Colon Colon Cancer Stem Cells Research Articles

ARL2 is required for homologous recombination repair and colon cancer stem cell survival

ARL2 regulates the dynamics of cytological components and is highly expressed in colon cancer tissues. Here, we report novel roles of ARL2 in the cell nucleus and colon cancer stem cells (CSCs). ARL2 is expressed at relatively low levels in K‐RAS active colon cancer cells, but its expression is induced in CSCs. Depletion of ARL2 results in M phase arrest exclusively in non‐CSC cultured cells; in addition, DNA break stress accumulates in CSCs leading to apoptosis. ARL2 expression is positively associated with the expression of all six RAD51 family genes, which are essential for homologous recombination repair (HRR). Furthermore, ARL2 is required for HRR and detected within chromatin compartments. These results demonstrate the requirement of ARL2 in colon CSC maintenance, which possibly occurs through mediating double‐strand break DNA repair in the nucleus.

Suppressive effect of \u03b1-mangostin for cancer stem cells in colorectal cancer via the Notch pathway

BackgroundSince colon cancer stem cells (CSCs) play an important role in chemoresistance and in tumor recurrence and metastasis, targeting of CSCs has emerged as a sophisticated strategy for cancer therapy. α-mangostin (αM) has been confirmed to have antiproliferative and apoptotic effects on cancer cells. This study aimed to evaluate the selective inhibition of αM on CSCs in colorectal cancer (CRC) and the suppressive effect on 5-fluorouracil (5-FU)-induced CSCs.MethodsThe cell viability assay was performed to determine the optimal concentration of αM. A sphere forming assay and flow cytometry with CSC markers were carried out to evaluate the αM-mediated inhibition of CSCs. Western blot analysis and quantitative real-time PCR were performed to investigate the effects of αM on the Notch signaling pathway and colon CSCs. The in vivo anticancer efficacy of αM in combination with 5-FU was investigated using a xenograft mouse model.ResultsαM inhibited the cell viability and reduced the number of spheres in HT29 and SW620 cells. αM treatment decreased CSCs and suppressed the 5-FU-induced an increase in CSCs on flow cytometry. αM markedly suppressed Notch1, NICD1, and Hes1 in the Notch signaling pathway in a time- and dose-dependent manner. Moreover, αM attenuated CSC markers CD44 and CD133, in a manner similar to that upon DAPT treatment, in HT29 cells. In xenograft mice, the tumor and CSC makers were suppressed in the αM group and in the αM group with 5-FU treatment.ConclusionThis study shows that low-dose αM inhibits CSCs in CRC and suppresses 5-FU–induced augmentation of CSCs via the Notch signaling pathway.

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.

Effects of β-carotene on Expression of Selected MicroRNAs, Histone Acetylation, and DNA Methylation in Colon Cancer Stem Cells.

BackgroundBeta-carotene (BC) is a carotenoid which exerts anti-cancer effects in several types of cancer, including colorectal cancer. Epigenetic modifications of genes, such as histone deacetylation and DNA hypermethylation, have also been detected in various types of cancer. To understand the molecular mechanism underlying cancer preventive and therapeutic effects of BC, microRNAs (miRNAs), histone acetylation, and global DNA methylation in colon cancer stem cells (CSCs) were investigated.MethodsHCT116 colon cancer cells positive for expression of CD44 and CD133 were sorted by flow cytometry and used in subsequent experiments. Cell proliferation was examined by the MTT assay and self-renewal capacity was analyzed by the sphere formation assay. The miRNA sequencing array was used to detect miRNAs regulated by BC. Histone acetylation levels were measured by the Western blot analysis. mRNA expression of DNA methyltransferases (DNMTs) was examined by qPCR and global DNA methylation levels were determined by enzyme-linked immunosorbent assay.ResultsTreatment of CD44+CD133+ colon CSCs with BC caused a reduction in both cell proliferation and sphere formation. Analysis of the miRNA sequencing array showed that BC regulated expression of miRNAs associated with histone acetylation. Histone H3 and H4 acetylation levels were elevated by BC treatment. In addition, BC treatment down-regulated DNMT3A mRNA expression and global DNA methylation in colon CSCs.ConclusionsThese results suggest that BC regulates epigenetic modifications for its anti-cancer effects in colon CSCs.

Effects of down-regulation of IGF2 gene on the biological characteristics of HCT116 colon cancer stem cells

Objective: To investigate the effect of down-regulation of insulin-like growth factor 2 (IGF2) gene on the biological characteristics of HCT116 colon cancer stem cells (CSCs). Methods: Flow cytometry sorting technology was used to isolate CSCs from colon cancer cell line HCT116 by a monoclonal antibody against CD133; serum free floating culture assay was used for the enrichment of CSCs. The proportion of CD133(+) cells was analyzed by flow cytometry; CSCs were identified by sphere culturing, immunofluorescence analysis and soft agar clone formation. RT-qPCR method was used to examine transcriptional level of IGF2 gene in CSCs. Western blotting was used to examine IGF2 protein expression in CSCs. siRNA was used to establish IGF2 transient knock down model in CSCs. Cell proliferation array, cell cycle and apoptosis analysis, cell invasion array and colony forming assay were used to further examine the role of IGF2 on the biological characteristics of colon CSCs. Results: CSCs were successfully isolated from HCT116 cell lines, which were cultured to form cell spheres in serum-free stem cell culture medium. We found that the morphology of sphere-forming-like cells after several passages maintained the same characteristics as that of the first passage. The results of immunofluorescence showed that CSC markers including CD133 and ALDH continued positively expressing on the cell surface of CSCs, and flow cytometry analysis showed that more than 90% of the spheroid cells remained CD133 positive. The clone formation rate of non-CSCs group and CSCs group were (28.10±2.66)% and (43.73±2.30)% respectively, with significant difference (P<0.01). The RT-qPCR results showed that the transcriptional level IGF2 gene in non-CSCs group and CSCs group were (1.06±0.24) and (2.17±0.51) respectively, with significant difference (P<0.05). The western blot results showed that the protein expression of IGF2 in CSCs group and non-CSCs group were (1.10±0.55) and (2.14±0.23) respectively, with significant difference (P<0.05). Knockdown of IGF2 significantly decreased the percentage of CD133(+) cells in CSCs and cell proliferation (P<0.01). Knockdown of IGF2 increased the percentage of G(2)/M phase (23.46% of siNC group vs 60.14% of siIGF2 group) and cell apoptosis (2.80% of siNC group vs 40.70% of siIGF2 group), while decreased the percentage of G(0)/G(1) phase (40.77% of siNC group vs 17.73% of siIGF2 group). The invasion results showed that the number of cells penetrating into the basement surface in siNC group and siIGF2 group was (109.00±16.37) and (54.00±8.19) respectively, with significant difference (P<0.01). The rate of sphere-forming of colon CSCs in siNC group and siIGF2 group were (51.70±7.42)% and (21.27±2.35)% respectively, with significant difference (P<0.01). The clone formation rate of siNC group and siIGF2 group were (37.20±3.87)% and (18.23±2.25)% respectively, with significant difference (P<0.01). Conclusion: IGF2 gene plays an important role in maintaining the biological characteristics of colon cancer stem cells and promoting self-renewal and stemness of colon CSCs.

Metabolic influence of walnut phenolic extract on mitochondria in a colon cancer stem cell model.

Walnut phenolic extract (WPE) reduces proliferation and enhances differentiation of colon cancer stem cells (CSCs). The present study investigated the metabolic influence of WPE on the mitochondrial function of colon CSCs to determine its underlying mechanism. CD133+CD44+ HCT116 colon cancer cells were selected by fluorescence-activated cell sorting and were treated with or without 40µg/mL WPE. RNA-sequencing (RNA-Seq) was performed to identify differentially expressed genes (DEGs), which were further validated with RT-PCR. WPE-induced alterations in mitochondrial function were investigated through a mitochondrial stress test by determining cellular oxygen consumption rate (OCR), an indicator of mitochondrial respiration, and extracellular acidification rate (ECAR), an indicator of glycolysis, which were further confirmed by glucose uptake and lactate production tests. RNA-Seq analysis identified two major functional clusters: metabolic and mitochondrial clusters. WPE treatment shifted the metabolic profile of cells towards the glycolysis pathway (ΔECAR = 36.98 mpH/min/ptn, p = 0.02) and oxidative pathway (ΔOCR = 29.18pmol/min/ptn, p = 0.00001). Serial mitochondrial stimulations using respiration modulators, oligomycin, carbonyl cyanide-4 (trifluoromethoxy) phenylhydrazone, and rotenone/antimycin A, found an increased potential of mitochondrial respiration (ΔOCR = 111.5pmol/min/ptn, p = 0.0006). WPE treatment also increased glucose uptake (Δ = 0.39pmol/µL, p = 0.002) and lactate production (Δ = 0.08nmol/µL, p = 0.005). WPE treatment shifts the mitochondrial metabolism of colon CSC towards more aerobic glycolysis, which might be associated with the alterations in the characteristics of colon CSC.

Opposing effects of low versus high concentrations of water soluble vitamins/dietary ingredients Vitamin C and niacin on colon cancer stem cells (CSCs).

Colorectal cancer is one of the global causes of cancer deaths. Cancer stem cells (CSCs) inside the tumour niche responsible for metastasis and relapses, and hence need to be targeted for cancer therapeutics. Although dietary fibre and lifestyle changes have been recommended as measures for colorectal cancer prevention, no such recommendations are available for using water soluble vitamins as prophylaxis measure for colorectal cancers. High dose of Vitamin C has been proven to selectively kill colon cancer cells having BRAF and KRAS mutations by inducing oxidative stress. In this study, we show for the first time the opposing effects of the low and high dose of Vitamin C and vitamin B3 on colon CSCs isolated from HT-29 and HCT-15 colorectal carcinoma cell lines. At small doses, both of these vitamins exerted a cell proliferative effect only on CSCs, while there was no change in the proliferation status of non-stem cancer cells and wild-type (WT) populations. On the other hand, the death effects induced by high doses of Vitamin C and B3 were of the order of 50-60% and ∼30% on CSCs from HT-29 and HCT15, respectively. Interestingly, the control fibroblast cell line (NIH3T3) was highly refractory all the tested concentrations of Vitamin C and B3, except for the highest dose - 10,000 μg of Vitamin C that induced only 15% of cell death. Hence, these results indicate the future scope of use of therapeutic doses of Vitamin C and B3 especially in patients with advanced colorectal cancer.

Knockdown of IRE1α inhibits colonic tumorigenesis through decreasing β-catenin and IRE1α targeting suppresses colon cancer cells.

The endoplasmic reticulum (ER) stress occurs frequently in cancers. The unfolded protein response (UPR) is activated to cope with ER stress. This has generated widespread interest in targeting UPR as therapeutic strategies. Inositol-requiring transmembrane kinase/endonuclease 1α (IRE1α), an ER stress sensor, is a key component of UPR. However, the role of IRE1α in tumorigenesis remains unclear. The purpose of this work is to investigate the role of IRE1α in colon cancer and to determine whether IRE1α could serve as a target for therapy. We found that knockdown of IRE1α suppressed the proliferation of colon cancer cells in vitro and xenograft growth in vivo. Inhibition of expression of IRE1α decreased stemness of colon cancer stem cells (CSCs) and attenuated growth of intestinal organoids. Genetic ablation of IRE1α prevented the colitis-associated colonic tumorigenesis in mice. The mechanistic study indicates that knockdown of IRE1α repressed the expression of β-catenin, a key factor that drives colonic tumorigenesis, through activating pancreatic ER kinase/eukaryotic translation initiation factor 2α signaling. We found that the IRE1a-specific inhibitor 4μ8C could suppress the production of β-catenin, inhibited the proliferation of colon cancer cells, repressed colon CSCs and prevented xenograft growth. The results suggest that IRE1α has a critical role in colonic tumorigenesis and IRE1α targeting might be a strategy for treatment of colon cancers.

Walnut Phenolic Extracts Induced Metabolic Reprogramming in Colon Cancer Stem Cells by Reinforcing the Mitochondrial Function: From RNA‐seq to Mitochondrial Stress Test

Previously we reported that walnut phenolic extracts (WPE) can reduce the stemness of colon cancer stem cells (CSCs). Since cancer cells commonly exhibit metabolic alterations, such as the Warburg effect, and CSCs also demonstrate impaired mitochondrial functions, we determined the metabolic influence of WPE on colon CSCs in the ramification of mitochondrial functions. From the HCT116 colon cancer cell line, CD133+CD44+ cells were sorted by FACS and treated with or without 20μg/mL WPE. To investigate the mechanism behind the effect of walnuts on colon CSCs, RNA‐sequencing (RNA‐seq) analysis was conducted using an Illumina Hiseq 2500 platform and differentially expressed genes (DEGs) were categorized into functional clusters based on Gene Ontology functional enrichment analysis. ClueGo and CluePedia plug‐ins in Cytoscape was used to construct inter‐connected functional networks including the relative expression patterns of the DEGs within the integrated metabolic and mitochondrial cluster. RT‐PCR method validated the RNA‐seq analysis data. To evaluate the bioenergetic profile of the WPE‐treated CD133+CD44+ HCT116 cells cellular oxygen consumption rate (OCR), an indicator of mitochondrial respiration, and extracellular acidification rate (ECAR), an indicator of glycolysis, were determined using XFp Analyzer (Seahorse Bioscience, North Billerica, MA), which can measure mitochondrial function and glycolytic activity through mitochondrial stress test. For the stress test 1 μmole/L oligomycin A, 0.4 μmole/L carbonyl cyanide‐4 phenylhydrazone, and the mixture of 1μmole/L rotenone and 1μmole/L antimycin A were added to the media sequentially to assess the global change in mitochondrial respiration. To confirm the data glucose uptake was analyzed using Colorimetric Glucose Uptake Kit (Abcam, Cambridge, UK) and lactate production was measured by L‐lactate Assay Kit (Abcam). RNA‐seq analysis found 1983 DEGs and up or down‐regulated DEGs were classified into two major functional clusters, the metabolic cluster (73 genes) and the mitochondria cluster (112 genes). WPE treatment shifted the bioenergetic profile of CD133+CD44+ HCT116 cells towards the more glycolysis pathway (ΔECAR=36.98 mpH/min/ptn, p=0.003). Mitochondrial stimulation demonstrated an increase in mitochondrial respiration potential (ΔOCR=111.5 pmol/min/ptn, p‐value=0.0006). We also confirmed that both glucose uptake (Δ=0.39 pmol/ul, p=0.002) and lactate production (Δ=0.08 nmol/ul, p=0.005) were elevated after WPE treatment. In the present colon cancer stem cell model WPE treatment elicited a broad range of transcriptional modulation associated with mitochondria and metabolic pathways. Enhanced glycolysis illustrated that WPE treatment may drive a Warburg effect‐like phenotypic metabolic shift. A high bioenergetic signature and increased mitochondrial capacity indicate the restoration and re‐maturation of mitochondrial function by WPE treatment, which may cause the diminished stemness in WPE‐treated colon CSCs.Support or Funding InformationCalifornia Walnut Commission

Grape compounds suppress colon cancer stem cells in vitro and in a rodent model of colon carcinogenesis.

BackgroundWe have previously shown that the grape bioactive compound resveratrol (RSV) potentiates grape seed extract (GSE)-induced colon cancer cell apoptosis at physiologically relevant concentrations. However, RSV-GSE combination efficacy against colon cancer stem cells (CSCs), which play a key role in chemotherapy and radiation resistance, is not known.MethodsWe tested the anti-cancer efficacy of the RSV-GSE against colon CSCs using isolated human colon CSCs in vitro and an azoxymethane-induced mouse model of colon carcinogenesis in vivo.ResultsRSV-GSE suppressed tumor incidence similar to sulindac, without any gastrointestinal toxicity. Additionally, RSV-GSE treatment reduced the number of crypts containing cells with nuclear β-catenin (an indicator of colon CSCs) via induction of apoptosis. In vitro, RSV-GSE suppressed - proliferation, sphere formation, nuclear translocation of β-catenin (a critical regulator of CSC proliferation) similar to sulindac in isolated human colon CSCs. RSV-GSE, but not sulindac, suppressed downstream protein levels of Wnt/β-catenin pathway, c-Myc and cyclin D1. RSV-GSE also induced mitochondrial-mediated apoptosis in colon CSCs characterized by elevated p53, Bax/Bcl-2 ratio and cleaved PARP. Furthermore, shRNA-mediated knockdown of p53, a tumor suppressor gene, in colon CSCs did not alter efficacy of RSV-GSE.ConclusionThe suppression of Wnt/β-catenin signaling and elevated mitochondrial-mediated apoptosis in colon CSCs support potential clinical testing/application of grape bioactives for colon cancer prevention and/or therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12906-016-1254-2) contains supplementary material, which is available to authorized users.

Abstract 2500: Grape compounds suppress colon cancer stem cells in vitro and in a rodent model of colon carcinogenesis

Abstract We have previously shown that the grape bioactive compound resveratrol (RSV) potentiates grape seed extract (GSE)-induced colon cancer cell apoptosis at physiologically relevant concentrations. However, RSV-GSE combination efficacy against colon cancer stem cells (CSCs), which play a key role in chemotherapy and radiation resistance, is not known. Hence, we tested the anti-cancer efficacy of the RSV-GSE in an azoxymethane-induced mouse model of colon carcinogenesis. RSV-GSE suppressed tumor incidence similar to sulindac, without any gastrointestinal toxicity. Additionally, RSV-GSE treatment reduced the number of crypts containing cells with nuclear β-catenin (an indicator of colon CSCs) via induction of apoptosis. In in vitro mechanistic studies, RSV-GSE suppressed proliferation and sphere formation properties of colon CSCs (positive for CD44, CD133 and ALDH1b1), suppressed pGSK3β and nuclear translocation of β-catenin, a critical regulator of CSC proliferation, similar to sulindac. RSV-GSE, but not sulindac, suppressed nuclear levels of downstream proteins of Wnt/β-catenin pathway, c-Myc and cyclin D1. RSV-GSE also induced mitochondrial-mediated apoptosis in colon CSCs characterized by elevated p53, Bax/Bcl-2 ratio and cleaved PARP. Furthermore, shRNA-mediated knockdown of p53, a tumor suppressor gene that is mutated in advanced stages of colon cancer, in the colon CSCs did not alter efficacy of RSV-GSE. The suppression of Wnt/β-catenin signaling and elevated mitochondrial-mediated apoptosis in colon CSCs support potential clinical testing/application of grape bioactives for colon cancer prevention and/or therapy. Citation Format: Lavanya Reddivari, Venkata Charepalli, Ramakrishna Vadde, Sridhar Radhakrishnan, Joshua D. Lambert, Ryan J. Elias, Jairam KP Vanamala. Grape compounds suppress colon cancer stem cells in vitro and in a rodent model of colon carcinogenesis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2500.

Highlights of This Issue

Inhibitors targeting Bromodomain and Extra-Terminal (BET) proteins are currently in clinical trials for a number of cancer types. In pre-clinical models, BET inhibitors have shown great promise as epigenetic therapies that alter a number of BET protein interactions, including interactions with transcription factors. In the Rapid Impact article, Asangani and colleagues develop clonal cell lines that are enzalutamide-resistant. In addition to evaluating androgen receptor (AR) signaling and transcriptional consequences in these enzalutamide-resistant model systems, BET inhibitor efficacy is also assessed. The data offer a strong rationale for combining BET inhibitors with AR antagonists for the treatment of advanced prostate cancer.Here a PCR-based miRNA (miR) profiling analysis identified miRNA-137 (miR-137) as downregulated in colon cancer stem cells (CSCs) compared to normal colon stem cells (NCSCs) and regulated doublecortin-like kinase 1 (DCLK1) gene expression. Moreover, experimental data demonstrated that miR-137 has the capacity to suppress the tumorigenicity of colon CSCs and that maintained expression of miR-137 in NCSCs contributes to suppressing uncontrolled cell proliferation through the inhibition of DCLK1 expression. The insight provided by this study concerning the miR-137/DCLK1 axis lends support for the development of potential gene therapeutic strategies targeting colon CSCs.miR-155 is an oncogenic microRNA (miR) associated with poor prognosis in a variety of cancer types including lymphoma, lung, breast, and colon cancer. Czochor and colleagues report that miR-155 over-expression drives an increase in mutation frequency in cell culture and in vivo in a transgenic mouse model. Mechanistic evidence suggests that miR-155 impacts DNA repair and genome stability by reducing expression of the high-fidelity DNA polymerase delta and by promoting the error-prone non-homologous end-joining (NHEJ) double-strand break (DSB) repair pathway. Elucidating the role of miR-155 in promoting tumorigenesis is an important step in developing therapies to target miR-155 over-expressing tumors.Multiple myeloma (MM) tumor cells are vulnerable to endoplasmic reticulum (ER) stress because of the huge amount of Ig protein they produce. One of the most successful anti-myeloma therapeutics, bortezomib, actually enhances ER stress and that may be the mechanism of its effect. This study shows that expression and activity of the serum and glucocorticoid-regulated kinase (SGK) is a resistance mechanism for sensitivity of MM cells to cytotoxicity of ER stress inducers including bortezomib. This was found in vitro in cell lines and primary cells as well as in vivo in xenografted mice. Mechanistically, resistance was mediated by restraint of a Jun kinase pro-apoptotic pathway in which SGK-induced phosphorylation of SEK down-regulated the pathway. These results support the development of SGK inhibitors in MM to be combined with bortezomib.

In vivo relevant mixed urolithins and ellagic acid inhibit phenotypic and molecular colon cancer stem cell features: A new potentiality for ellagitannin metabolites against cancer

Colon cancer stem cells (CSCs) offer a novel paradigm for colorectal cancer (CRC) treatment and dietary polyphenols may contribute to battle these cells. Specifically, polyphenol-derived colon metabolites have the potential to interact with and affect colon CSCs. We herein report the effects against colon CSCs of two mixtures of ellagitannin (ET) metabolites, ellagic acid (EA) and the gut microbiota-derived urolithins (Uro) at concentrations detected in the human colon tissues following the intake of ET-containing products (pomegranate, walnuts). These mixtures reduce phenotypic and molecular features in two models of colon CSCs: Caco-2 cells and primary tumour cells from a patient with CRC. The mixture containing mostly Uro-A (85% Uro-A, 10% Uro-C, 5% EA) was most effective at inhibiting the number and size of colonospheres and aldehyde dehydrogenase activity (ALDH, a marker of chemoresistance) whereas the mixture containing less Uro-A but IsoUro-A and Uro-B (30% Uro-A, 50% IsoUro-A, 10% Uro-B, 5% Uro-C, 5% EA) had some effects on the number and size of colonospheres but not on ALDH. These data support a role for polyphenols metabolites in the control of colon cancer chemoresistance and relapse and encourage the research on the effects of polyphenols against CSCs.

Rapid Recognition and Isolation of Live Colon Cancer Stem Cells by Using Metabolic Labeling of Azido Sugar and Magnetic Beads.

New approach for colon cancer stem cells (CSCs) recognition and isolation is reported. Colon CSCs are responsible for colonic tumor growth, metastasis, and resistance for radio-/chemotherapies. An accurate identification and isolation method is critical for understanding and characterization of these cells. In our work, we recognized CSCs' population from colon cancer cells by using metabolic labeling of azido sugar based on the quiescent nature of these cells, which differed fundamentally from previously described methods by using specific cellular markers to recognize and isolate CSCs. Later the putative CSCs were isolated by using commercially available magnetic beads. The isolated cells population had much higher sphere formation efficiency, soft-agar colony formation efficiency, and an mRNA level of colon stem cells marker Lgr5 than the leftover population. Our method provides a new avenue and a general strategy for recognition and isolation of CSCs, which shows great potential for further use in both the fundamental research of CSCs and clinical tests.

ErbB-3 activation by NRG-1β sustains growth and promotes vemurafenib resistance in BRAF-V600E colon cancer stem cells (CSCs).

Approximately 5-10% of metastatic colorectal cancers harbor a BRAF-V600E mutation, which is correlated with resistance to EGFR-targeted therapies and worse clinical outcome. Vice versa, targeted inhibition of BRAF-V600E with the selective inhibitor PLX 4032 (Vemurafenib) is severely limited due to feedback re-activation of EGFR in these tumors. Mounting evidence indicates that upregulation of the ErbB-3 signaling axis may occur in response to several targeted therapeutics, including Vemurafenib, and NRG-1β-dependent re-activation of the PI3K/AKT survival pathway has been associated with therapy resistance.Here we show that colon CSCs express, next to EGFR and ErbB-2, also significant amounts of ErbB-3 on their membrane. This expression is functional as NRG-1β strongly induces AKT/PKB and ERK phosphorylation, cell proliferation, clonogenic growth and promotes resistance to Vemurafenib in BRAF-V600E mutant colon CSCs. This resistance was completely dependent on ErbB-3 expression, as evidenced by knockdown of ErbB-3. More importantly, resistance could be alleviated with therapeutic antibody blocking ErbB-3 activation, which impaired NRG-1β-driven AKT/PKB and ERK activation, clonogenic growth in vitro and tumor growth in xenograft models. In conclusion, our findings suggest that targeting ErbB-3 receptors could represent an effective therapeutic approach in BRAF-V600E mutant colon cancer.

Potential role for the Metnase transposase fusion gene in colon cancer through the regulation of key genes.

The Metnase fusion gene consists of a SET histone methyltransferase domain and a transposase domain from Mariner transposase. This transposable element is involved in chromosome decatenation, enhances DNA repair, promotes foreign DNA integration, and assists topoisomerase II function. This study investigates the role of Metnase in colon cancer homeostasis and maintenance of the stemness phenotype in colon cancer stem cells (CSCs). Silencing of Metnase was performed in human cancer cell lines before and after treatment with cisplatin, and in colon CSCs. Subsequent changes in the expression of genes involved in repair mechanisms, DNA synthesis, topoisomerase II function, and metastasis as well stemness transcription factors were studied with RT-qPCR experiments. Cellular viability and apoptosis were evaluated by flow cytometry. The results suggest that Metnase influences the expression of many genes involved in the above processes. Furthermore, Metnase levels appear to impact upon expression of NANOG, OCT3/4, and SOX2. Suppression of Metnase also led to an increase in apoptosis. Therefore, Metnase may possess an important role in DNA repair, topoisomerase II function, and the maintenance of stemness during colon cancer development.

Abstract 3903: A sub-set of DCLK1+ve colon cancer stem cells (CSCs) survive curcumin induced autophagy, while co-treatment with curcumin +DCLK1-siRNA eliminates CSCs: Role of long and short isofoms of DCLK1

Abstract Curcumin induces apoptotic/autophagic cell death of colon cancer cells, and targets cancer stem cells (CSC). In here, we examined for the first time, possible apoptotic/autophagic effects of curcumin on colon CSCs, positive for DCLK1/CD44/LGR5. Curcumin (20-25µM) caused significant loss in the expression of stem cell markers DCLK1/CD44/ALDHA1/LGR5, along with down-regulation of several pluripotency factors, in tumorspheres and xenografts derived from human colon cancer cells. Surprisingly, curcumin induced proliferation and autophagic survival of a small subset of DCLK1+ve CSCs. Colon cancer tumorspheres were disintegrated by curcumin, but regrew as secondary tumorspheres after 30-40 days, suggesting the novel possibility that autophagy-associated survival may allow CSCs to survive and regrow as tumorspheres. We next examined inhibitory effects of DCLK1-siRNA. Unlike curcumin, DCLK1-siRNA only induced apoptotic cell death, with no autophagic response. Combination of curcumin+DCLK1-siRNA caused massive apoptotic/autophagic cell death of colon cancer cells growing as tumorpsheres or xenografts, and reversed the transformed phenotype of CSCs, as previously reported by us (Int.J.Cancer, 2012). Co-treatment with curcumin+DCLK-siRNA was more effective that either agent alone in: 1)attenuating growth of tumorspheres, 2)shrinking the pre-formed xenografts in vivo, and 3)eliminating relapse (re-growth) of tumorspheres. We recently discovered that colon cancer cells and adenocarcinomas from patients express DCLK1-short (S) isoform , transcribed from intron-V promoter; long (L) isoform was silenced by promoter methylation. Normal intestinal cells and non-transformed epithelial cells, on the other hand, mainly expressed DCLK1-L isoform. Promoter-reporter studies and ChIP assays demonstrated that curcumin significantly reduced expression levels of DCLK1-S isoform in colon cancer cells but paradoxically increased its expression in non-transformed/normal intestinal cells. Thus, the differential effects of curcumin on DCLK1 expression in cancer vs normal cells may explain the underlying mechanisms by which curcumin targets CSCs while sparing normal stem cells. Conclusion. Our studies strongly suggest that, 1) DCLK1 represents a functional protein for colon cancers, 2) combination of curcumin+DCLK1-siRNA may target and eradicate colon cancer stem cells., and 3) identifying small molecules that inhibit expression of S-isoform may allow to specifically target cancer stem cells, while sparing normal stem cells for cancer treatment purposes. This work was supported by NIH Granst to PS (R01CA09795909 and RO1CA0975909-S1). Citation Format: Shubhashish Sarkar, Malaney O'Connell, Carla, Kantara, Pomila Singh. A sub-set of DCLK1+ve colon cancer stem cells (CSCs) survive curcumin induced autophagy, while co-treatment with curcumin +DCLK1-siRNA eliminates CSCs: Role of long and short isofoms of DCLK1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3903. doi:10.1158/1538-7445.AM2014-3903

Effect of the ADAPT strategy on dormant CD133+ colon cancer stem cells (CSC) and molecular complete remission measured by peripheral blood mononuclear (PBMC) CD133 mRNA.

e14153 Background: The median survival for patients with unresectable metastatic colorectal cancer (CRC) is ~2 years with modern chemotherapy which yields only 5-10% complete responses (CR) including metastasectomy. Recurrences after CR are very common thanks to presence of dormant CSC that are best targeted by our proposed two-step ADAPT strategy: activate from dormancy and potentiate targeting. We examine this strategy in various CRC models and reviewed the impact on stemess including CD133 mRNA, a circulating CSC marker that predict colon cancer relapse. Methods: Different CRC models (in vitro and in vivo) were interrogated similar to clinical ADAPT treatment protocol using capecitabine (or 5FU) plus celecoxib. We also conducted IRB approved retrospective review of unresectable metastatic CRC patients treated ADAPT therapy and in those who also had PBMC CD133 mRNA measured. Results: Contrary to 5FU, which eliminates proliferating CRC cells via apoptosis but also stimulates stemness, celecoxib preferentially deplete CD133+ colon cells and exert potent stemness inhibition via rapid tumor necrosis by perturbing hypoxia and energy metabolism via CA-IX. Following response to first-line chemotherapy, ADAPT strategy plus radiation improved CR or near CR rate to 49/126 (40%) in unresectable CRC patients whose median survival had reached 92.7 months (95% CI, 53.5 months - not reached). Paradoxically, none surgical CR patients (n= 16) enjoyed 100% 5-year relapse free survival compared to 42% of surgical patients (p = 0.04). The PBMC CD133 mRNA in five long-term CR patients were 0.0024, 0.29, 0.5, 0.56, 2.96 respectively, all below previously reported cutoff value of 4.79 for recurrence and far below CD133 mRNA levels (28, 375, 3997, 15662, 83240) in none CR patients. Conclusions: ADAPT plus radiation preferentially targets colon CSC via hypoxia/CA-IX and improves clinical CR rate and molecular CR as measured by PBMC CD133 mRNA. We are actively interrogating the effects of ADAPT strategies in a phase II study funded by Gateway in CRC patients and in genetic CRC animal models.

CD133 expression is an independent prognostic marker for low survival in colorectal cancer

Colon cancer cells have previously been demonstrated to contain a subpopulation of CD133+ tumour cells that have the ability to initiate tumour growth and are thus referred to as colon cancer-initiating cells or colon cancer stem cells (CSCs). As CD133 is currently one of the best markers to characterise colon CSCs, we analysed CD133+ tumour cells in colorectal cancer specimens using immunohistochemistry. We show that CD133 detection is specific and that the CD133 antigen is localised on the glandular-luminal surface of colon cancer cells, whereas undifferentiated tumour cells at the front of invasion are CD133−. In addition, CD133+ cells are characterised in situ by lack of CK20 expression, whereas they are positive for EpCAM. Moreover, we show that CD133 expression in colorectal cancer is an independent prognostic marker that correlates with low survival in a stratified patient collective. Our results indicate that in colorectal cancer, the CD133+ tumour cells can be detected by immunohistochemistry, which facilitates their further characterisation in situ.

Human colon cancer stem cells locate in hypoxic niche

22209 Background: It has been proposed that a small number of undifferentiated tumorigenic CD133+ cells from human colon carcinoma be defined as colon cancer stem cells (CSCs) based on their self-renewal and propagation properties. In order to further investigate the CSCs niche, we applied an approach to resolve the spatial organization of human colon cancer stem cells according to hypoxia and the impact of hypoxia on irradiation. Methods: Eight fresh samples of primary human colon cancers were implanted in immunocompromised mice. The percentage of hypoxic CD133+ cell population was detected and quantified by use of hypoxic maker pimonidazole (PIM) via flow-cytometry. Measurements were also made of CD133+ cell population and its change of hypoxic situation following irradiation with a single dose of 2 Gy, 5 Gy, 10 Gy, and 20 Gy, respectively. Results: CD133+ cells were found to be the most positive for binding of PIM. The percentage of hypoxic population of CD133+ cells were ranged from 51.6% to 87.0% with an average of 70.2%, whereas the other colon cancer cells labeled CD133- demonstrated an average of 20.2% ranged from 5.3% to 49.1%. Hypoxic population of CD133+ cells was 4.5 times (from 2 to 12 times) than that of CD133- cells. Compared with control, irradiation at 2 Gy to 20 Gy with a single fraction reduced the CD133+ cell population in period of 1 to 6 hours after treatment. Interestingly, it resulted in an increase of the percentage of hypoxic CSCs to different extent rather than other hypoxic colon cancer cells. Conclusions: Most population of human colon CSCs locate in hypoxic niche, and the hypoxic human colon CSCs resist radiotherapy to some extent. It is implicated that regionally defined hypoxia plays a fundamental role in both regulating human colon CSCs function and diminishing therapeutic response. No significant financial relationships to disclose.