection (CKI) is a traditional Chinese medicine (TCM) which has been used for the treatment of various tumors. However, whether the dendritic-colon cancer stem cell fusion cells (DC-cCSC FCs) could activate the CIK cells and kill the colon cancer stem cells is unknown. And whether the CKI could enhance the lethal effect is still unclear. In this study, we collected peripheral blood samples from healthy participants to acquire mononuclear cells and induced DC and CIK cells. Meanwhile, the CD44+ cells (cCSCs) were screened from SW480 cells. Next, the DC-cCSC FCs were established for the next experiments. At last, CCK-8 assays were performed to determine the effect of DC-cCSC FCs and CKI on the viability of cCSCs. We found that DC-cCSC FCs enhanced the proliferation of CIK cells and induce the CIK cells to secrete more IL-12. The DC-cCSC FCs enhanced the inhibitory effect of CIK cells on cCSCs. Furthermore, application of CKI enhanced the killing rates of DC-cCSC FCs and CIK cells on cCSCs. CIK cells activated by the DC-cCSC FCs had the lethal effect on the cCSCs. Furthermore, CKI enhanced this lethal effect of DC-cCSC FCs and CIK cells.

A Preliminary Study of miR-144 Inhibiting the Stemness of Colon Cancer Stem Cells by Targeting Krüppel-Like Factor 4.

Colon cancer is a prevalent clinical malignant tumor of the digestive system. The current study aims to explore the miR-144 expression in colorectal cancer (CRC) cell lines and CRC stem cells (CSCs) and to explore its effect on the stemness of CSCs and the targeted regulation of Krüppel-like factor 4 (KLF4). Use qRT-PCR to detect the expression level of miR-144 in CRC cells SW480, HCT116, and H129 and the healthy colon cell NCM460. The CSCs that were used were cultured in HCT116 cells. Use western blot to explore the expressions of Nanog, SOX2, and OCT4 stemness marker protein. After it was transfected with miR-144 mimics or KLF4 plasmid, use MTT to explore the cell viability of CSCs, use flow cytometry to evaluate apoptosis, and use transwell assay to evaluate the ability of invasive of CSCs. The targeting effect of miR-144 on the KLF4 gene was verified using TargetScan prediction and the double-luciferase reporter gene test. Use qRT-PCR to evaluate the role of miR-144 mimics on KLF4 mRNA expression in CSCs. The qRT-PCR results exhibited that the miR-144 expression in CRC cells was higher than that in the healthy colon cell line. The expressions of OCT4, Nanog, and SOX2 stem cell markers were up-regulated in CSCs, and the expression of miR144 increased in CSCs. The cell viability, apoptosis, and invasion of CSCs increased after miR-144 was transfected. The TargetScan prediction and double-luciferase reporter gene assay confirmed that miR-144 was targeted by KLF4, and the expression of KLF4 mRNA in the miR-144 mimics group reduced. Moreover, the overexpression of KLF4 could partially reverse the role of miR-144 mimics on CSCs. In summary, miR-144 was highly expressed in CRC cell lines and CSCs, and the overexpression of miR-144 in CSCs significantly promoted the proliferation of CSCs, inhibited its apoptosis, and promoted its invasion ability. In addition, its preliminary mechanism, possibly through negative regulation KLF4, promotes the stemness of CSCs, and miR-144 is likely to be a potential target for eliminating CSC from CRC treatment.

Diet-Derived Phytochemicals Targeting Colon Cancer Stem Cells and Microbiota in Colorectal Cancer.

Colorectal cancer (CRC) is a fatal disease caused by the uncontrolled propagation and endurance of atypical colon cells. A person’s lifestyle and eating pattern have significant impacts on the CRC in a positive and/or negative way. Diet-derived phytochemicals modulate the microbiome as well as targeting colon cancer stem cells (CSCs) that are found to offer significant protective effects against CRC, which were organized in an appropriate spot on the paper. All information on dietary phytochemicals, gut microbiome, CSCs, and their influence on CRC were accessed from the various databases and electronic search engines. The effectiveness of CRC can be reduced using various dietary phytochemicals or modulating microbiome that reduces or inverses the progression of a tumor as well as CSCs, which could be a promising and efficient way to reduce the burden of CRC. Phytochemicals with modulation of gut microbiome continue to be auspicious investigations in CRC through noticeable anti-tumorigenic effects and goals to CSCs, which provides new openings for cancer inhibition and treatment.

Colon Cancer Heterogeneity: Welcome to the RiboZone.

In this issue of Cell Stem Cell, Morral etal. (2020) shed new light on the hierarchical organization of cells within colorectal cancer. They show that tumor cells at the apex of this hierarchy reside in particular tumor zones and possess high protein synthesis activity. Interference with their biosynthetic activity results in an irreversible growth arrest of CRC.

Syndecan-1-Dependent Regulation of Heparanase Affects Invasiveness, Stem Cell Properties, and Therapeutic Resistance of Caco2 Colon Cancer Cells.

The heparan sulfate proteoglycan Syndecan-1 binds cytokines, morphogens and extracellular matrix components, regulating cancer stem cell properties and invasiveness. Syndecan-1 is modulated by the heparan sulfate-degrading enzyme heparanase, but the underlying regulatory mechanisms are only poorly understood. In colon cancer pathogenesis, complex changes occur in the expression pattern of Syndecan-1 and heparanase during progression from well-differentiated to undifferentiated tumors. Loss of Syndecan-1 and increased expression of heparanase are associated with a change in phenotypic plasticity and an increase in invasiveness, metastasis and dedifferentiation. Here we investigated the regulatory and functional interplay of Syndecan-1 and heparanase employing siRNA-mediated silencing and plasmid-based overexpression approaches in the human colon cancer cell line Caco2. Heparanase expression and activity were upregulated in Syndecan-1 depleted cells. This increase was linked to an upregulation of the transcription factor Egr1, which regulates heparanase at the promoter level. Inhibitor experiments demonstrated an impact of focal adhesion kinase, Wnt and ROCK-dependent signaling on this process. siRNA-depletion of Syndecan-1, and upregulation of heparanase increased the colon cancer stem cell phenotype based on sphere formation assays and phenotypic marker analysis (Side-population, NANOG, KLF4, NOTCH, Wnt, and TCF4 expression). Syndecan-1 depletion increased invasiveness of Caco2 cells in vitro in a heparanase-dependent manner. Finally, upregulated expression of heparanase resulted in increased resistance to radiotherapy, whereas high expression of enzymatically inactive heparanase promoted chemoresistance to paclitaxel and cisplatin. Our findings provide a new avenue to target a stemness-associated signaling axis as a therapeutic strategy to reduce metastatic spread and cancer recurrence.

Pancreatic and colon cancer stem cells escape NK cell effector function via PCNA–NKp44 interaction

Abstract Natural killer (NK) cells play an important role in the innate immune response against cancer. Unlike B or T lymphocytes, NK cells do not require prior sensitization and can immediately respond to target cells. NK cell function depends on a balance of signals transmitted from activating and inhibitory receptors interacting with ligands on the surface of cells. Cancer cells may escape NK-mediated responses by expressing inhibitory ligands. Proliferating cell nuclear antigen (PCNA), in association MHC I, forms the inhibitory ligand for NKp44. Cancer stem cells (CSC), a unique subset of tumor cells, possess a stem-cell-like phenotype and are thought to facilitate metastasis by escaping NK cell effector function. CSC can be identified by a variety of markers such as surface co-expression of CD44 and CD133. In both Panc-1 and HCT 116 cell lines, surface PCNA is associated with co-expression of CD44 and CD133. Triple positive (PCNA+CD44+CD133+) cells have increased CSC transcription factor (NANOG, SOX2, and Oct-4) expression compared to double positive (PCNA−CD44+CD133+) or negative cells. Additionally, blocking the PCNA–NKp44 interaction alters IFN-g secretion and increases specific lysis of cancer cells by NK cells. Taken together, these data suggest that surface co-expression of PCNA, CD44, and CD133 are markers of pancreatic and colon CSC and blocking the PCNA–NKp44 interaction may provide an immunotherapeutic approach to target pancreatic and colon CSC and prevent metastasis.

Inhibition of scutellarin on differentiation of colonic cancer stem cells via hedgehog signaling pathway

The objective of this study was to investigate the inhibitory effect of scutellarin on the differentiation of colonic cancer stem cells in vitro and in vivo and to explore its underlying hedgehog signaling-based mechanism. The effect of scutellarin on the growth in vitro of HT-29 cells-derived cancer stem-like cells(HT-29 CSC) was observed with 3 D cell culture. The effect of scutellarin on the transformation of HT-29 CSC cells was assessed by soft agar colony formation assay. Fetal calf serum was used to induce differentiation of stem cells and observe the effect of scutellarin on HT-29 CSC cells differentiation in vitro. The effects of scutellarin on mRNA expressions of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells were determined by quantitative Real-time polymerase chain reaction(qRT-PCR). The effects of scutellarin on protein expressions of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells were examined by Western blot. After subcutaneous implantation of HT-29 CSC cells in nude mice, the effect of scutellarin on the mouse body weight and the growth of HT-29 CSC-derived tumor were explored. qRT-PCR was used for evaluating the effect of scutellarin on mRNA levels of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog in tumor. Western blot and immunohistochemistry analysis were used to detect the effect of scutellarin on protein expressions of c-Myc, Gli1, Lgr5, CD133 and Ki-67 in tumor. The in vitro experiments showed that scutellarin inhibited the growth, transformation and differentiation of HT-29 CSC cells, significantly down-regulated the mRNA levels of Lgr5, c-Myc, CK20 and Nanog in HT-29 CSC cells as well as the protein expression levels of c-Myc, Gli1 and Lgr5 in HT-29 CSC cells. Additionally, animal experiments showed that scutellarin significantly inhibited the growth of subcutaneous xenografts in nude mice, and down-regulated the mRNA expressions of CD133, Lgr5, Gli1, Ptch1, c-Myc, Ki-67, CK20 and Nanog as well as the protein levels of c-Myc, Gli1, Lgr5, CD133 and Ki-67 of xenografts in nude mice. Taken together, scutellarin could inhibit the differentiation of colo-nic cancer stem cells in vitro and in vivo, potentially by down regulation of hedgehog signaling pathway activity.

A Prospective for the Potential Effect of Local Anesthetics on Stem-Like Cells in Colon Cancer

Colorectal cancer is the third most prevalent cancer and the second most frequent cause of cancer-related death in the world. Surgical resection of the primary tumor is the central aspect of the current multiple modes of treatment and has been associated with better prognosis. The process of surgery, including anesthetic regimens, has increasingly been recognized to affect colon cancer recurrence and metastasis. Both retrospective clinical studies and laboratory studies have reported that colon cancer cells are inhibited by some local anesthetics. However, the application of local anesthetics in colon cancer treatment is limited by our understanding of the mechanisms underlying their effects on cancer biology. Local anesthetics have been proved to preferentially inhibit cancer stem cells which imply that local anesthetics target colon cancer stem cell to suppress cancer progressing. Here this paper will review and propose several potential studies, including using colon cancer cell lines and animal models to test the effect of local anesthetics on population, viability, and migration of colon cancer stem-like cell, and screen and search for potential molecular targets underlying these effects.

Mesenchymal stem cells maintain the stemness of colon cancer stem cells via interleukin-8/mitogen-activated protein kinase signaling pathway.

Mesenchymal stem cells (MSCs) can act as a carrier in tumor therapy, and tumor suppressor gene-modified MSCs can reach and suppress the tumor. However, in the colon cancer microenvironment, MSCs could promote tumor growth and create the environment that is conducive to the survival of cancer stem cells (CSCs). This study discovered MSCs from three sources (bone marrow, adipose, placenta) could induce the stemness and epithelial–mesenchymal transition (EMT) of HCT116 in vitro, meanwhile adipose- and placenta-derived MSCs increase the proportion of CD133+/CD44+ HCT116. Then, we explored the interaction mechanism between CD133+/CD44+ HCT116 and MSCs by the bioinformatics and in vitro assays. After CD133+/CD44+ HCT116 were co-cultured with MSCs, many cytokines in MSCs were stimulated, including interleukin-8 (IL-8). The binding of IL-8/CXCR2 activates the downstream mitogen-activated protein kinase (MAPK) signaling pathway in colon CSCs, thereby promoting the stemness and EMT. However, inhibition of IL-8/CXCR2/Erk1/2 could reverse the effect of MSCs on CSC stemness. In addition, MSCs co-cultured with CD133+/CD44+ HCT116 produce a carcinoma-associated fibroblast phenotype via intracellular FGF10–PKA–Akt–β-catenin signaling, which can be attenuated by IL-8 peptide inhibitor. To conclude, IL-8 promotes the interaction between colon CSCs and MSCs, and activates the MAPK signaling pathway in colon CSCs, which provides a theoretical basis for the application of MSCs in clinical practice. Impact statement MSCs have the property of chemotaxis and they can migrate to the tumor site by paracrine pathway in the tumor environment, and then interact with tumor cells. Although a mass of studies have been conducted about the impact of MSCs on tumors, it is still controversial whether the exogenous MSCs promote or inhibit tumor growth. In this work, we evaluated the effects of MSCs from three sources (bone marrow, adipose, placenta) on the proliferation, stemness, and metastasis of the colon cancer cells both in vitro and in vivo. Then, we proved the IL-8/CXCR2/MAPK and FGF10–PKA–Akt–β-catenin signaling pathway which mediate the interplay between MSC and CD133+/CD44+ colon cancer cell. This research aims to provide a theoretical basis for the safe application of MSCs in the clinical treatment of colon cancer.

WBSCR22 Competes with Long Non-coding RNA Linc00346 for miR-509-5p Binding Site to Regulate Cancer Stem Cell Phenotypes of Colorectal Cancer.

Various Long non-coding RNAs (lncRNAs) and MicroRNAs (miRNAs) have been demonstrated to be involved in colorectal cancer stem cells (CSCs). WBSCR22 is a key gene we previously found that functions in colorectal cancer (CRC). This paper aims to investigate the effects of WBSCR22 and its corresponding miRNA and lncRNA in CRC. The expression of WBSCR22 was detected by Western blot and qRT-PCR analysis. Tumor sphere formation assays, CCK-8 analysis, and Transwell assays were applied to examine the colorectal cancer stem cell properties. Luciferase assay and biotin RNA pulldown assay were used to detect the interaction between miR-509-5p and WBSCR22/Linc00346. WBSCR22 is highly expressed in CRC and correlates with poor prognosis. Knockdown of WBSCR22 decreased the sphere-forming capacities, cell proliferation, and invasion abilities. WBSCR22 was negatively regulated by miR-509-5p. Linc00346 promoted the expression of WBSCR22 by adsorbing miR-509-5p. The Linc00346/miR-509-5p/WBSCR22 signal axis regulated the characteristics of colon cancer stem cells. Linc00346 regulated the expression of WBSCR22 by binding to miR-509-5p, thereby monitoring the characteristics of colorectal cancer stem cells. Thus, WBSCR22, Linc00346, and miR-509-5p might be utilized as potential targets for clinical diagnosis and treatment of CRC.

Cancer stemness contributes to cluster formation of colon cancer cells and high metastatic potentials.

The ability of cancer cells to form clusters is a characteristic feature in the development of metastatic tumours with drug resistance. Several studies demonstrated that clusters of circulating tumour cells (CTCs) have a greater metastatic potential to establish new tumours at secondary sites than single CTCs. However, the mechanism of cluster formation is not well understood. In this study, we investigated whether cancer stemness would contribute to cluster formation. We used a tumour sphere culture method to enrich cancer stem cells (CSCs) from colon cancer cells and found that during the second generation of sphere culture, clusters (between 3 and 5 cells) formed within the first 24hours, whereas the rest remained as single cells. The clusters were analysed for stemness and metastatic potential, including gene expressions for cancer stemness (CD133 and Lgr5), epithelial-mesenchymal transition (E-cadherin and TGF-β 1-3) and hypoxia-induced factors (HIF-1α and HIF-2α). The results showed that the clusters expressed higher levels of these genes and colon CSC surface markers (including CD24, CD44 and CD133) than the single cells. Among these markers, CD24 seemed the major contributor linking the cells into the clusters. These clusters also showed a stronger ability to both form colonies and migrate. Our data collectively suggest that colon cancer stemness contributes to cluster formation and that clustered cells exhibit a great metastatic potential. Our study thus provides a method to study the CTC clusters and derive insight into oncogenesis and metastasis.

CD147 Promotes Cell Small Extracellular Vesicles Release during Colon Cancer Stem Cells Differentiation and Triggers Cellular Changes in Recipient Cells.

Cancer cells secrete small extracellular vesicles (sEVs) that are involved in the remodeling of tumor microenvironment (TME) and can promote tumor progression. The role of sEVs and their molecular key players in colon cancer stem cells differentiation are poorly understood. This study aimed to analyze the role and content of sEVs released during the differentiation of colorectal cancer stem cells. Here we show that sEVs secretion during colon cancer stem cells differentiation is partially controlled by CD147, a well-known player involved in colon cancer tumorigenesis. CD147 + sEVs activate a signaling cascade in recipient cells inducing molecular invasive features in colon cancer cells. CD147 knockdown as well as anti-CD147 antibodies impaired sEVs release and downstream effects on recipient cells and blocking multivesicular body maturation prevented sEVs release during the differentiation. Our findings reveal a functional role of CD147 in promoting sEVs release during the differentiation of colon cancer stem cells and in triggering cellular changes in recipient cells.

Quercetin pretreatment enhances the radiosensitivity of colon cancer cells by targeting Notch-1 pathway

Cancer stem-like cells are rare immortal cells within tumor, which are thought to play important roles in ionizing radiation (IR) therapy-resistance. Quercetin is a natural flavonoid with potential anti-cancer properties without significant cytotoxicity in normal tissues. In this study, we demonstrated that quercetin-IR combination treatment exhibited more dramatic anti-cancer effect than either quercetin or IR treatment alone via targeting colon cancer stem cells (CSCs) and inhibiting the Notch-1 signaling. These effects were further verified by in vivo studies which showed remarkable decrease of the CSCs markers and the expression of Notch-1 signaling proteins in human colon cancer xenografts in nude mice. Co-treatment with quercetin and low dose of radiation significantly reduced the expressions of all five proteins of γ-secretase complex in HT-29 and DLD-1 cells. In addition, ectopic expression of the Notch intracellular domain (NICD) partly reversed the inhibition effects by the combination therapy. In conclusion, our results indicated that the combination of quercetin (20 μM) and IR (5Gy) might be a promising therapeutic strategy for colon cancer treatment by targeting colon cancer stem-like cells and inhibiting the Notch-1 signaling. In future studies, we intend to further explore the potential therapeutic efficacy of the quercetin-radiation combination treatment in clinical trials.