Colorectal Colorectal Cancer Stem Cells Research Articles

Metformin inhibits self-renewal of colorectal cancer stem cells by inhibiting mitochondrial oxidative phosphorylation

To investigate the mechanism of metformin for inhibiting self-renewal of colorectal cancer stem cells (CSCs). CSCs were sorted from Wnt reporter- transfected colorectal cancer patient-derived organoids (PDOs) by fluorescence-activated cell sorting (FACS) and treated with metformin. The changes in self-renewal of the cells were assessed using sphere formation, colony formation and limiting dilution assays. The mRNA expressions of genes related with stemness and differentiation and Wnt target genes was detected by qRT-PCR. Wnt activity was assessed using flow cytometry in the CSCs. Seahorse analysis was used to evaluate cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) after metformin treatment. Mitochondrial membrane potential levels were detected with TMRE staining, and reactive oxygen species (ROS) levels were detected using MitoSOX staining. Galactose (10 mmol/L), metformin (10 μmol/L), NAC (5 mmol/L), and galactose+metformin were used to modulate ROS levels in the CSCs, and sphere-formation assay and flow cytometry were used to assess the changes in self- renewal capacity and Wnt activity. The effect of lentiviral transfection of yeast NADH dehydrogenase NDI1 on TMRE staining, MitoSOX staining and Wnt activity in the CSCs were analyzed with flow cytometry. Metformin significantly decreased the capacities of CSCs to form spheres, colonies and xenografts and reduced Wnt activity in the cells (P < 0.01). The mRNA levels of stemness-related genes and Wnt target genes decreased significantly while those of differentiation-related genes increased in metformin-treated CSCs (P < 0.05), which also showed significantly decreased OCR, TMRE and ROS levels with enhanced ECAR (P < 0.001). Galactose significantly increased sphereforming capacity, ROS levels and Wnt activity of the cells, and these effects were significantly inhibited by metformin (P < 0.05). Transfection of the CSCs with NDI1 significantly attenuated the inhibitory effects of metformin on proportion of CSCs and Wnt signaling pathway activity. Metformin reduces mitochondrial oxidative phosphorylation and ROS levels by inhibiting mitochondrial complex Ⅰ, thereby suppressing Wnt signaling pathway to reduce selfrenewal ability of colorectal CSCs.

Knockdown of hnRNPAB reduces the stem cell properties and enhances the chemosensitivity of human colorectal cancer stem cells.

Heterogeneous ribonucleoproteinAB (hnRNPAB) is one of the main members of the nuclear heterogeneous ribonucleoprotein family and plays a crucial role in the occurrence and development of tumours. A previous study by the authors demonstrated that hnRNPAB was highly expressed in colorectal cancer tissues and was closely associated with a poor prognosis of patients. However, the contribution of hnRNPAB to the tumorigenesis and drug resistance of colorectal cancer (CRC) stem cells (CSCs) remains elusive. The aim of the present study was thus to examine whether hnRNPAB can enhance the characteristics of colorectal CSCs and chemotherapeutic drug resistance by altering the cell cycle and the apoptosis of colorectal CSCs. The results revealed that the expression of hnRNPAB in colorectal CSCs was increased compared with that in their parental cells. The knockdown of hnRNPAB reduced the sphere formation of and the levels of CSC markers in colorectal CSCs, enhanced sensitivity to 5‑fluorouracil and oxaliplatin chemotherapy and increased apoptosis. Taken together, these data indicate the role of hnRNPAB in maintaining CSC properties and provide a novel therapeutic target for the treatment of CRC and particularly, drug resistance.

EpCAM is critical for tumor proliferation and oxaliplatin chemoresistance in EpCAMhigh/CD44+ colorectal cancer stem cells

Abstract Objectives A small subpopulation of colorectal cancer stem cells (CSCs) possess the ability to self-renew and the capacity to initiate the original tumor. EpCAMhigh/CD44+ cells are regarded as CSCs in colorectal cancer. The present study was undertaken to investigate the significance of EpCAM in the in vitro proliferation ability and oxaliplatin chemoresistance of EpCAMhigh/CD44+ colorectal CSCs. Methods We applied fluorescence-activated cell sorting (FACS) to separate the EpCAMhigh/CD44+ subset from human colorectal cancer cell line HCT116. We also used siRNA targeting EpCAM to create EpCAM−/CD44+ subpopulation. Then we compared EpCAMhigh/CD44+ cells and EpCAM−/CD44+ cells for proliferation ability and the chemoresistance to oxaliplatin by CCK8 assay. Results The EpCAMhigh/CD44+ subset comprises almost 6.25 ± 0.09% in cell line HCT116, and the EpCAM−/CD44+ cells displayed a significantly lower proliferation ability and weaker oxaliplatin chemoresistance than the EpCAMhigh/CD44+ cells. Conclusions EpCAM is critical for tumor proliferation and oxaliplatin chemoresistance in EpCAMhigh/CD44+ colorectal CSCs.

5-hydroxytryptamine produced by enteric serotonergic neurons initiates colorectal cancer stem cell self-renewal and tumorigenesis

Colorectal cancer stem cells (CSCs) contribute to colorectal tumorigenesis and metastasis. Colorectal CSCs reside within specialized niches and harbor self-renewal and differentiation capacities. However, the niche regulations of CSCs remain unclear. Here, we show that intestinal nerve cells are required for CSC self-renewal and colorectal tumorigenesis. Enteric serotonergic neurons produce 5-hydroxytryptamine (5-HT) to function as a modulator of CSC self-renewal. 5-HT receptors HTR1B/1D/1F are highly expressed in colorectal CSCs and engage with 5-HT to initiate Wnt/β-catenin signaling. Mechanistically, colorectal cancer (CRC)-enriched microbiota metabolite isovalerate suppresses the enrichment of the NuRD complex onto Tph2 promoter to initiate Tph2 expression, leading to 5-HT production. 5-HT signaling is correlated with CRC severity. Blocking 5-HT signaling in mice not only inhibits the self-renewal of colorectal CSCs but also displays therapeutic efficacy against CRC tumors. Our findings reveal a cross talk between intestinal neurons and tumor cells that serves as an additional layer for CSC regulation.

Withdrawn: circLgr4 drives colorectal tumorigenesis and invasion through Lgr4-targeting peptide.

Colorectal cancer is one of the most common cancers all over the world. Colorectal cancer stem cells (CSCs) drive colorectal tumorigenesis and metastasis, while, the biology of colorectal CSCs remains unclear. Circular RNAs (circRNAs) emerge as critical regulators in various biological processes, but their roles in colorectal CSCs remain indistinct. Here we identified circLgr4 as a highly expressed circRNA in colorectal tumors and colorectal CSCs. circLgr4 knockdown inhibited colorectal CSC self-renewal, colorectal tumorigenesis and invasion, and circLgr4 overexpression played opposite roles. With peptide-coding capacity, circLgr4 drove the self-renewal of colorectal CSCs through peptide-dependent manner. circLgr4-derived peptide interacted with and activated Lgr4, which further promoted the activation of Wnt/β-catenin signaling. circLgr4-peptide-Lgr4 axis could be used to target colorectal CSCs and colorectal tumorigenesis. Our work revealed a functional circRNA in colorectal CSCs, adding a novel layer for circRNA function and colorectal CSC regulation. This article is protected by copyright. All rights reserved.

Curcumin suppresses LGR5(+) colorectal cancer stem cells by inducing autophagy and via repressing TFAP2A-mediated ECM pathway

Colorectal cancer stem cells (CSCs) have the potential for self-renewal, proliferation, and differentiation. And LGR5 is a stem cell marker gene of colorectal cancer. Curcumin can suppress oncogenicity of many cancer cells, yet the effect and mechanism of curcumin in LGR5(+) colorectal cancer stem cells (CSCs) have not been studied. In this study, we studied the effect of curcumin on LGR5(+) colorectal CSCs using the experiments of tumorsphere formation, cell viability and cell apoptosis. Then autophagy analysis, RNA-Seq, and real-time PCR were used to identify the mechanism responsible for the inhibition of LGR5(+) colorectal CSCs. Our results showed that curcumin inhibited tumorsphere formation, decreased cell viability in a dose-dependent manner, and also promoted apoptosis of LGR5(+) colorectal CSCs. Next, we found curcumin induced autophagy of LGR5(+) colorectal CSCs. When LGR5(+) colorectal CSCs were co-treated with curcumin and the autophagy inhibitor (hydroxychloroquine), curcumin-induced cell proliferation inhibition decreased. In addition, we also found that curcumin inhibited the extracellular matrix (ECM)-receptor interaction pathway via the downregulation of the following genes: GP1BB, COL9A3, COMP, AGRN, ITGB4, LAMA5, COL2A1, ITGB6, ITGA1, and TNC. Further, these genes were transcriptionally regulated by TFAP2A, and the high expression of TFAP2A was associated with poor prognosis in colorectal cancer. In conclusion, curcumin suppressed LGR5(+) colorectal CSCs, potentially by inducing autophagy and repressing the oncogenic TFAP2A-mediated ECM pathway.Graphic abstract

Dichloroacetate attenuates the stemness of colorectal cancer cells via trigerring ferroptosis through sequestering iron in lysosomes.

Colorectal cancer stem cell (CSC) has been regarded to be the root of colorectal cancer progression. However, there is still no effective therapeutic method targeting colorectal CSC in clinical application. Here, we investigated the effects of dichloroacetate (DCA) on colorectal cancer cell stemness. We showed that DCA could reduce colorectal cancer cell stemness in a dose-dependent manner, which is evident by the decreased expression of stemness markers, tumor cell sphere-formation and cell migration ability. In addition, it was found that DCA trigerred the ferroptosis of colorectal CSC, which is characterized as the upregulation of iron concentration, lipid peroxides, and glutathione level, and decreased cell viability. Mechanistic studies demonstrated that DCA could sequester iron in lysosome and thus trigger ferroptosis, which is necessary for DCA-mediated attenuation on colorectal cancer cell stemness. Taken together, this work suggests that DCA might be a colorectal CSC-killer.

Molecular Immunotherapy: Promising Approach to Treat Metastatic Colorectal Cancer by Targeting Resistant Cancer Cells or Cancer Stem Cells.

The immune system is able to recognize and eliminate tumor cells. Some tumors, including colorectal cancer (CRC), induce immune tolerance via different mechanisms of “immunoediting” and “immune evasion” and can thus escape immune surveillance. The impact of immunotherapy on cancer has been investigated for many years, but so far, the application was limited to few cancer types. Immuno-oncological therapeutic strategies against metastatic colorectal cancer (mCRC), the adaptive immune system activating approaches, offer a high potential for adaptation to the great heterogeneity of CRC. Moreover, novel treatment approaches are currently being tested that might specifically target the disease initiating and maintaining population of colorectal cancer stem cells (CSCs). In this review, we aim to summarize the current state of immune-oncology and tumor immunotherapy of patients with mCRC and discuss different therapeutic modalities that focus on the activation of tumor-specific T-cells and their perspectives such as tumor vaccination, checkpoint inhibition, and adoptive T-cell transfer or on the eradication of colorectal CSCs.

Targeting SLC7A11 specifically suppresses the progression of colorectal cancer stem cells via inducing ferroptosis

Recent studies have revealed the critical roles of ferroptosis in different physiological and pathological processes, however, its effects on the progression of colorectal cancer stem cells (CSCs) are still unclear. Here, we found that colorectal CSCs exhibited a remarkably lower level of reactive oxygen species (ROS), a higher level of cysteine, glutathione and SLC7A11 compared to colorectal cancer cells. Knockout of SLC7A11 increased the ROS level and reduced the levels of cysteine and glutathione, subsequently attenuating the viability of colorectal CSCs. Erastin, an inhibitor of SLC7A11, was found to hold a remarkably stronger cytotoxic effect on colorectal CSCs via in vitro and in vivo experiments. Finally, it was found that Erastin attenuated the chemoresistance of colorectal CSCs. This work indicates that colorectal CSCs are more sensitive to ferroptosis, which could be targeted to attenuate colorectal cancer progression and chemoresistance.

Bcl-3 promotes Wnt signaling by maintaining the acetylation of \u03b2-catenin at lysine 49 in colorectal cancer

Wnt/β-catenin signaling plays a critical role in colorectal cancer (CRC) tumorigenesis and the homeostasis of colorectal cancer stem cells (CSCs), but its molecular mechanism remains unclear. B-cell lymphoma 3 (Bcl-3), a member of the IκB family, is overexpressed in CRC and promotes tumorigenicity. Here, we report a novel function of Bcl-3 in maintaining colorectal CSC homeostasis by activating Wnt/β-catenin signaling. Silencing Bcl-3 suppresses the self-renewal capacity of colorectal CSCs and sensitizes CRC cells to chemotherapeutic drugs through a decrease in Wnt/β-catenin signaling. Moreover, our data show that Bcl-3 is a crucial component of Wnt/β-catenin signaling and is essential for β-catenin transcriptional activity in CRC cells. Interestingly, Wnt3a increases the level and nuclear translocation of Bcl-3, which binds directly to β-catenin and enhances the acetylation of β-catenin at lysine 49 (Ac-K49-β-catenin) and transcriptional activity. Bcl-3 depletion decreases the Ac-K49-β-catenin level by increasing the level of histone deacetylase 1 to remove acetyl groups from β-catenin, thus interrupting Wnt/β-catenin activity. In CRC clinical specimens, Bcl-3 expression negatively correlates with the overall survival of CRC patients. A significantly positive correlation was found between the expression of Bcl-3 and Ac-K49-β-catenin. Collectively, our data reveal that Bcl-3 plays a crucial role in CRC chemoresistance and colorectal CSC maintenance via its modulation of the Ac-K49-β-catenin, which serves as a promising therapeutic target for CRC.

Krüppel-like factor 4 regulates stemness and mesenchymal properties of colorectal cancer stem cells through the TGF-β1/Smad/snail pathway.

Krüppel‐like factor 4 (KLF4) was closely associated with epithelial‐mesenchymal transition and stemness in colorectal cancer stem cells (CSCs)‐enriched spheroid cells. Nonetheless, the underlying molecular mechanism is unclear. This study showed that KLF4 overexpression was accompanied with stemness and mesenchymal features in Lgr5+CD44+EpCAM+ colorectal CSCs. KLF4 knockdown suppressed stemness, mesenchymal features and activation of the TGF‐β1 pathway, whereas enforced KLF4 overexpression activated TGF‐β1, phosphorylation of Smad 2/3 and Snail expression, and restored stemness and mesenchymal phenotypes. Furthermore, TGF‐β1 pathway inhibition invalidated KLF4‐facilitated stemness and mesenchymal features without affecting KLF4 expression. The data from the current study are the first to demonstrate that KLF4 maintains stemness and mesenchymal properties through the TGF‐β1/Smad/Snail pathway in Lgr5+CD44+EpCAM+ colorectal CSCs.

PD-L1 promotes colorectal cancer stem cell expansion by activating HMGA1-dependent signaling pathways

PD-L1 is critical for tumor cell escape from immune surveillance by inhibiting T cell function via the PD-1 receptor. Accumulating evidence demonstrates that anti-PD-L1 monoclonal antibodies might potently enhance antitumor effects in various tumors, but the effect of PD-L1 on colorectal cancer stem cells (CSCs) remains unclear. We observed high PD-L1 expression in CD133+CD44+ colorectal CSCs and CSC-enriched tumorspheres. Altering PD-L1 expression promoted colorectal CSC self-renewal by increasing the expression of stemness genes, the CD133+CD44+ cell population sizes and the ability to form tumorspheres. Additionally, PD-L1 expression was markedly increased in chemoresistant colorectal cancer (CRC) cells in vitro and in vivo. More importantly, PD-L1 enhanced CRC cell tumorigenicity in nude mice; the inoculation of 1 × 104 cells resulted in high tumor formation efficiency. Mechanistically, PD-L1 directly interacted with HMGA1, and HMGA1 upregulation by PD-L1 activated HMGA1-dependent pathways, including the PI3K/Akt and MEK/ERK pathways, and promoted CSC expansion. HMGA1 downregulation rescued the PD-L1-induced phenotypes, highlighting the role of HMGA1 in PD-L1-mediated colorectal CSC self-renewal. Moreover, PD-L1 expression was correlated with the expression of CSC markers and HMGA1 in clinical CRC specimens. Thus, PD-L1 could crucially contribute to the maintenance of CSC self-renewal by activating HMGA1-dependent signaling pathways.

SIRT6 inhibits colorectal cancer stem cell proliferation by targeting CDC25A

Silent information regulator 6 (SIRT6) is broadly considered as a tumor suppressor due to its function in the suppression of oncogene expression. However, the role of SIRT6 in colorectal cancer stem cells (CSCs) remains uncharacterized. In the present study, it was demonstrated that SIRT6 expression was reduced in colorectal CSCs. Overexpression of SIRT6 in colorectal CSCs did not induce cell apoptosis. However, SIRT6 significantly inhibited cell proliferation, colony formation and induced G0/G1 phase arrest in colorectal CSCs. In addition, SIRT6 repressed the expression of cell division cycle 25A (CDC25A), an oncogenic phosphatase. Chromatin immunoprecipitation experiments indicated that SIRT6 directly bound to the CDC25A promoter and decreased the acetylation level of histone H3 lysine 9. Altogether, these data indicated that SIRT6 inhibits colorectal cancer stem cell proliferation by targeting CDC25A.

Effects and mechanisms of shRNA interfered with expression of leucine-rich repeat containing G-protein coupled receptor 5 on the malignant behaviors of colorectal cancer stem cells

Objective To investigate the effects and mechanisms of shRNA interfered with expression of leucine-rich repeat containing G-protein coupled receptor 5 (Lgr5) on the malignant behaviors of colorectal cancer stem cells (CSCs). Methods The experimental study was conducted. The CSCs expressing Lgr5+ were sorted by fluorescence activated cell sorting. Lgr5+ cells that were transfected with Lgr5-shRNA lentiviral vector and non-target shRNA lentiviral vector were respectively allocated into the experimental group and control group. The percentage of Lgr5+ cells was analyzed by flow cytometery. The relative expression of Lgr5 mRNA was detected by fluorescence quantitative real-time polymerase chain reaction (qRT-PCR). The capacity of self-renewal was detected by sphere forming assay. The tumorigenesis in vitro and in vivo were respectively measured by colony formation assay and xenografting experiment. The mRNA expressions of stem cells related genes (Oct4, Sox2, Nanog, KLF4), CSCs genes (CD133, CD44, ALDH) and Wnt/β-catenin pathway key genes (Axin2, Wnt5a, Wnt3a, Fzd3, c-myc, VEGF, Ascl2, claudin-1) were detected by qRT-PCR. Measurement data with normal distribution were represented as ±s. Comparison between groups was analyzed using the t test. Results (1) Transfection efficiency of shRNA lentiviral vector induced Lgr5 by flow cytometery was respectively 6.8%±1.0% in the experimental group and 92.7%±3.3% in the control group, with a statistically significant difference (t=43.148, P 0.05). Conclusion The malignant behaviors of colorectal CSCs are suppressed after shRNA lentivirus interfered with expression of Lrg5, and its mechanism is related to inhibiting activity of Wnt/β-catenin pathway. Key words: Colorectal neoplasms; Cancer stem cells; Self-renewal; Wnt/β-catenin pathway

Commensal-infected macrophages induce dedifferentiation and reprogramming of epithelial cells during colorectal carcinogenesis.

The colonic microbiome contributes to the initiation of colorectal cancer through poorly characterized mechanisms. We have shown that commensal-polarized macrophages induce gene mutation, chromosomal instability, and endogenous transformation through microbiome-induced bystander effects (MIBE). In this study we show that MIBE activates Wnt/β-catenin signaling and pluripotent transcription factors associated with dedifferentiation, reprogramming, and the development of colorectal cancer stem cells (CSCs). Exposure of murine primary colon epithelial cells (YAMC) to Enterococcus faecalis-infected macrophages increased Wnt3α expression while suppressing Wnt inhibitor factor 1 (Wif1). Wnt/β-catenin activation was confirmed by increased active β-catenin and Tcf4. in vivo, active β-catenin was evident in colon biopsies from E. faecalis-colonized Il10 knockout mice compared to sham-colonized mice. This effect was mediated, in part, by 4-hydroxy-2-nonenal and tumor necrosis factor α. MIBE also activated pluripotent transcription factors c-Myc, Klf4, Oct4, and Sox2 in YAMC cells and colons from E. faecalis-colonized Il10 knockout mice. These transcription factors are associated with cellular reprogramming, dedifferentiation, and induction of colorectal CSC progenitors. In support of this was an increase in the expression of Dclk1 and CD44, two colorectal CSC markers, in YAMC cells that were exposed to MIBE. Finally, compared to normal colon biopsies and hyperplastic polyps, DCLK1 expression increased in human tubular adenomas and invasive colorectal cancers. Blocking β-catenin/TCF4 signaling using FH535 and CTNNB1-specific small interfering RNA decreased DCLK1 expression in HCT116 human colon cancer cells. These findings provide mechanism for microbiome-induced colorectal cancer and identify new potential targets for colorectal cancer prevention.

Roles of microRNAs and RNA-Binding Proteins in the Regulation of Colorectal Cancer Stem Cells.

Colorectal cancer stem cells (CSCs) are responsible for the initiation, progression and metastasis of human colorectal cancers, and have been characterized by the expression of cell surface markers, such as CD44, CD133, CD166 and LGR5. MicroRNAs (miRNAs) are differentially expressed between CSCs and non-tumorigenic cancer cells, and play important roles in the maintenance and regulation of stem cell properties of CSCs. RNA binding proteins (RBPs) are emerging epigenetic regulators of various RNA processing events, such as splicing, localization, stabilization and translation, and can regulate various types of stem cells. In this review, we summarize current evidences on the roles of miRNA and RBPs in the regulation of colorectal CSCs. Understanding the epigenetic regulation of human colorectal CSCs will help to develop biomarkers for colorectal cancers and to identify targets for CSC-targeting therapies.

KDM3 epigenetically controls tumorigenic potentials of human colorectal cancer stem cells through Wnt/\u03b2-catenin signalling

Human colorectal cancer stem cells (CSCs) are tumour initiating cells that can self-renew and are highly tumorigenic and chemoresistant. While genetic mutations associated with human colorectal cancer development are well-known, little is known about how and whether epigenetic factors specifically contribute to the functional properties of human colorectal CSCs. Here we report that the KDM3 family of histone demethylases plays an important role in tumorigenic potential and survival of human colorectal CSCs by epigenetically activating Wnt target gene transcription. The depletion of KDM3 inhibits tumorigenic growth and chemoresistance of human colorectal CSCs. Mechanistically, KDM3 not only directly erases repressive H3K9me2 marks, but also helps to recruit histone methyltransferase MLL1 to promote H3K4 methylation, thereby promoting Wnt target gene transcription. Our results suggest that KDM3 is a critical epigenetic factor in Wnt signalling that orchestrates chromatin changes and transcription in human colorectal CSCs, identifying potential therapeutic targets for effective elimination of CSCs.

Evaluation of circulating cellular DCLK1 protein, as the most promising colorectal cancer stem cell marker, using immunoassay based methods.

DCLK1, as the most potential colorectal cancer stem cell (CSC) marker has been the core of many recent investigations. However, no study has been performed to evaluate the circulating cellular DCLK1 protein (CCDP) that might reflect the presences of colorectal CSC in circulation. We aimed to evaluate CCDP in the peripheral blood mononuclear cells (PBMCs) of colorectal cancer (CRC) patients applying immunoassay based methods including PLA, IPCR and ELISA in order to introduce the method of choice for clinical detection of CCDP. PBMCs were extracted from blood samples of 58 CRC patients along with 58 blood samples of tumor free controls. Total protein of PBMC was extracted and the CCDP level was evaluated. The results of three applied immunoassay tests were compared and the best approach for clinical application was introduced, accordingly. In addition, the correlation of CCD Plevel with clincopathologic findings of CRC patients was assessed. The results of three immuneassay methods confirmed each other. Based on our finding, ELISA could be the most judicious method for clinical evaluation of CCDP considering its simplicity for clinical implications. Our results also showed a significant higher amount of CCDP in peripheral blood of CRC patients compared to control group which was also correlated with patients' clinicopathologic finding such as stage, grade and neoadjuvant history. CCDP could be applied for monitoring purposes in CRC patients. However, its application needs to be more elucidated in future investigations implementing larger samples.

Abstract 3012: ONC201 induces cell death in triple negative, BRCA1-deficient and non-triple negative breast cancer cells

Abstract Breast cancer is a major cause of cancer related death. TRAIL has been of interest as a cancer therapeutic, but resistance has been observed in breast cancer and only a subset of triple negative breast cancers (TNBC) is sensitive to TRAIL. Small molecule ONC201 functions through the ATF4/CHOP pathway to upregulate TRAIL receptor DR5 (Kline et. al., Sci. Sig., in press, 2015), and through dual inhibition of Akt/ERK signaling to upregulate TRAIL (Allen et. al Sci. Trans. Med., 2013). We recently reported that ONC201 depletes colorectal cancer stem cell (CSC) markers and prevents formation of colonospheres. ONC201 also prevents colorectal CSCs from initiating growth of xenografted tumors (Prabhu et. al Can. Res., 2015). ONC201 has completed its first-in-human clinical trial in advanced solid tumors (Stein et al., 2015 AACR-NCI-EORTC meeting) and is being tested in multiple phase I/II clinical trials (NCT02250781, NCT02324621, NCT02420795, NCT02392572, NCT02609230, NCT02525692, NCT02038699). We investigated ONC201 efficacy in TNBC (TRAIL-sensitive), BRCA1-deficient, and non-TNBC (TRAIL-resistant) cells. We demonstrate IC50 values for ONC201 in the low μM range for TNBC or BRCA1-deficient (n = 6) and non-TNBC cells (n = 5), doses that are achievable based on human PK. Importantly, ONC201 induces apoptotic death in both TNBC and non-TNBC cells. ONC201 depletes Aldefluor+ putative breast CSCs in vitro whereas paclitaxel does not. ONC201 inhibits growth of TNBC, BRCA1-deficient and non-TNBC CSC-like mammospheres while paclitaxel chemotherapy does not. We show ONC201 is well tolerated and efficacious in vivo against the MDA-MB-231 TNBC xenograft model. We investigated effects of ONC201 on mechanisms of TRAIL resistance in breast cancer cells, including expression of inhibitor of apoptosis (IAP) family proteins. We found that ONC201 mediates a decrease in XIAP, c-IAP1, and c-IAP2 expression in the breast cancer cells used. We also examined levels of DR5 using flow cytometry, as a mechanism of TRAIL resistance in breast cancer cells involves receptor endocytosis from the cell surface (Zhang et al., Mol Cancer Res., 2008). Interestingly, we observed greater increases in cell surface DR5 in TNBC cells than non-TNBC cells after ONC201 treatment. Our findings suggest that ONC201 exerts cytotoxic effects against a broad range of breast cancer cells, including TNBC, BRCA1-deficient, and non-TNBC subtypes. ONC201 is efficacious against breast cancer cells regardless of the TRAIL sensitivity of the cells, and this efficacy may be mediated through mechanisms involving downregulation of IAP proteins and upregulation of cell surface death receptors. Furthermore, ONC201 may target paclitaxel-resistant breast CSCs. Our findings suggest unique targeting of multiple non-TNBC, BRCA1-deficient and TNBC cells and develop a preclinical rationale for the use of ONC201 as a treatment for breast cancer. Citation Format: Marie D. Baumeister, Jessica Wagner, Varun V. Prabhu, Christina LB Kline, Bora Lim, Josh E. Allen, David T. Dicker, Wafik S. El-Deiry. ONC201 induces cell death in triple negative, BRCA1-deficient and non-triple negative breast cancer cells. [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 3012.

Lgr5 Methylation in Cancer Stem Cell Differentiation and Prognosis-Prediction in Colorectal Cancer.

ObjectiveLeucine-rich-repeat-containing G-protein-coupled receptor 5 (lgr5) is a candidate marker for colorectal cancer stem cells (CSC). In the current study, we investigated the methylation status within thelgr5 promoter and evaluated its relationship with CSC differentiation, prognosis for colorectal cancer, and its clinicopathological features.MethodsThe methylation status within Lgr5 promoter was detected with a methylation-specific PCR in six colorectal cancer cell lines as well as 169 primary colorectal tumor tissues. Differentiation of CSC was examined with immunofluorescence and immunocytochemistry. Down-regulation of lgr5 was achieved with gene-specific siRNA. The associations between lgr5 methylation and the clinicopathological features as well as survival of patients were analyzed with statistical methods.ResultsThe lgr5 promoter was methylated to different degrees for the six colorectal cell lines examined, with complete methylation observed in HCT116 cells in which the lgr5 expression was partially recovered following DAC treatment. The stem-cell sphere formation from HCT116 cells was accompanied by increasing methylation within the lgr5 promoter and decreasing expression of lgr5. Knocking down lgr5 by siRNA also led to stem-cell spheres formation. Among primary colorectal tumors, 40% (67/169) were positive for lgr5 methylation, while none of the normal colon tissues were positive for lgr5 methylation. Furthermore, lgr5 methylation significantly associated with higher tumor grade, and negative distant metastasis (p < 0.05), as well as better prognosis (p = 0.001) in patients with colorectal cancer.ConclusionsOur data suggests that lgr5 methylation, through the regulation of lgr5 expression and colorectal CSC differentiation, may constitute a novel prognostic marker for colorectal cancer patients.