Colorectal Cancer Stem Cell Marker Research Articles

QPH-FR: A Novel Quinoa Peptide Enhances Chemosensitivity by Targeting Leucine-Rich Repeat-Containing G Protein-Coupled Receptor 5 in Colorectal Cancer.

Chemoresistance is one of the difficulties in the treatment of colorectal cancer (CRC), and the enhanced stemness of tumor cells is the underlying contributing factor. Leucine-rich repeat-containing G-protein-coupled receptor 5 (LGR5) is a classical marker of CRC stem cells and can be an important potential target for CRC chemotherapy. Quinoa, a protein-rich plant, offers potential as a source of high-quality active peptides. Novelly, the study obtained quinoa protein hydrolysate (QPH) from whole quinoa grains by simulated digestion. In vivo experiments revealed that the tumor volume in the 5-FU+QPH group decreased from 145.90 ± 13.35 to 94.49 ± 13.05 mm3 in the 5-FU group, suggesting that QPH enhances the chemosensitivity of CRC. Further, the most effective peptide QPH-FR from 631 peptides in QPH was screened by activity prediction, molecular docking, and experimental validation. Mechanistically, QPH-FR competitively suppressed the formation of the LGR5/RSPO1 complex by binding to LGR5, causing RNF43/ZNRF3 to ubiquitinate the FZD receptor, thereby suppressing the Wnt/β-catenin signaling pathway and exerting stemness inhibition. In summary, the study proposes that a novel peptide QPH-FR from quinoa elucidates the mechanism by which QPH-FR targets LGR5 to enhance chemosensitivity, providing theoretical support for the development of chemotherapeutic adjuvant drugs based on plant peptides.

Colorectal Cancer Stem Cell Biomarkers: Biological Traits and Prognostic Insights.

Due to self-renewal, differentiation, and limitless proliferation properties, Cancer Stem Cells (CSCs) increase the probability of tumor development. These cells are identified by using CSC markers, which are highly expressed proteins on the cell surface of CSCs. Recently, the therapeutic application of CSCs as novel biomarkers improved both the prognosis and diagnosis outcome of colorectal Cancer. In the present review, we focused on a specific panel of colorectal CSC markers, including LGR5, ALDH, CD166, CD133, and CD44, which offers a targeted and comprehensive analysis of their functions. The selection criteria for these markersCancer were based on their established significance in Colorectal Cancer (CRC) pathogenesis and clinical outcomes, providing novel insights into the CSC biology of CRC. Through this approach, we aim to elevate understanding and stimulate further research for developing effective diagnostic and therapeutic strategies in CRC.

Abstract 2784: Fatty acid synthase enhances stem-like properties of normal intestinal and colorectal cancer cells via an increase in notum expression and secretion

Abstract Introduction: Overexpression of lipogenic enzymes has been associated with a poor clinical outcome in colorectal cancer (CRC). Fatty acid synthase (FASN) synthesizes palmitate which can be utilized for post-translational modifications of various proteins. Notum, a palmitoleoyl-protein, prevents the downstream activation of Wnt signaling by de-palmitoylating Wnt ligands. Notum is also a CRC stem cell marker, and its expression correlates with poor prognosis in CRC. We found that upregulation of FASN is associated with an increase in Notum expression in mouse adenoma tissues. Therefore, the purpose of this study is to elucidate the mechanisms and contribution of FASN/Notum axis in CRC. Methods: Adenoma and normal intestinal organoids, established from ApcMin/VillinCre-ERT2 and Apc/VillinCre-ERT2 mouse models with hetero- and homozygous deletion of FASN. 4-hydroxytamoxifen (4-OHT) and TVB-3664 (FASN inhibitor) treatments were used to assess the effect of FASN deletion/inhibition on organoid growth and viability. LIVE/DEAD™ Viability/Cytotoxicity and Cell Titer-Glo® 3D Cell Viability Assays were used for quantitative analysis of growth. Human NOTUM/Protein notum homolog ELISA Kit was used to analyze Notum secretion. CRC cells, NTC and FASN shRNA, and control and FASN overexpression, were used for Notum analysis. Results: The TCGA data analysis shows that Notum is significantly upregulated in CRC and its expression correlates with expression of FASN in tumor tissue. Utilizing qRT-PCR and Western blot analyses, we show that downregulation of FASN leads to a decrease in expressions of active β-catenin, Notum, and other stem cell markers in transgenic mouse models. Moreover, 4-OHT-mediated inhibition of FASN results in decrease viability and size in ApcMin/FASN/VillinCre-ERT2 and Apc/FASN/VillinCre-ERT2 organoids. Consistently, shRNA-mediated deletion of FASN decreases expression of Notum in CRC cells. In contrast, overexpression of FASN increases the expression levels of active and total β-catenin, Notum, and other stem cell markers in SW480 cells. We show that normal epithelial cells exposed to CRC cell medium exhibit stem-like phenotype which is reduced when cells are exposed to medium collected from Notum shRNA knockdown cells. Moreover, the addition of recombinant Notum restores this phenotype. Conclusion: In summary, downregulation of FASN leads to a decrease in expression of Notum and is associated with morphological changes and significant decrease in viability in organoid models. Conversely, FASN overexpression upregulates Notum expression suggesting a potential cross talk between de novo lipid synthesis and Notum. Delineating the role of FASN regulation of stem-like properties via upregulation of β-catenin signaling and expression of Notum and other stem cell markers will provide the rationale for targeting the FASN/Notum axis in CRC. Citation Format: Courtney Olivia Kelson, Josiane Weber Tessmann, Daheng He, Chi Wang, Yekaterina Zaytseva. Fatty acid synthase enhances stem-like properties of normal intestinal and colorectal cancer cells via an increase in notum expression and secretion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2784.

ZNF217-activated Notch signaling mediates sulforaphane-suppressed stem cell properties in colorectal cancer

Cancer stem cells (CSCs) are known to contribute to the progression of colorectal cancer (CRC). However, understanding of the molecular mechanisms and key factors involved in CRC is still insufficient to identify therapeutic targets against colorectal CSCs. In an effort to identify such mechanisms, we conducted bioinformatics analyses to evaluate the expression patterns in tumor and normal colorectal tissues, leading us to focus on the role of the ZNF217/Notch1 axis in mediating stem cell properties in CRC. Our findings revealed that ZNF217 overexpression activated self-renewal ability, expression of colorectal CSC markers, and Notch signaling in CRC. Dual-luciferase reporter assay suggested a role for ZNF217 in targeting Notch1 to activate Notch signaling. We observed that the promotional effects of Notch signaling, as well as CSC markers, under ZNF217 overexpression were attenuated after Notch1 knockdown. In addition to in vitro data, our in vivo results confirmed the inhibitory effect of sulforaphane on the tumorigenicity of CSCs, depicted the suppressive role of sulforaphane on colorectal CSCs mediated by the ZNF217/Notch1 axis, thereby providing new targetable vulnerabilities and therapeutic strategies for CRC.

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.

Transcriptional Regulation of the Colorectal Cancer Stem Cell Markers, Nanog and Oct4, Induced by a Thermodynamic-Based Therapy Approach.

Cancer stem cells (CSC), as responsible issues to cancer development and progression, play a crucial role in tumorigenesis, recurrence, metastasis, and chemoresistance. Both hyperthermia and photodynamic therapy (PDT) may be effective for cancer treatment, particularly when combined with other therapeutic approaches. This study aimed to evaluate the effect of hyperthermia combined with PDT on colorectal CSC and the gene expression of the CSC markers, presenting a more effective approach for cancer therapy. The study was conducted in the Pasteur institute of Iran, Tehran, Iran in 2018. We evaluated the anticancer role of hyperthermia, Gold nanoparticles coated with curcumin (Cur-GNPs) in PDT and combination of the two approaches on cell viability and the expression of CSC markers, Nanog and Oct4 in colorectal cancer cell line HT-29. The cytotoxicity effect of Cur-GNPs against the cells was assessed in vitro. The cell viability was assessed using MTT assay, and the expression analysis of the CSC genes was evaluated using a q-real-time PCR. Cell viability was decreased by PDT (P=0.015) and the combination therapy (P=0.006) but not by hyperthermia alone (P=0.4), compared to control. Also, the expression of CSC markers, Nanog and Oct4 was shown to significantly down-regulate in all hyperthermia, PDT and combination groups. Hyperthermia combined with PDT was indicated to be more efficient in eliminating tumors than hyperthermia or PDT alone.

Abstract 2581: Fatty acid synthase regulates expression of Notum in colorectal cancer

Abstract Background: Upregulation of lipid synthesis has been associated with poor clinical outcomes in colorectal cancer (CRC). Fatty acid synthase (FASN) synthesizes 16-carbon fatty acid palmitate which can be utilized for post-translational modifications of various proteins. Notum, a palmitoleoyl-protein carboxylesterase, is involved in the negative regulation of the Wnt signaling via its role in de-palmitoylation of Wnt ligands and has been identified as a marker for poor prognosis in CRC. However, the crosstalk between FASN and Notum has not been reported. Our preliminary data suggest that FASN regulates β-catenin signaling and Notum expression. Therefore, the purpose of this study is to elucidate (I) the mechanisms of how FASN regulates expression of Notum and (II) the contribution of the FASN/Notum axis to CRC. Methods: Tumor and normal intestinal organoids were established from transgenic mice models, ApcMin and Apc/VillinCre-ERT2, with inducible hetero- and homozygous deletion of FASN. The effect of genetic deletion and pharmacological inhibition of FASN on organoid growth and viability was assessed by 4-hydroxytamoxifen (4-OHT) and TVB-3664 (a FASN inhibitor) treatments, respectively. LIVE/DEAD™ Viability/Cytotoxicity Cell Viability Kit and Cell Titer-Glo® 3D Cell Viability Assays were used for quantitative analysis of growth and viability. HCT116, NTC and FASN shRNA, and SW480, control and FASN overexpression, cells were used for analysis. Results: ERT2-mediated deletion of Apc leads to upregulation of FASN and Notum expression in mouse intestinal tissues and organoids. RNA-seq analysis of adenomas from Apc/VillinCre mice showed that hetero- and homozygous germline deletion of FASN is associated with a significant decrease in the number of adenomas, expression of Notum and CRC stem cell markers. Using qRT-PCR and Western blot, we confirmed that FASN downregulation is associated with a decrease in active β-catenin, Notum, and stem cell markers. Consistently with Apc/VillinCre model, downregulation of FASN results in a decrease in bud formation in Apc/VillinCre-ERT2 organoids, and viability and size in ApcMin organoids. Furthermore, overexpression of FASN increases the levels of active and total β-catenin, Notum, and stem cell markers expression in SW480 cells. In contrast, shRNA-mediated deletion of FASN decreases expression of Notum in HCT116 cells. Conclusion: Downregulation of FASN leads to a decrease in expression of Notum and is associated with morphological changes and a significant decrease in viability in organoid models. Conversely, FASN overexpression upregulates Notum expression suggesting a potential cross-talk between de novo lipid synthesis and Notum. Delineating the role of FASN regulation of stemness via altered β-catenin signaling and expression of Notum and other stem cell markers will provide the rationale for targeting the FASN/Notum axis as a preventative or early-stage therapeutic approach in CRC. Citation Format: Courtney Olivia Kelson, James Drury, Daheng He, Chi Wang, Yekaterina Zaytseva. Fatty acid synthase regulates expression of Notum in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2581.

Abstract B021: Claudin-1 expression promotes chemoresistance and cancer stem cell characteristics in colorectal cancer

Abstract Background: Claudins are integral components of tight junction complexes. Beyond just passive cell adhesion molecules, they influence structural dynamics, molecular trafficking, and important cell signaling pathways. Claudins are often dysregulated in cancer, leading to changes in adhesion and metastatic potential. Claudin-1 (Cldn1) is expressed at low levels in normal colon but is highly expressed in colorectal cancer (CRC) and promotes epithelial-mesenchymal transition and metastasis. Cancer stem cells (CSCs) are a small subset of cancer cells that display increased capacity for self-renewal and can undergo symmetric and asymmetric division to reform tumors after treatment. CSCs are resistant to cytotoxic drugs and are thought to be responsible for treatment failure, metastasis, and relapse. Significance of problem: CRC is the second leading cause of cancer-related deaths in the United States with an estimated 151,030 new cases in 2022. Although CRC has an overall relative 5-year survival rate of 64%, prognosis varies dramatically with the stage at diagnosis. Localized CRC can be treated surgically and has a 5-year survival rate of 91%, while the survival rate of metastatic CRC falls to 15%. The primary problem in treating metastatic CRC is a combination of intrinsic and acquired chemoresistance. Therefore, understanding the mechanisms of chemoresistance is vital to developing effective therapies. Hypothesis: Claudin-1 expression in colorectal cancer increases resistance to chemotherapeutic drugs and promotes cancer stem cell characteristics. Experimental design: Bioinformatic analysis was performed on a database of responders and non-responders to standard-of-care CRC chemotherapy. Cldn1 expression was manipulated in CRC cell lines, and assays were performed to evaluate chemoresistance and CSC characteristics. Results: Bioinformatic analysis of a patient dataset of responders and non-responders to chemotherapy revealed that Cldn1 expression was positively correlated with resistance to FOLFOX treatment. It was also significantly correlated with colorectal CSC marker expression including CD44, CD133, and Lgr5. Overexpression of Cldn1 in SW480 cells led to a significant increase in resistance to 5-FU, side population and ALDH-high percentage, colorectal CSC markers, and tumorigenic potential, while knockout of Cldn1 in SW620 and DLD-1 cells led to a decreased response in these assays. Follow-up experiments identified the PDZ-binding domain of Cldn1 as essential for the chemoresistant phenotype. Specifically, both CD44 and its downstream target phospho-AKT are increased with the expression of full-length but not mutated Cldn1. Conclusion: Cldn1 expression induces chemoresistance and cancer stem cell properties in colorectal cancer. Because of this, Cldn1 may serve as a biomarker in CRC for prescribing more aggressive adjuvant therapy or as a molecule for targeted treatment aimed at the resistant tumor cells responsible for treatment failure and recurrence. Citation Format: Mark Primeaux, Saiprasad Gowrikumar, Dhundy Bastola, Punita Dhawan, Amar Singh. Claudin-1 expression promotes chemoresistance and cancer stem cell characteristics in colorectal cancer [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B021.

The cAMP/PKA/CREB and TGFβ/SMAD4 Pathways Regulate Stemness and Metastatic Potential in Colorectal Cancer Cells.

This study identifies signaling pathways essential for maintaining the stemness and metastatic potential of colorectal cancer cells and proposes CREB as a therapeutic target in metastatic colorectal cancer.

Niclosamide induces miR-148a to inhibit PXR and sensitize colon cancer stem cells to chemotherapy.

SummaryTumor recurrence is often attributed to cancer stem cells (CSCs). We previously demonstrated that down-regulation of Pregnane X Receptor (PXR) decreases the chemoresistance of CSCs and prevents colorectal cancer recurrence. Currently, no PXR inhibitor is usable in clinic. Here, we identify miR-148a as a targetable element upstream of PXR signaling in CSCs, which when over-expressed decreases PXR expression and impairs tumor relapse after chemotherapy in mouse tumor xenografts. We then develop a fluorescent reporter screen for miR-148a activators and identify the anti-helminthic drug niclosamide as an inducer of miR-148a expression. Consequently, niclosamide decreased PXR expression and CSC numbers in colorectal cancer patient-derived cell lines and synergized with chemotherapeutic agents to prevent CSC chemoresistance and tumor recurrence in vivo. Our study suggests that endogenous miRNA inducers is a viable strategy to down-regulate PXR and illuminates niclosamide as a neoadjuvant repurposing strategy to prevent tumor relapse in colon cancer.

Relationship between CD 163 Tumor-Associated Macrophages and Colorectal-Cancer Stem Cell Markers

BACKGROUND: Colorectal-cancer stem cells (CR-CSCs) represent a specific subpopulation of colorectal cancer (CRC) cells, which are characterized by the expression of CD133 and CD166. Tumor-associated macrophages (TAMs), found near CSCs may represent polarized macrophages, which are characterized by CD163 expression. In most tumors, TAMs may promote aggressive tumor development, leading to poor prognoses. AIM: The aim of this study was to determine whether any association exists between CD163 expression in TAMs and CD133 and CD166 expression in CR-CSCs. METHODS: This study used a cross-sectional design that was conducted at the General Hospital and affiliates in Medan, from September 2018 to July 2019. CRC tissues were collected from colonoscopy biopsies and surgical resections performed on CRC patients, who fulfilled all necessary inclusion and exclusion criteria and provided informed consent. Subjects were divided into high- and low-CD163-level groups. We analyzed the expression levels of CD163, CD133, and CD166 using immunohistochemical (IHC) assays. RESULTS: A total of 118 CRC patients were enrolled in this study, of whom 58.5% were male. No significant differences in hemoglobin, leukocyte, or platelet levels were observed between high- and low-level CD163 expression. We didn’t find any significant association of CD163 TAM with CRC histological grade and TNM stagings. Significant associations were found between the CD 163 expression level and the CD133 expression level (p < 0.001) and between the CD 163 expression level and the CD166 expression level (p< 0.001). Increased TAM levels of CD163 was associated with 2.770-fold and 2.616-fold increased risks of elevated CD133 and CD166 levels, respectively. CONCLUSION: An association was found between the expression levels of CD163 in TAMs and the expression levels of CD133 and CD166 in CR-CSCs.

PCGF1 promotes epigenetic activation of stemness markers and colorectal cancer stem cell enrichment

Colorectal cancer (CRC) stem cells are resistant to cancer therapy and are therefore responsible for tumour progression after conventional therapy fails. However, the molecular mechanisms underlying the maintenance of stemness are poorly understood. In this study, we identified PCGF1 as a crucial epigenetic regulator that sustains the stem cell-like phenotype of CRC. PCGF1 expression was increased in CRC and was significantly correlated with cancer progression and poor prognosis in CRC patients. PCGF1 knockdown inhibited CRC stem cell proliferation and CRC stem cell enrichment. Importantly, PCGF1 silencing impaired tumour growth in vivo. Mechanistically, PCGF1 bound to the promoters of CRC stem cell markers and activated their transcription by increasing the H3K4 histone trimethylation (H3K4me3) marks and decreasing the H3K27 histone trimethylation (H3K27me3) marks on their promoters by increasing expression of the H3K4me3 methyltransferase KMT2A and the H3K27me3 demethylase KDM6A. Our findings suggest that PCGF1 is a potential therapeutic target for CRC treatment.

Correlation between Lgr5 Expression and 5-FU based Chemotherapy Response in Stage IV Colorectal Cancer Patients

Background. Colorectal cancer is the third commonest malignancy and the second leading cause of cancer death in the world. 5-Fluorouracyl-based chemotherapy is the primary treatment modality for colorectal cancer. Cancer stem cells are known to be responsible for chemotherapy resistance. Lgr5 is a colorectal cancer stem cell marker that is the target gene for Wnt signaling. Lgr5 potentiates the Wnt signaling pathway through inhibition of a regulator that inhibits Wnt signaling. Lgr5 overexpression is associated with a worse prognosis and chemotherapy resistance. This study was aimed to investigate the correlation between Lgr5 expression and 5-FU-based chemotherapy response in stage IV colorectal cancer patients at Dr. Mohammad Hoesin Hospital Palembang.Methods. This study used a correlative analysis study with a retrospective design using secondary data from medical records and paraffin blocks of stage IV colorectal cancer patients who received 5-FU-based chemotherapy from September 2018 to September 2020. The number of samples was 30 subjects consisting of 22 cases of negative responses and eight positive responses. All samples were stained with Lgr5 immunohistochemistry. Data analysis used the contingency coefficient correlation test.Results. Of the 30 research subjects, 20 subjects (66.7%) had high Lgr5 expression and ten subjects (33.3%) with low Lgr5 expression. Correlation analysis using the contingency coefficient test showed a weak correlation between Lgr5 expression and 5-FU based chemotherapy response with a positive direction, which means the higher the Lgr5 expression, the less response to chemotherapy.Conclusion. There is a weak correlation between Lgr5 expression and 5-FU based chemotherapy response in stage IV colorectal cancer patients at dr.Mohammad Hoesin Hospital Palembang.

Correlation between Lgr5 Expression and 5-FU based Chemotherapy Response in Stage IV Colorectal Cancer Patients

Background. Colorectal cancer is the third commonest malignancy and the second leading cause of cancer death in the world. 5-Fluorouracyl-based chemotherapy is the primary treatment modality for colorectal cancer. Cancer stem cells are known to be responsible for chemotherapy resistance. Lgr5 is a colorectal cancer stem cell marker that is the target gene for Wnt signaling. Lgr5 potentiates the Wnt signaling pathway through inhibition of a regulator that inhibits Wnt signaling. Lgr5 overexpression is associated with a worse prognosis and chemotherapy resistance. This study was aimed to investigate the correlation between Lgr5 expression and 5-FU-based chemotherapy response in stage IV colorectal cancer patients at Dr. Mohammad Hoesin Hospital Palembang.Methods. This study used a correlative analysis study with a retrospective design using secondary data from medical records and paraffin blocks of stage IV colorectal cancer patients who received 5-FU-based chemotherapy from September 2018 to September 2020. The number of samples was 30 subjects consisting of 22 cases of negative responses and eight positive responses. All samples were stained with Lgr5 immunohistochemistry. Data analysis used the contingency coefficient correlation test.Results. Of the 30 research subjects, 20 subjects (66.7%) had high Lgr5 expression and ten subjects (33.3%) with low Lgr5 expression. Correlation analysis using the contingency coefficient test showed a weak correlation between Lgr5 expression and 5-FU based chemotherapy response with a positive direction, which means the higher the Lgr5 expression, the less response to chemotherapy.Conclusion. There is a weak correlation between Lgr5 expression and 5-FU based chemotherapy response in stage IV colorectal cancer patients at dr.Mohammad Hoesin Hospital Palembang.

Downregulation of Ascl2 promotes cell apoptosis by enhancing autophagy in colorectal cancer cells.

Colorectal cancer (CRC) is the third most common cancer, according to recently published literature. While the incidence and the mortality of CRC has decreased due to effective cancer screening measures, there has been an increase in the number of young patients diagnosed with colon cancer due to unclear reasons. As a target molecule of the Wnt signaling pathway, Ascl2 is an important marker of CRC stem cells and plays an important role in maintaining the nature of colon cancer stem/precursor cells. However, the role of Ascl2 in autophagy in CRC cells is rarely elucidated. In this study, we found that Ascl2 was increased in CRC compared with adjacent tissue. Downregulation of Ascl2 in CRC cells could suppress proliferation and invasion, and induce apoptosis, of CRC cells. Moreover, we found that autophagy-relative protein LC3 increased after Ascl2 knockdown. Furthermore, we treated CRC cells with autophagy inhibitors 3-MA (3-Methyladenine) and CQ (Chloroquine). The results showed that autophagy inhibitors could prevent apoptosis, which was induced by Ascl2 knockdown. Finally, we confirmed that the downregulation of Ascl2 in CRC cells could alleviate the pathological process in vivo by xenograft experiment. Our findings indicated that si-Ascl2 (small/short interfering) exerted a tumor suppression function in CRC by inducing autophagic cell death, and suggest that Ascl2 targeted therapy represents a novel strategy for CRC treatment.

The Role of KDM2B and EZH2 in Regulating the Stemness in Colorectal Cancer Through the PI3K/AKT Pathway.

Background: The incidence of colorectal cancer (CRC) has been increasing worldwide in recent years. Targeting cancer stem cells (CSCs) in CRC remains a difficult challenge. KDM2B and EZH2 play important role in the maintenance of CSCs' self-renewal capacity and tumorigenic ability; however, the biological functions of those genes in CRC remain unclear. In this study, we aimed to define the contribution of the expression of KDM2B in the features of CRC and establish the relationship between KDM2B and EZH2 in colorectal CSCs.Methods: The expression of KDM2B and EZH2 in the specimens of CRC and CRC cell lines were analyzed by immunohistochemistry, Western blot, and immunofluorescence. The underlying mechanisms of altered expressions of KDM2B and EZH2 and their impact on the biologic features of CRC and stemness in CRC were investigated.Results: The KDM2B gene was highly expressed in CRC tissues, and its overexpression positively correlated with tumor stages and tumor/node/metastasis (TNM) classification. The downregulation of KDM2B retarded cell proliferation, induced DNA damage, reduced spheroid formation, and decreased CRC stem cell markers: CD44, CD133, and ALDH-1. Moreover, the downregulation of KDM2B decreased the expression of EZH2 and both regulated cell migration, invasion, and stemness in the CRC cell line. Additionally, the interaction between KDM2B and EZH2 significantly increased the components of the PI3K/AKT pathway including AKT and PI3K. The high expression of KDM2B positively correlated with EZH2 in CRC tissues.Conclusion: This study shows that the downregulation of KDM2B and EZH2 can regulate CRC cell stemness, and their interaction may serve as a novel prognostic marker and therapeutic target for patients with CRC.

The prognostic value of combining CD133 and mismatch repair proteins in patients with colorectal cancer.

The prognostic value of cancer stem cells (CSCs) is a hot topic in colorectal carcinoma (CRC) research. CD133 has been identified as an important colorectal CSC marker, but its prognostic significance remains controversial. Recently, studies have reported a possible functional link between CSCs and DNA mismatch repair (MMR) system. However, the relationship between CRC stemness and MMR proteins remains little explored, and whether the predictive role of CD133 is affected by MMR proteins is still unknown. The aim of our study is to investigate the influence of MMR proteins on the predictive significance of CD133 in terms of CRC patient survival and to further analyze the correlation between MMR proteins and cancer stemness. In our study, we didn't observe the prognostic value of CD133 in CRC patients. However, we demonstrated that in patients with low expression of MSH6, MSH2, PMS2 and MLH1, especially MSH6, CD133 was an effective prognostic biomarker. Moreover, correlation analysis revealed a positive correlation between MSH6 and CD133 expression. In vitro studies supported our clinical data and showed that the expression of cancer-associated stemness markers CD133, BMI-1, OCT-4 and SOX-2 was significantly decreased in siRNA-MSH6/MLH1 CRC cells. Thus, our results demonstrated that MMR proteins might play an important role in modulating the stemness of CRC cells. MMR proteins might be a crucial determinant that can help to accurately identify tumour subclones that may benefit from using the CSC marker CD133 as a prognostic marker.

DCLK1, a promising colorectal cancer stem cell marker, regulates tumor progression and invasion through miR-137 and miR-15a dependent manner.

Cancer stem cells (CSCs) are thought to be a major player in tumor initiation, progression, and metastasis. Targeting CSCs for elimination presents a promising therapeutic strategy; however, this approach will require a stronger understanding of CSC biology and identification of CSC-specific markers. The present study was conducted to examine the correlation between DCLK1 and miR-137 and miR-15a levels in colorectal cancer. A total of 222 samples, including 181 colorectal cancer specimens, 24 adenomatosis, and 17 non-adenomatosis colonic polyps, were stained for DCLK1 expression using immunohistochemistry. Also, expression of miR-137 and miR-15a was assessed in colorectal cancer with high and low DCLK1 expression levels. Most colorectal cancer specimens (76%) showed strong expression of DCLK1, whereas only 21% of adenomatous and none of non-adenomatous colonic polyps showed strong DCLK1 expression. A significant difference in DCLK1 expression was found between colorectal cancer, adenomatous, and non-adenomatous colonic polyps (P < 0.001). Higher expression of DCLK1 was more frequently detected in colorectal cases with larger tumor size (P = 0.03), poor differentiation (P = 0.03), and lymph node involvement (P = 0.04). Comparison of miR-137 and miR-15a in colorectal cancer cases revealed a significant inverse correlation with DCLK1 expression (P = 0.03 and P = 0.04, respectively). DCLK1 may act as a candidate marker for colorectal cancer stem cells. The critical role of DCLK1 in colorectal cancer suggests that it may represent an early diagnostic marker and therapeutic target; however, further investigation is warranted.

Abstract C036: Cohesins and colorectal cancer (CRC): Modulation of CRC stem cells and chemoresistance by STAG1 with race-specific implications

Abstract Despite advances in treatment and early detective measures for colorectal cancer (CRC), African-Americans (AAs) suffer disproportionately from CRC (~25% higher incidence and ~50% higher mortality) than Caucasians. Furthermore, AAs tend to receive earlier diagnoses in CRC and AA CRCs with high-grade differentiation were 3xs more likely to die within 5 yrs post-surgery as compared to whites (Alexander et al., Cancer 2005). The underlying mechanisms behind this more aggressive disease in AAs are poorly understood. Tomassati and Vogelstein postulate stem cell division correlates with risk of cancer (Science 2015). Addressing disease progression/aggressiveness, CRC stem cell markers are linked to chemoresistance; stem cell markers LGR5 and ALDH1a are established CRC stem cell markers. We have shown that the cohesin family member STAG1, a chromatin remodeler, is lost in the colonic epithelium of patients with premalignant lesions and AAs had a more profound loss (Cancer Prev Research 2016) through AA-specific STAG1 SNPs (Neoplasia 2018) and hypothesize that STAG1 loss is associated with poorer prognosis. Therefore, we wanted to investigate if STAG1 loss may lead to CRC stem cell induction as a potential mechanism of the racial disparities in CRC. RNA from rectal biopsies from 100 patients undergoing screening colonoscopies was processed for real-time PCR for CRC stem cell markers LGR5 and ALDH1a, as well as STAG1. To determine chemoresistance, STAG1 siRNA was transfected in CRC cell line HT29 and a CRISPR transfection of STAG1 SNP rs34149860 (found only in AAs) was transfected in CRC cell line RKO (possessing wildtype to SNP). Cell were treated with 5-FU and oxaliplatin and subjected to Annexin V Assay. Real-time PCR and analysis of STAG1 and CRC stem cell markers were performed for causation. AAs harboring neoplasias displayed a more robust loss of STAG1 mRNA (~50%, p&amp;lt;0.007) vs Caucasians with neoplasias (~25%, p=0.1). In regard to CRC stem cell markers, AAs with adenomas showed a stronger increase of LGR5 (67%, p=0.14), and ALDH1a (73%, p&amp;lt;0.007) vs Caucasians with neoplasias (LGR5 increased 59%, p=0.2; ALDH1a increased ~47%, p&amp;lt;0.05). HT29 cells transfected with STAG1 siRNA showed 30% less apoptotic response to 5-FU vs scramble vector and a marked 74% less response with oxaliplatin. CRIPSR SNP transfection in RKO showed similar effects, with a 2-fold less apoptotic response to 5-FU and &amp;gt;90% less response to oxaliplatin. PCR showed RKO STAG1-SNP transfected cells displayed a loss of STAG1(~40%, p&amp;lt;0.05) and an upregulation of LGR5 (~50%, p&amp;lt;0.05) and ALDH1a (~30%, p&amp;lt;0.05). This shows, for the first time, that STAG1 loss is implicated in colon carcinogenesis through potentiation of cancer stem cells through early carcinogenesis/initiation as well as disease progression and aggressiveness as shown through chemoresistance. Our work provides a potential mechanism in CRC, thus providing a biomarker for cancer screening and therapeutics that could mitigate the racial disparity of CRC in AAs. Citation Format: Mart Dela Cruz, Caroline Zaworski, Somenath Datta, Sanjib Chowdhury, Hemant K. Roy. Cohesins and colorectal cancer (CRC): Modulation of CRC stem cells and chemoresistance by STAG1 with race-specific implications [abstract]. In: Proceedings of the Eleventh AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2018 Nov 2-5; New Orleans, LA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl):Abstract nr C036.

In silico Analysis, Molecular Docking, Molecular Dynamic, Cloning, Expression and Purification of Chimeric Protein in Colorectal Cancer Treatment.

IntroductionColorectal cancer (CRC) is a type of cancer in humans that leads to high mortality and morbidity. CD166 and CD326 are immunoglobulins that are associated with cell migration. These molecules are included in tumorigenesis of CRC and serve a great marker of CRC stem cells. In the present study, we devised a novel chimeric protein including the V1-domain of the CD166 and two epitopes of CD326 to use in diagnostic or therapeutic applications.MethodsIn silico techniques were launched to characterize the properties and structure of the protein. We have predicted physicochemical properties, structures, stability, MHC class I binding properties and ligand-receptor interaction of this chimeric protein by means of computational bioinformatics tools and servers. The sequence of chimeric gene was optimized for expression in prokaryotic host using online tools and cloned into pET-28a plasmid. The recombinant pET28a was transformed into the E. coli BL21DE3. Expression of recombinant protein was examined by SDS-PAGE and Western blotting.ResultsThe designed chimeric protein retained high stability and the same immunogenicity as of the original proteins. Bioinformatics data indicated that the epitopes of the synthetic chimeric protein might induce B-cell- and T-cell-mediated immune responses. Furthermore, a gene was synthesized using the codon bias of a prokaryotic expression system. This synthetic gene expressed a bacterial expression system. The recombinant protein with molecular weights of 27kDa was expressed and confirmed by anti-his Western blot analysis.ConclusionThe designed recombinant protein may be useful as a CRC diagnostic tool and for developing a protective vaccine against CRC.