Human CRC Cell Research Articles

Gingerenone A induces ferroptosis in colorectal cancer via targeting suppression of SLC7A11 signaling pathway

BackgroundColorectal cancer (CRC) is one of the most common and fatal diseases, yet effective therapeutic drugs are lacking in clinical settings. Gingerenone A (GA) is an active compound derived from ginger, has demonstrated anti-tumor properties. However, the efficacy of GA against CRC and its primary mechanism of action remain unclear. Materials and methodsMTT assay and colony formation assay were employed to evaluate cell viability. Transwell assays were utilized to assess the migratory and invasive capabilities of the cells. The effects of GA on ferroptosis related proteins were analyzed using Western blot. Levels of glutathione (GSH), malondialdehyde (MDA), Fe2+, and 4-hydroxynonenal (4-HNE) levels were measured with a biochemical index determination kit. Cellular reactive oxygen species (ROS) were quantified using flow cytometry. CETSA, pull-down, and co-immunoprecipitation (Co-IP) assays confirmed the interactions between GA and SLC7A11, as well as the ubiquitination promoted by SLC7A11. A xenograft mouse model was employed to validate the anticancer effect of GA in vivo. ResultsWe observed that GA significantly suppressed proliferation in human CRC cells. Additionally, GA treatment inhibited the migration, invasion, and colony formation of CRC cells. Subsequently, through the use of specific inhibitors, we discovered that the suppression of CRC cells by GA was dependent on ferroptosis rather than autophagy or apoptosis. Previous research has demonstrated that GA treatment significantly triggers ferroptosis. Mechanistically, GA treatment promotes the degradation of the SLC7A11 protein, which plays a crucial role in ferroptosis. Notably, the knockdown of SLC7A11 abolished the detrimental effects of GA on the proliferation of CRC cells and reversed GA-induced ferroptosis in CRC cells both in vivo and in vitro. Further research has shown that GA can directly bind to the SLC7A11 protein and promote its ubiquitination. ConclusionOur research provides compelling evidence that GA may serve as a potential agent for suppressing the progression of CRC by inducing ferroptosis and promoting the ubiquitination and degradation of SLC7A11.

P53 genotype-independent anticancer effects of olibanum extract and 11-keto-beta-boswellic acid on human colorectal cancer cells

BackgroundColorectal cancer (CRC) is the third most common cancer, and CRC-related mortality is increasing annually. A key regulator of the cell cycle and cell death, the p53 gene, is frequently found in patients with CRC as mutants. Since tumors with abnormal p53 sometimes show resistance to anticancer drugs, it is important to develop anticancer drugs according to genetic characteristics. Although evidence shows that boswellic acids (BAs) might be potential anticancer agents, the anticancer effects and mechanisms involved in their activities in CRC are unclear. Methods and ResultsHere, we investigated whether olibanum (Boswellia serrata Roxb.) extract and 11-keto-β-boswellic acid exerted cytotoxic effects against p53 wildtype, p53 mutated-, and p53-deleted human CRC cell lines. Results show that 11-keto-β-boswellic acid considerably reduced cell viability and induced cell cycle arrest in HCT116, HT29, and SW1417 cells. And increase of apoptotic cell population and morphological changes in apoptotic cells induced by 11-keto-β-boswellic acid were observed. The 11-keto-β-boswellic acid mediated cell cycle arrest and apoptosis were accompanied by changes in the expression of factors linked to apoptosis, regardless of p53 genotype. In addition, 11-keto-β-boswellic acid-treated cells dissociated from their spheroidal structure and appeared in an irregular form. ConclusionsAlthough further investigations are required to fully understand the underlying mechanisms, these results may provide insight into the potential of using olibanum extract and 11-keto-β-boswellic acid as therapeutic agents, with no difference in sensitivity depending on the p53 genotype.

MiRNAs: possible regulators of toll like receptors and inflammatory tumor microenvironment in colorectal cancer

BackgroundColorectal cancer (CRC) is ranked as the third most commonly diagnosed cancer and the third cause of cancer related deaths. CRC is greatly attributed to genetic and epigenetic mutations and immune dysregulation. Tumor aberrant expression of Toll-like Receptors (TLRs) can contribute to tumorigenesis. Recent studies suggested that microRNAs act as direct ligands of TLRs altering their expression and signaling pathways.AimTo prove our concept that specific miRNA mimics may act as antagonists of their specific toll like receptors inhibiting their expression that could limit the release of pro-inflammatory and pro-tumorigenic cytokines leading to apoptosis of tumor cells.MethodsFrom public microarray databases, we retrieved TLRs and miRNAs related to CRC followed by in silico docking of the selected miRNA ligands into the TLRs. Clinical validation after co-immunoprecipitation of TLRs and their interacting miRNA ligands was done. Expression of TLRs 1, 7,8 was determined by ELISA while miRNAs was measured by RT-qPCR. In addition, microRNA mimics of the down regulated miRNAs were transfected into human CRC cell lines.ResultsOur data demonstrate that TLRs 1, 7, 8 are up regulated in CRC compared to controls. Further, three miRNAs (-122, -29b and -15b) are relatively downregulated, while 4 miRNAs (-202, miRNA-98, -21 and -let7i) are upregulated in CRC patients compared to those with benign tumor and healthy controls. Transfection of down regulated miRNA mimics into CRC cell lines resulted in a significant reduction of the number and viability of cells as well as down regulating the expression of TLRs 1, 7 and 8 with ultimate reduction of downstream effector IL6 protein, suggesting that these miRNAs are negative regulators of carcinogenesis.ConclusionMicroRNAs could act as antagonistic ligands of TLRs limiting the inflammatory tumor microenvironment.

Zosterabisphenone B, a new diarylheptanoid heterodimer from the seagrass Zostera marina, induces apoptosis cell death in colon cancer cells and reduces tumour growth in mice.

Colorectal cancer (CRC) is one of the most common malignant tumours worldwide. Diarylheptanoids, secondary metabolites isolated from Zostera marina, are of interest in natural products research due to their biological activities. Zosterabisphenone B (ZBP B) has recently been shown to inhibit the viability of CRC cells. The aim of this study was to investigate the therapeutic potential of ZBP B for targeting human CRC cells. Cell viability was determined using the MTT assay. Flow cytometry and Western blot analyses were used to assess apoptosis and autophagy. A CRC xenograft model was used to evaluate the in vivo effect of ZBP B. No cytotoxic effect on HCEC cells was observed in the in vitro experiments. ZBP B caused morphological changes in HCT116 colon cancer cells due to an increase in early and late apoptotic cell populations. Mechanistically, ZBP B led to an increase in cleaved caspase-3, caspase-8, caspase-9, PARP and BID proteins and a decrease in Bcl-2 and c-Myc proteins. In the xenograft model of CRC, ZBP B led to a reduction in tumour growth. These results indicate that ZBP B exerts a selective cytotoxic effect on CRC cells by affecting apoptotic signalling pathways and reducing tumour growth in mice. Taken together, our results suggest that ZBP B could be a lead compound for the synthesis and development of CRC drugs.

Abstract 2167: Microbial bile acid, 3-oxo-LCA, inhibits colorectal cancer progression

Abstract Colorectal cancer (CRC) affects a significant number of individuals each year, ranking as a leading cause of cancer-related deaths in the U.S. Bile acids (BAs), natural compounds crucial for digesting dietary fats, not only influence the gut microbiota but are also involved in gut health through their interaction with the Farnesoid X Receptor (FXR). Recent findings have highlighted certain microbiota-generated BAs like 3-oxo-lithocholic acid (LCA) and IsoLCA, modulate host immunity, hinder the expansion of intestinal pathogens, and potentially anti-aging. Despite the advancements in identifying the gut microbes behind these BAs, their precise impact on intestinal cells (IECs) and their role in disease progression are largely unexplored. Our pilot investigation unveiled 3oxoLCA as an FXR agonist, restoring FXR signaling in both cancer cells and APCmin/+ mice (a classic CRC mice model). As a result, 3-oxo-LCA inhibits the growth of both human and mouse CRC cells, such as MC38, CT26 and HCT116. Additionally, 3-oxo-LCA also restrained the intestinal stem cells (ISCs)’ proliferation in organoids from both wild-type and APCmin/+ mice, and patient-derived CRC organoids (PDCOs). Remarkably, treatment with 3-oxo-LCA decreased BAs levels, improved gut barrier function, reduced tumor load, and inhibited tumor progression in APCmin/+ mice. Furthermore, 3-oxo-LCA significantly suppressed tumor growth in cancer cell-derived xenograft model mice. Crucially, the 3-oxo-LCA-FXR interaction transcriptionally regulated key apoptotic genes, encouraging cancer cell death. These discoveries highlight the therapeutic promise of incorporating 3-oxo-LCA into strategies for treating CRC. Citation Format: Fei Sun, Xingchen Dong, Ming Qi, Ting Fu. Microbial bile acid, 3-oxo-LCA, inhibits colorectal cancer progression [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 2167.

Abstract 2086: Effect of XPO1 inhibition on colorectal cancer tumorigenesis

Abstract Colorectal Cancer (CRC) is the second leading cause of cancer-related death in the U.S. A subset of individuals faces a notably higher likelihood of developing CRC within their lifetime. Hence, there is a compelling need for innovative chemopreventive treatments aimed at minimizing CRC tumorigenesis. Exportin 1 (XPO1; also referred to as CRM1) plays a pivotal role in transporting proteins from the nucleus to the cytoplasm. The overexpression of XPO1 has been identified across several cancers, including CRC. XPO1 interacts with a diverse spectrum of more than 1000 proteins. The overexpression of XPO1 leads to the excessive removal of tumor suppressors from the nucleus, rendering these proteins functionally inert. To counteract the overexpression of XPO1 in tumors, a novel class of drugs known as Selective Inhibitors of Nuclear Export (SINE) has been developed. Among these, Eltanexor (KPT-8602) has emerged as a promising therapeutic agent, demonstrating fewer side effects compared to its precursors, and is currently under evaluation in Phase 1/2 clinical trials. The treatment effects of SINE compounds on CRC remain largely unknown. We have assessed Eltanexor's ability to limit CRC tumorigenesis using a genetic CRC model in Apcmin/+ mice. Our findings indicate that Eltanexor substantially reduces tumor burden by approximately 3-fold and induces a significant reduction of tumors exceeding 1mm in size. Furthermore, a xenograft experiment using the human CRC cell line, HCT116, reveals nearly a 3-fold reduction in tumor size upon Eltanexor treatment. In both CRC models, Eltanexor exhibits noteworthy tolerability. Additionally, XPO1 protein levels are consistently diminished in both models, aligning with our in vitro observations. To further ascertain Eltanexor's specificity for CRC tumors versus normal tissue, we conducted drug sensitivity assays using organoids derived from Apcmin/+ tumors and wild-type mice small intestine tissue. We found that Apcmin/+ tumoroids have heightened sensitivity to Eltanexor-treatments, when compared to wild-type organoids. In an endeavor to elucidate Eltanexor's potent in vivo tumorigenesis inhibitory effects, we conducted multiple assays which revealed a reduction in COX-2 RNA and protein expression levels in Eltanexor-treated cells and reduced COX-2 protein in the tumors of Eltanexor-treated Apcmin/+ mice. In CRC, COX-2 is implicated in CRC development and is currently the target of multiple CRC chemopreventive agents. Our observations reveal that Eltanexor-treated CRC cells exhibit reduced COX-2 gene expression through both inhibiting COX-2’s promoter and its mRNA post-transcriptional stability. Collectively, our findings underscore XPO1 as a potent target for inhibiting CRC tumorigenesis. Future studies will aim to further assess how Eltanexor is impacting COX-2’s promoter, while additionally testing Eltanexor’s tumorigenesis-inhibiting potential in a colitis-induced CRC mouse model. Citation Format: Andrew E. Evans, Dan A. Dixon. Effect of XPO1 inhibition on colorectal cancer tumorigenesis [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 2086.

Abstract 1566: Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer

Abstract Colorectal cancer (CRC) is one of the leading causes of cancer-related deaths worldwide. The high mortality is directly associated with metastatic disease which is thought to be initiated by colon cancer stem cells according to cancer stem cell (CSC) model. Consequently, early identification of those patients who are at high risk for metastasis is crucial for improved treatment and patient outcome. Metastasis-associated in colon cancer 1 (MACC1) is a novel prognostic biomarker for tumor progression and metastasis formation independent of tumor stage. We previously showed an involvement of MACC1 in cancer stemness in the mouse intestine of our transgenic mouse models. However, the expression of MACC1 in human CSCs and possible implications remain elusive. Here, we explored the molecular mechanisms by which MACC1 regulates stemness and CSC-associated invasive phenotype based on patient-derived 3D cell culture models (PD3D), patient-derived xenografts (PDX) and human CRC cell lines. We showed that CD44-enriched CSCs from PD3D models express significantly higher levels of MACC1 and display higher tumorigenicity in immunocompromised mice. Similarly, RNA sequencing performed on PD3D and PDX models demonstrated significantly increased MACC1 expression in ALDH1(+) CSCs, highlighting its involvement in cancer stemness. We further showed the correlation of MACC1 with CSC markers CD44, NANOG and LGR5 in PD3D models as well as established cell lines. Additionally, MACC1 increased stem cell gene expression, clonogenicity and sphere formation. Strikingly, we showed that MACC1 binds as a transcription factor to the LGR5 gene promoter uncovering the long-known CSC marker LGR5 as novel essential signaling mediator used by MACC1 to induce CSC-like properties in human CRC patients. Our in vitro findings were further substantiated by significant positive correlation of MACC1 and LGR5 in CRC cell lines as well as CRC patient tumors. Taken together, this study indicates that the metastasis-inducer MACC1 act as a cancer stem cell-associated marker. Interventional approaches targeting MACC1 would potentially improve further targeted therapies for colorectal cancer patients to eradicate CSCs, prevent cancer recurrence and distant metastasis formation. Citation Format: Müge Erdem, Kyung Hwan Lee, Markus Hardt, Joseph Regan, Dennis Kobelt, Wolfgang Walther, Christian Regenbrecht, Ulrike S. Stein. Cancer metastasis and stemness: The metastasis-inducer MACC1 regulates LGR5 to promote cancer stem cell-like properties in colorectal cancer [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 1566.

A54 SHP2 FUNCTION IN NORMAL AND APC-MUTANT INTESTINAL EPITHELIAL CELLS

Abstract Background Somatic SHP2 gain-of-function mutations were found in some colorectal tumours. However, its function in colorectal epithelium remains to be determined. SHP2 is a tyrosine phosphatase necessary for RAS/MAPK pathway activation by most, if not all, tyrosine kinase receptors, as well as by GPCRs and cytokine receptors. SHP2 can also regulate the PI3K, Jak/Stat, RhoA and Hippo pathways in different cell settings. As a result, SHP2 can control cellular processes including growth, migration and differentiation. Previously, we observed that SHP2 levels are enhanced in human colorectal tumours, notably in late adenomas. Likewise, SHP2 silencing inhibited ERK activation and tumour properties of established human CRC cell lines. Hence, these findings support a model in which SHP2 activation promotes tumorigenesis in the intestinal epithelium. But how? Aims To analyze the exact cellular and molecular mechanisms underlying SHP2 pro-oncogenic function in intestinal epithelial cells. Methods The impact of SHP2 pharmaco-inhibition (RMC-4550 and/or SHP099) on intracellular signaling, cell cycle, proliferation and survival was evaluated in three complementary models: 1) HIEC-6 cells, normal human fetal intestinal crypt-like cells; 2) organoids derived from normal mouse small intestine or ApcMin/+ tumors; 3) Caco-2/15 cells, colorectal cancer cells with APC mutation. Results 1- Pharmaco-inhibition of SHP2 blocks proliferation of HIEC-6 cells (loss of pRb phosphorylation, decreased EdU staining) and induces cell enlargement and darkening of the outside of perinuclear area. This phenotype was reminiscent of cellular senescence, and we indeed observed increased senescence-associated β-galactosidase activity (SA-β-Gal) and INK4A gene expression. 2- As expected, SHP2 inhibition in normal mouse organoids resulted in a reduction in the number of protrusions and EdU positive cells. RNA sequencing was performed, and gene set enriched revealed significant down regulation of several Ras signaling-dependant genes including cell cycle regulated genes (E2F targets, G2/M checkpoint) and Myc targets 48h after SHP2 inhibition when compared to DMSO treated organoids. Genes associated with PI3K signaling were also diminished in SHP099 treated organoids but no significant modulation of genes associated with Wnt/b-catenin signaling was observed. 3- Notably, SHP2 pharmaco-inhibition in Caco-2/15 cells as well as APC-mutant organoids rapidly induced cell death associated with increased expression of stem cell markers. Conclusions These results suggest that SHP2 is required for proliferation of normal IEC and protects against senescence. Notably, its inhibition restrains the proliferative and tumorigenic potential conferred by APC inactivation. Overall, our data suggest that SHP2 could represent a potential target to counter colorectal tumour initiation. Funding Agencies CIHRFRQS

Abstract C111: A novel human monoclonal antibody targeting active ADAM10 demonstrates anti-tumor effects in colorectal cancer

Abstract Colorectal cancer (CRC) is the third most diagnosed cancer in the USA and accounts for more than 600,000 deaths annually worldwide, primarily due to relapse with highly aggressive, chemo-resistant disease characterized by poorly differentiated cancer cells with stem cell-like properties. A common signature of these chemo-resistant tumors is dysregulation of Notch receptor signaling, as well as upregulation of its metalloprotease activator, ADAM10. Although small molecule inhibition of either Notch or ADAM10 has been shown to produce potent anti-tumor effects, these therapeutic strategies have failed in clinical trials primarily due to systemic toxicities, especially cytotoxic effects on the gastrointestinal tract and musculoskeletal system, highlighting the need for development of more targeted approaches. We have previously demonstrated that ADAM10 predominantly exists in an inhibited state in normal tissues but is activated in tumor cells through a conformational change in the extracellular domain, thus providing a potential target for tumor-specific modulation of ADAM10 activity. Here, we look to characterize a novel human monoclonal antibody agent (1H5) that selectively targets an exposed extracellular region of activated ADAM10 on tumor cells. We previously demonstrated that treatment with a murine version of the antibody (8C7), specific for the activated form of both mouse and human ADAM10, conferred a significant reduction in tumor burden against human CRC cell lines in cell culture and transplants in xenograft models, and relapse was prevented when 8C7 was combined with chemotherapy. Here, we demonstrate that the 1H5 monoclonal antibody effectively reduces cell viability in numerous colorectal cancer cell lines in vitro and induces marked tumor regression in established xenograft models through inhibition of Notch signaling. Through the use of additional genetic tools combined with in vitro and in vivo models, our preclinical study aims to fully evaluate 1H5 as novel therapeutic agent to treat aggressive colorectal cancer. We aim to propel this novel agent toward clinical-stage development. Citation Format: Pargol Mashati, Dan Sun, Eduardo Garcia Reino, Jessica A. Blandino, Ben Mason, Nayanendu Saha, Dimitar B. Nikolov, Prem Premsrirut. A novel human monoclonal antibody targeting active ADAM10 demonstrates anti-tumor effects in colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C111.

Abstract C043: Discovery of new drug combinations for metastatic colorectal cancer

Abstract Colorectal cancer (CRC) is the second most common cause of cancer-related death worldwide. Despite a range of therapeutic options, drug resistance remains a significant challenge in CRC treatment. Drug combination therapy offers an opportunity to enhance therapeutic responses. There are several combination drug options that have been used for CRC patients such as FOLFOX (5-FU + Oxaliplatin) and FOLFIRI (5-FU + Irinotecan). This study aimed to discover novel drug combinations for CRC by conducting high-throughput screening of 800 pharmacologically active compounds in combination with the three main chemotherapy backbones used in CRC treatment: 5-FU, SN-38, and Oxaliplatin. Initially, we performed a primary drug screen using 11 human CRC cell lines, resulting in the identification of 72 candidate drug combinations (24 candidates x 3 backbones) exhibiting synergistic effects. Subsequently, we validated these candidates in a subset of 5 cell lines. Among them, 24 drug combinations (8 candidates x 3 backbones) were selected and further evaluated in an expanded panel of 44 CRC cell lines to determine the spectrum of drug responses and identify potential mutation, gene and protein expression biomarkers, that could predict the efficacy of the drug combinations. We investigated the molecular mechanisms underlying the synergistic effects of final 4 candidates combined with chemotherapeutic agents. Chk1 inhibitor MK-8776 showed significantly higher synergy scores in combination with SN-38 in TP53 mutated cell lines, which was further validated by in vitro apoptosis analysis and in vivo xenograft models using TP53 wildtype and knockout isogenic CRC cell lines. MK-8776 abrogated SN-38-mediated G2 cell cycle arrest, resulting in an increased cytotoxicity. These findings highlight the potential of the combination treatment involving SN-38 and MK-8776 as a promising strategy for CRC patients with TP53 mutations. Citation Format: Eun-Jung In, Tao Tan, Dmitri Mouradov, Anuratha Sakthianandeswaren, Amanda Au, Kym Lowes, Oliver Sieber. Discovery of new drug combinations for metastatic colorectal cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C043.

Ethyl acetate fraction of Osmanthus fragrans var. aurantiacus and its triterpenoids suppress proliferation and survival of colorectal cancer cells by inhibiting NF-κB and COX2

Ethnopharmacological relevanceColorectal cancer (CRC) remains a significant global health concern, and targeting inflammation has emerged as a promising approach for its prevention and treatment. Medicinal plants and phytochemicals have garnered attention for their potential efficacy against inflammation with minimal toxicity. Osmanthus fragrans var. aurantiacus Makino (O. fragrans) has a history of traditional use in Korea and China in treating various inflammation-related conditions, but its potential use for CRC has not been uncovered. Aim of the studyThis study aims to explore the potential anti-proliferative and pro-apoptotic properties of O. fragrans, focusing on its impact on CRC treatment. By investigating O. fragrans, we aim to uncover its anti-proliferative and apoptotic effects in human CRC cells, potentially paving the way for effective and well-tolerated therapeutic strategies for CRC patients. Materials and methodsEthanol (EtOH) extracts of O. fragrans leaf and flower, along with specific fractions (n-hexane, ethyl acetate (EtOAc), n-butanol, and the aqueous residue) were evaluated for their anti-proliferative effects in human CRC cells using MTT assays, and compared to normal colon cells. Mechanistic insights and chemical profiling were obtained through flow cytometry, colorimetric assays, western blotting, and molecular docking, and high-performance liquid chromatography (HPLC) system. ResultsBoth flower and leaf EtOH extracts of O. fragrans exhibited significant anti-proliferative effects in human CRC cells, with the leaf extract demonstrating higher potency. The EtOAc fraction from the leaf extract displayed the strongest anti-CRC cell proliferative effects while no cytotoxic effects in normal colon cells. Chemical profiling of these fractions identified triterpenoids as significant components in the EtOAc fractions. The leaf EtOAc fraction caused cell cycle arrest and apoptosis, accompanied by elevating intracellular reactive oxygen species and mitochondrial dysfunction in CRC cells. Additionally, it inhibited NF-κB and ERK1/2 signaling, leading to reduced COX2 expression. Notably, two triterpenoids isolated from the leaf EtOAc fraction, maslinic acid and corosolic acid, displayed potent anti-cancer activity in CRC cells without affecting normal colon cells. Corosolic acid exhibited a strong binding affinity to COX2 and reduced its expression, supporting its role in the anti-inflammatory and anti-cancer effects. ConclusionsOur findings suggest that O. fragrans, particularly its triterpenoid-rich EtOAc fraction, holds promise as a novel therapeutic agent for CRC prevention and therapy. These results provide valuable insights into the potential application of O. fragrans and its bioactive compounds in combating CRC.

PEGylated Fluorescent Anti-carcinoembryonic Antigen Antibody Labels Colorectal Cancer Tumors in Orthotopic Mouse Models

IntroductionColorectal cancer (CRC) patients often develop liver metastasis. However, curative resection of liver metastasis is not always possible due to poor visualization of tumor margins. The present study reports the characterization of a humanized anti-carcinoembryonic antigen monoclonal antibody conjugated to a PEGylated near-infrared dye, that targets and brightly labels human CRC tumors in metastatic orthotopic mouse models. MethodsThe hT84.66-M5A (M5A) monoclonal antibody was conjugated with a polyethylene glycol (PEG) chain that incorporated a near infrared (NIR) IR800 dye to establish M5A-IR800 Sidewinder (M5A-IR800-SW). Nude mice with CRC orthotopic primary tumors and liver metastasis both developed from a human CRC cell line, were injected with M5A-IR800-SW and imaged with the Pearl Trilogy Imaging System. ResultsM5A-IR800-SW targeted and brightly labeled CRC tumors, both in primary-tumor and liver-metastasis models. M5A-IR800-SW at 75 μg exhibited highly-specific tumor labeling in a primary-tumor orthotopic model with a median tumor-to-background ratio of 9.77 and in a liver-metastasis orthotopic model with a median tumor-to-background ratio of 7.23 at 96 h. The precise labeling of the liver metastasis was due to lack of hepatic accumulation of M5A-IR800-SW in the liver. ConclusionsM5A-IR800-SW provided bright and targeted NIR images of human CRC in orthotopic primary-tumor and liver-metastasis mouse models. The results of the present study suggest the clinical potential of M5A-IR800-SW for fluorescence-guided surgery including metastasectomies for CRC. The lack of hepatic NIR signal is of critical importance to allow for precise labeling of liver tumors.

2-Nucleobase-substituted 4,6-Diaminotriazine Analogs: Synthesis and Anti-cancer Activity in 5-Fluorouracil-sensitive and Resistant Colorectal Cancer Cells.

Cancer continues to be the second leading cause of death worldwide, with colorectal cancer (CRC) being the third most common type. Despite significant advances in cancer therapies, the current treatment of CRC remains suboptimal. In addition, the effectiveness of available chemotherapeutic drugs such as 5-Fluorouracil (5-FU) is limited by CRC-acquired resistance. In this study, we provide innovative approaches employed in synthesizing four novel nucleobase analogs. Equally, we describe the effects of these compounds on proliferation, migration, aggregation, and adhesion of 5-FU-sensitive (HCT116) and -resistant (5-FU-R-HCT116) human CRC cells. In either cell type, our synthesized novel analogs significantly inhibited cell viability in a concentration- and time-dependent manner. This highlights the higher potency of these novel analogs. In addition, these compounds attenuated migration and adhesion of both cell types while they promoted homotypic cell-cell interaction. These changes were reflected by the downregulation of matrix metalloproteases (MMP-2 and MMP-9). Furthermore, our analogs exhibited potent anti-angiogenic activity in vivo. These novel nucleobase analogs reduced the level of secreted vascular endothelial growth factor (VEGF) and nitric oxide (NO) production in both 5-FU-sensitive and -resistant CRC cells. Taken together, our data highlight the potential chemotherapeutic properties of our novel analogs against CRC, including the 5-FU-resistant form.

Blastocystis sp. reduces the efficacy of 5-fluorouracil as a colorectal cancer chemotherapeutic treatment

Blastocystis is an enteric protozoan parasite with extensive genetic variation and unclear pathogenicity. It is commonly associated with gastrointestinal symptoms such as nausea, diarrhea, vomiting and abdominal pain in immunocompromised individuals. In this study, we explored the in vitro and in vivo effects of Blastocystis on the activity of a commonly used CRC chemotherapeutic agent, 5-FU. The cellular and molecular effects of solubilized antigen of Blastocystis in the presence of 5-FU were investigated using HCT116, human CRC cell line and CCD 18-Co, normal human colon fibroblast cells. For the in vivo study, 30 male Wistar rats were divided into six groups, as follows; Control Group: oral administration of 0.3 ml Jones' medium, Group A: rats injected with azoxymethane (AOM), Group A-30FU: Rats injected with AOM and administered 30 mg/kg 5-FU, Group B-A-30FU: rats inoculated with Blastocystis cysts, injected with AOM and administered 30 mg/kg 5-FU, Group A-60FU: rats injected with AOM and administered 60 mg/kg 5-FU and Group B-A-60FU: rats inoculated with Blastocystis cysts, injected with AOM and administered 60 mg/kg 5-FU. The in vitro study revealed that the inhibitory potency of 5-FU at 8 μM and 10 μM was reduced from 57.7% to 31.6% (p < 0.001) and 69.0% to 36.7% (p < 0.001) respectively when co-incubated with Blastocystis antigen for 24 h. However, the inhibitory potency of 5-FU in CCD-18Co cells was not significantly affected in the presence of Blastocystis antigen. The reduced inhibitory potency of 5-FU against cancer cell proliferation due to the presence of Blastocystis is consistent with the upregulation of expression of type 2 cytokines, transforming growth factor (TGF-β) and nuclear factor E2-related factor 2 (Nrf2) gene expression. Increased inflammation and abnormal histopathological findings along with a significant cancer multiplicity and adenoma incidence were evident in the intestine of the B-A-30FU and B-A-60FU groups when compared with the A-30FU and A-60FU groups respectively. Our in vitro and in vivo findings indicate that Blastocystis infection could potentially interfere with chemotherapy regimens such as 5-FU in CRC patients undergoing chemotherapy.

Abstract 3481: A local tumor microenvironment acquired super-enhancer induces an oncogenic driver in colorectal carcinoma

Abstract Tumors exhibit widespread enhancer landscape reprogramming compared to normal tissue. The etiology is believed to be largely cell-intrinsic in non-hormonal cancers, attributed to such genomic alterations as focal amplification of non-coding regions, aberrant activation of transcription factors, and non-coding mutations creating de novo transcription factor binding sites. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon epithelia, we find divergent epigenetic landscapes between primary CRC tumors and CRC cell lines. We identify a unique super-enhancer signature largely absent in cell culture. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over patient-matched normal epithelium suggesting novel insight into the etiology of enhancer reprogramming in CRC and its downstream relevance to tumor biology. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. CRISPR-dcas9-KRAB interference of this super-enhancer identifies PDZK1IP1 as its target gene. PDZK1IP1 is previously observed to be highly up-regulated in CRC. However, the mechanism behind its transcriptional activation is not fully understood. We restore both the super-enhancer and PDZK1IP1 levels following treatment with cytokines or xenotransplantation into nude mice, thus demonstrating its etiology via local tumor microenvironment acquisition. Deletion of inflammatory transcription factors RELA and STAT3 in human CRC cells inhibits PDZK1IP1 induction in xenografts. PDZK1IP1 appears to be critical for CRC growth in the setting of its super-enhancer induction as xenografts, but not in cell culture where the super-enhancer is absent and expression is largely silent. Building on its known role in glucose uptake via SGLT receptors, we demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity CRC cancer cells via the pentose phosphate pathway using polar metabolomic profiling. We show this activation enables efficient growth under oxidative conditions both in vitro and in vivo, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the biologic significance of epigenomic profiling on patient-matched primary specimens and identify this microenvironment-acquired super-enhancer as an oncogenic driver in the setting of the inflamed tumor. Citation Format: Royce Zhou, Jia Xu, Tiphaine Martin, Alexis Zachem, John He, Sait Ozturk, Deniz Demircioglu, Ankita Bansal, Andrew Trotta, Bruno Giotti, Berkley Gryder, Yao Shen, Saul Carcamo, Xuewei Wu, Kaitlyn Bosch, Benjamin Hopkins, Alexander Tsankov, Randolph Steinhagen, Drew Jones, John Asara, Jerry Chipuk, Rachel Brody, Steven Itzkowitz, Iok In Christine Chio, Dan Hasson, Emily Bernstein, Ramon Parsons. A local tumor microenvironment acquired super-enhancer induces an oncogenic driver in colorectal carcinoma [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 3481.

Abstract 1819: Integrin inhibitor GLPG-0187 promotes T-cell killing of mismatch repair-deficient colorectal cancer cells by suppression of SMAD/TGF-β signaling

Abstract Colorectal cancer is the third leading cause of cancer-related death and the third most common cause of cancer. As the five-year survival with advanced metastatic colorectal cancer (mCRC) is 14%, new treatment strategies are needed. Immune checkpoint blockade which takes advantage of an individual's immune system in order to fight cancer, is a promising future direction of research, although it is only effective and approved for the treatment of mismatch repair (MMR)-deficient CRCs. Long-term outcomes even in MMR-deficient mCRC suggest that most patients are not cured but eventually develop therapy resistance. We hypothesized that targeting TGF- β signaling may enhance immune-mediated T-cell killing by MMR-deficient CRC cells. Using GLPG-0187, an inhibitor of multiple integrin receptors and TGF- β, we demonstrate minimal cytotoxicity against MMR-deficient HCT116 or p53-null HCT116 human CRC cells. However, GLPG-0187 promoted significant immune cell killing of the CRC cells by TALL-104 T lymphoblast cells. GLPG-0187 reduced pSMAD2 in HCT116 p53-null cells either in the absence or presence of exogenous TGF- β. We observed a reduction in CCL20, CXCL5, prolactin, and TRAIL-R3 while GDF-15 was increased in TALL-104 cells treated with a T-cell activating dose of GLPG-0187 (4 μM). Our results suggest that TGF- signaling inhibition by a general integrin receptor inhibitor may boost T-cell killing of MMR-deficient colorectal cancer cells, and suggest that a combination of anti-GDF-15 in combination with TGF- β blockade should be further investigated in the treatment of MMR-deficient mCRC. Our results support the development of a novel immune-based therapeutic strategy to treat colorectal cancer by targeting the TGF-β signaling pathway through integrin receptor blockade. Citation Format: Brooke Verschleiser, William MacDonald, Lindsey Carlsen, Kelsey E. Huntington, Lanlan Zhou, Wafik El-Deiry. Integrin inhibitor GLPG-0187 promotes T-cell killing of mismatch repair-deficient colorectal cancer cells by suppression of SMAD/TGF-β signaling [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 1819.

α-hederin regulates glucose metabolism in intestinal epithelial cells by increasing SNX10 expression

BackgroundSorting nexin 10 (SNX10) has recently been identified as a critical regulator of colorectal carcinogenesis, whose deletion promoted cell proliferation and survival in human CRC cells, and promoted colorectal tumor growth and upregulated amino-acid metabolism in mice. However, what happens when silencing SNX10 in normal human intestinal epithelial cells (IECs) remains unknown, and no drugs targeting SNX10 have been reported. Here, we first investigated the biological function and underlying mechanisms of SNX10 in normal human IECs, and found that α-hederin, a pentacyclic triterpenoid saponin, has a regulatory effect on SNX10 expression. PurposeThis study aimed to explore the function of SNX10 in IECs to provide a new target for the prevention and treatment of malignant transformation and the intervention mechanism of α-hederin for further development of potential novel agents targeting SNX10. MethodsThe transfection approach was used to construct SNX10 stable knockdown cells. Cell proliferation was detected by CCK8, clone formation, EdU, flow cytometry, and wound healing assays. Enzyme activity assays for glucose metabolism, qRT-PCR, western blotting, and immunofluorescence staining were performed to investigate the protein expression of signaling pathways. ResultsSilencing SNX10 promoted cell proliferation and cycle transition in IECs and increased the activity of key enzymes involved in glucose metabolism. Moreover, DEPDC5 expression was significantly decreased following SNX10 knockdown, followed by activation of the mTORC1 pathway. α-hederin reversed the accelerated cell proliferation, cycle progression, and glucose metabolic activity, as well as the activated mTORC1 pathway caused by SNX10 knockdown, by notably increasing SNX10 expression in a dose-dependent manner. ConclusionWe first reported that knockdown of SNX10 in normal human IECs promoted cell proliferation and activated glucose metabolism by activating the mTORC1 pathway. Meanwhile, we first found that α-hederin down-regulated glucose metabolism activity and slowed cell proliferation by increasing SNX10 expression in IECs.

SPATA2 and CYLD inhibit T cell infiltration into colorectal cancer via regulation of IFN-γ/STAT1 axis.

Colorectal cancer (CRC) is largely refractory to currently available immunotherapies such as blockade of programmed cell death protein-1 (PD-1). In this study, we identified SPATA2 and its protein partner CYLD as novel regulators of CXC-ligand 10 (CXCL10), a T-cell-attractant chemokine, in CRC. By specifically deleting SPATA2 and CYLD in human and mouse CRC cell lines, we showed that these two proteins inhibit STAT1 accumulation and activation and subsequently CXCL10 expression in tumor cells. At steady-state, STAT1 is highly ubiquitinated in a SPATA2/CYLD-dependent manner. Finally, we demonstrated that tumor-specific deletion of SPATA2 and CYLD enhances anti-PD-1 response in vivo. Our data suggest that SPATA2 and CYLD represent two potential novel targets for treatment of immune-excluded, PD-1-resistant tumors.

Abstract PR007: A local tumor microenvironment acquired super-enhancer induces an oncogenic driver for efficient growth under oxidative conditions in colorectal carcinoma

Abstract Tumors exhibit widespread enhancer landscape reprogramming compared to normal tissue. The etiology is believed to be largely cell-intrinsic in non-hormonal cancers, attributed to such genomic alterations as focal amplification of non-coding regions, aberrant activation of transcription factors, and non-coding mutations creating de novo transcription factor binding sites. Here, using freshly resected primary CRC tumors and patient-matched adjacent normal colon epithelia, we find divergent epigenetic landscapes between primary CRC tumors and CRC cell lines. We identify a unique super-enhancer signature largely absent in cell culture. Intriguingly, this phenomenon extends to highly recurrent aberrant super-enhancers gained in CRC over patient-matched normal epithelium suggesting novel insight into the etiology of enhancer reprogramming in CRC and its downstream relevance to tumor biology. We find one such super-enhancer activated in epithelial cancer cells due to surrounding inflammation in the tumor microenvironment. CRISPR-dcas9-KRAB interference of this super-enhancer identifies PDZK1IP1 as its target gene, a gene previously observed to be highly up-regulated in CRC. However, the mechanism behind its transcriptional activation and contribution to tumor growth is not fully understood. We restored both the super-enhancer and PDZK1IP1 expression levels following treatment with cytokines or xenotransplantation into nude mice, which was also associated with high levels of cytokines, thus demonstrating its etiology via local tumor microenvironment acquisition. Deletion of inflammatory transcription factors RELA and STAT3 in human CRC cells inhibits PDZK1IP1 induction in xenografts. PDZK1IP1 appears to be critical for CRC growth in the setting of its super-enhancer induction in xenografts, but not in cell culture where the super-enhancer is absent and expression is largely silent. Building on its known role in glucose uptake via SGLT receptors, we demonstrate mechanistically that PDZK1IP1 enhances the reductive capacity of CRC cancer cells via the pentose phosphate pathway using polar metabolomic profiling. We show this activation enables efficient growth under oxidative conditions both in vitro and in vivo, challenging the previous notion that PDZK1IP1 acts as a tumor suppressor in CRC. Collectively, these observations highlight the biologic significance of epigenomic profiling on patient-matched primary specimens and identify this microenvironment-acquired super-enhancer as an oncogenic driver in the setting of the inflamed tumor that is commonly upregulated in CRC. Citation Format: Royce Zhou, Ramon Parsons. A local tumor microenvironment acquired super-enhancer induces an oncogenic driver for efficient growth under oxidative conditions in colorectal carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr PR007.

Lactate promotes metastasis of normoxic colorectal cancer stem cells through PGC-1α-mediated oxidative phosphorylation

Uneven oxygen supply in solid tumors leads to hypoxic and normoxic regions. Hypoxic cells exhibit increased secretion of lactate, which creates an acidic tumor microenvironment (TME). This acidic TME is positively associated with tumor metastasis. Despite the increased metastatic capacity of hypoxic cells, they are located relatively further away from the blood vessels and have limited access to the circulatory system. Studies have shown that cancer stem cells (CSCs) are enriched for tumor metastasis-initiating cells and generally undergo aerobic respiration, which could be enhanced by lactate. We therefore hypothesized that TME-derived lactate may promote the metastasis of normoxic CSCs. In the present study, the abundance of hypoxic and normoxic CSCs was analyzed in primary CRC tumors. It was found that the proportion of normoxic CSCs was positively associated with tumor stage. Using two human CRC cell lines, LoVo and SW480, and a patient-derived xenograft (XhCRC), it was found that treatment with lactate promoted normoxic CSC metastasis. Metabolism analysis indicated that, upon treatment with lactate, oxidative phosphorylation (OXPHOS) activity in normoxic CSCs was enhanced, whereas hypoxic CSCs were rarely altered. At the molecular level, the expression of peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), a master regulator of lactate oxidation, was found to be elevated in normoxic CSCs. Furthermore, PGC-1α knockdown markedly reduced the metastatic potential of normoxic CSCs. Notably, both the PGC-1α-mediated OXPHOS activity and metastatic potential were impaired when hypoxia-inducible factor-1α (HIF-1α) was activated in normoxic CSCs. Together, these findings provide a therapeutic strategy against tumor metastasis through the targeting of PGC-1α and, thus, the suppression of lactate-feeding OXPHOS in normoxic CSCs may improve the therapeutic benefit of patients with cancer, particularly CRC.