Human Colorectal Cancer Patients Research Articles

Squalene epoxidase promotes the chemoresistance of colorectal cancer via (S)-2,3-epoxysqualene-activated NF-κB

BackgroundWhile de novo cholesterol biosynthesis plays a crucial role in chemotherapy resistance of colorectal cancer (CRC), the underlying molecular mechanism remains poorly understood.MethodsWe conducted cell proliferation assays on CRC cells with or without depletion of squalene epoxidase (SQLE), with or without 5-fluorouracil (5-FU) treatment. Additionally, a xenograft mouse model was utilized to explore the impact of SQLE on the chemosensitivity of CRC to 5-FU. RNA-sequencing analysis and immunoblotting analysis were performed to clarify the mechanism. We further explore the effect of SQLE depletion on the ubiquitin of NF-κB inhibitor alpha (IκBα) and (S)-2,3-epoxysqualene on the binding of IκBα to beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC) by using immunoprecipitation assay. In addition, a cohort of 272 CRC patients were selected for our clinical analyses.ResultsMechanistically, (S)-2,3-epoxysqualene promotes IκBα degradation and subsequent NF-κB activation by enhancing the interaction between BTRC and IκBα. Activated NF-κB upregulates the expression of baculoviral IAP repeat containing 3 (BIRC3), sustains tumor cell survival after 5-FU treatment and promotes 5-FU resistance of CRC in vivo. Notably, the treatment of terbinafine, an inhibitor of SQLE commonly used as antifungal drug in clinic, enhances the sensitivity of CRC to 5-FU in vivo. Additionally, the expression of SQLE is associated with the prognosis of human CRC patients with 5-FU-based chemotherapy.ConclusionsThus, our finding not only demonstrates a new role of SQLE in chemoresistance of CRC, but also reveals a novel mechanism of (S)-2,3-epoxysqualene-dependent NF-κB activation, implicating the combined potential of terbinafine for 5-FU-based CRC treatment.

Engineering of an EPHA2-Targeted Monobody for the Detection of Colorectal Cancer.

Colorectal cancer (CRC) is the third most common cancer worldwide, and is second only to lung cancer with respect to cancer-related deaths. Noninvasive molecular imaging using established markers is a new emerging method to diagnose CRC. The human ephrin receptor family type-A 2 (hEPHA2) oncoprotein is overexpressed at the early, but not late, stages of CRC. Previously, we reported development of an E1 monobody that is specific for hEPHA2-expressing cancer cells both in vitro and in vivo. Herein, we investigated the ability of the E1 monobody to detect hEPHA2 expressing colorectal tumors in a mouse model, as well as in CRC tissue. The expression of hEPHA2 on the surface of CRC cells was analyzed by western blotting and flow cytometry. The targeting efficacy of the E1 monobody for CRC cells was examined by flow cytometry, and immunofluorescence staining. E1 conjugated to the Renilla luciferase variant 8 (Rluc8) reporter protein was used for in vivo imaging in mice. Additionally, an enhanced green fluorescence protein (EGFP) conjugated E1 monobody was used to check the ability of the E1 monobody to target CRC tissue. The E1 monobody bound efficiently to hEPHA2-expressing CRC cell lines, and E1 conjugated to the Rluc8 reporter protein targeted tumor tissues in mice transplanted with HCT116 CRC tumor cells. Finally, E1-EGFP stained tumor tissues from human CRC patients, showing a pattern similar to that of an anti-hEPHA2 antibody. The E1 monobody has utility as an EPHA2 targeting agent for the detection of CRC.

Abstract 287: Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of colorectal cancer

Abstract A new subset of cancer associated fibroblasts (CAFs) called antigen presenting CAF (apCAF) was recently identified, and modulation of the tumor immunity might be associated with tumor progression. We examined the markers of apCAF, such as IRF5, HLA-DRA and HLA-DQA1 by bioinformatics analyses of human colorectal cancer (CRC) datasets. The expression of apCAF markers were upregulated in CAFs isolated from human CRCs and liver metastases compared with their normal counterparts. Kaplan-Meir analysis of 172 human CRC samples clarified that the patients with apCAF marker positive expression in the tumor stroma had worse prognosis than that with negative expression. In addition, the origin of apCAFs was reported to be mesothelial cells, therefore, we established cancer-associated mesothelial cells (CAmes) from ascites of human CRC patients with peritoneal dissemination. Consistently, the expression of antigen-presentation markers was highly upregulated in CAmes, however, allo-mixed lymphocyte reaction analysis demonstrated that their stimulating activity of CD4+ T lymphocytes was diminished. In an in vivo study, we applied a rectal orthotopic transplant model with mouse tumor organoid (MTO) to investigate the effect of apCAF on tumor progressions. Using fluorescence-activated cell sorting (FACS), we examined the ratios of apCAF, myCAF, and iCAF in orthotopic rectal tumors. The percentage of apCAF was highest in the 6-week-tumors and the percentage of myCAF was highest in the 9-week-tumors. Interestingly, the percentage of apCAF was higher in liver metastases than in primary tumor, consistent with bioinformatics analysis. The apCAF increased during the stage of tumor growth, and the percentage of myCAF increase in the later stage of tumor progression with fibrosis. In summary, apCAF might be associated with tumor progression in CRC. Citation Format: Yasuhiro Fukui, Hiroaki Kasashima, Zizhou Wang, Ken Yonemitsu, Kishu Kitayama, Yuichiro Miki, Mami Yoshii, Tatsunari Fukuoka, Tatsuro Tamura, Masatsune Shibutani, Takahiro Toyokawa, Hiroaki Tanaka, Shigeru Lee, Masakazu Yashiro, Kiyoshi Maeda. Analysis for the role of antigen-presenting cancer-associated fibroblasts in tumor microenvironment of 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 287.

Cancer-derived exosomal Alu RNA promotes colorectal cancer progression

Inflammation plays a crucial role in cancer progression, but the relevance of the inflammasome remains unclear. Alu RNA was the first endogenous nucleic acid shown to activate the NLRP3 (nucleotide-binding domain leucine-rich repeat containing 3) inflammasome. Here, we showed that Alu RNA can induce epithelial-to-mesenchymal transition (EMT) through NLRP3 inflammasome activation and IL-1β release in colorectal cancer (CRC) cells. Alu RNA is stored, transported and transferred to CRC cells by exosomes. Exosomal Alu RNA promotes tumorigenesis by inducing invasion, metastasis and EMT via NLRP3 inflammasome activation. Consistent with these data, we found that significantly increased Alu RNA expression correlates with the induction of NLRP3 priming in human CRC patients. Furthermore, the level of Alu RNA in circulating exosomes correlates with CRC progression in a preclinical model. These findings reveal the direct involvement of Alu RNA in cancer pathogenesis, and its presence in CRC cell-derived exosomes could be used as a noninvasive diagnostic biomarker.

The microbial genotoxin colibactin exacerbates mismatch repair mutations in colorectal tumors

Certain Enterobacteriaceae strains contain a 54-kb biosynthetic gene cluster referred to as “pks” encoding the biosynthesis of a secondary metabolite, colibactin. Colibactin-producing E. coli promote colorectal cancer (CRC) in preclinical models, and in vitro induce a specific mutational signature that is also detected in human CRC genomes. Yet, how colibactin exposure affects the mutational landscape of CRC in vivo remains unclear. Here we show that colibactin-producing E. coli-driven colonic tumors in mice have a significantly higher SBS burden and a larger percentage of these mutations can be attributed to a signature associated with mismatch repair deficiency (MMRd; SBS15), compared to tumors developed in the presence of colibactin-deficient E. coli. We found that the synthetic colibactin 742 but not an inactive analog 746 causes DNA damage and induces transcriptional activation of p53 and senescence signaling pathways in non-transformed human colonic epithelial cells. In MMRd colon cancer cells (HCT 116), chronic exposure to 742 resulted in the upregulation of BRCA1, Fanconi anemia, and MMR signaling pathways as revealed by global transcriptomic analysis. This was accompanied by increased T>N single-base substitutions (SBS) attributed to the proposed pks+E. coli signature (SBS88), reactive oxygen species (SBS17), and mismatch-repair deficiency (SBS44). A significant co-occurrence between MMRd SBS44 and pks-associated SBS88 signature was observed in a large cohort of human CRC patients (n=2,945), and significantly more SBS44 mutations were found when SBS88 was also detected. Collectively, these findings reveal the host response mechanisms underlying colibactin genotoxic activity and suggest that colibactin may exacerbate MMRd-associated mutations.

Abstract 6525: Colon cancer screening and treatment with engineered probiotics

Abstract Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment strategies. Here, we demonstrate selective colonization of CRC tumors with the orally-delivered probiotic E. coli Nissle 1917 (EcN) in genetically-engineered and orthotopic murine models of CRC, and human CRC patients. We subsequently engineer EcN to produce screening and therapeutic molecules, resulting in non-invasive detection of precursor CRC lesions and reduction of tumor burden through local induction of robust anti-tumor immunity. Citation Format: Candice R. Gurbatri, Georgette Radford, Jongwon Im, Courtney Coker, Nicholas Arpaia, Susan L. Woods, Daniel L. Worthley, Tal Danino. Colon cancer screening and treatment with engineered probiotics [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 6525.

Abstract 465: The preclinical characterization of TST003, a first-in-class mAb targeting gremlin-1 overexpressed by cancer-associated fibroblasts and tumor cells

Abstract Cancer associated fibroblast (CAF) is a rich source of factors for immune suppression and promotes cancer progression and metastasis via epithelial to mesenchymal transition (EMT). It is well known that tumors with mesenchymal phenotype have significantly poor prognosis and reduced response to checkpoint inhibitors. Targeting CAF becomes an increasingly attractive cancer therapeutic approach. Gremlin-1 is a member of the TGFβ superfamily, expressed by CAFs and tumor cells, and known to play a key role in EMT transition, cancer cell proliferation and stroma maintenance. Gremlin-1 overexpression in CAF or tumor cells often correlates with poor clinical prognosis in multiple cancers including prostate, pancreatic, gastric cancer etc. Here we describe the characterization of TST003 (14E3), a novel humanized IgG1 monoclonal antibody targeting Gremlin-1. TST003 bound to human Gremlin-1 with high affinity and high selectivity, and blocked Gremlin-1 binding to BMP2/4 with an EC50 of 3.68 nM to BMP2 and 4.53 nM to BMP4 respectively. TST003 could dose-dependently reverse Gremlin-1 inhibition of the BMP4-mediated Smad1/5/9 phosphorylation in tumor cells as measured by Western blot. In an ex vivo assay using prostate cancer patient-derived organoids (PDOs), TST003 inhibited the growth of 9 out of 12 human PDOs significantly. TST003 also demonstrated its single agent tumor growth inhibition (TGI>50%) at 10 mg/kg in a human prostate cancer PC3 xenograft model on castrated male nude mice and a human colorectal cancer patient derived xenograft (PDX) mouse model in the presence of human PBMC. Both tumor models expressed Gremlin-1 as detected by IHC analysis. In addition, a significantly better anti-tumor activity was observed when TST003 combined with an anti-VEGFR2 antibody in this CRC PDX (MSS, Kras G12D, PD-L1 negative) model. The safety profiles of TST003 were characterized in the single and repeated dose toxicology studies integrated with safety pharmacology, local tolerance, and in vitro hemolysis study, TCR study, and cytokine release study. TST003 was well tolerated in cynomolgus monkeys without noteworthy abnormalities following a single dose or repeated doses. In summary, TST003 is a first-in-class therapeutic mAb targeting Gremlin-1 overexpressed by CAFs and tumor cells. Our preclinical characterization results provided the rationale for on-going clinical evaluation of TST003 in patients with advanced solid tumors with high unmet medical need either as monotherapy or in combination with SoC. Citation Format: Di Sun, Huanhuan Guo, Chaping Cheng, Hongjun Li, Xinlai Yao, Shuang Lu, Jie Ding, Shenjie Zhang, Shijie Zhou, Steven Yu, Yi Gu, Xueming Qian. The preclinical characterization of TST003, a first-in-class mAb targeting gremlin-1 overexpressed by cancer-associated fibroblasts and tumor cells [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 465.

Peptidomic changes in human serous colorectal cancer patients.

Colorectal cancer (CRC) is the third most common cancer worldwide and one of the leading causes of cancer-related death. Peptidomics, considered a novel branch of proteomics, has an increasing range of applications in the screening, diagnosis, prognosis, and even monitoring of cancer. However, there is little information for peptidomics analysis in CRC. In this study, a comparative peptidomic profiling was analyzed in 3 CRC tissue samples and 3 adjacent intestinal epithelial tissue samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among the total 133 nonredundant peptides identified, 59 were significantly differentially expressed in the CRC samples and benign colonic epithelium conditions [fold change (FC) >2, P<0.05]. Totals of 25 up-regulated and 34 down-regulated peptides were detected, respectively. Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were applied to predict the possible functions of these relevant precursor proteins. To clearly identify the potential interaction network of peptide precursors, the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) was used to determine protein interactions and a possible central role in CRC. Our results for the first time revealed the differentially expressed peptides between the serous CRC tissue and the adjacent intestinal epithelial tissue samples, and these prominently variable peptides might have an important potential role in occurrence and development of CRC.

5-Fluorouracil Suppresses Colon Tumor through Activating the p53-Fas Pathway to Sensitize Myeloid-Derived Suppressor Cells to FasL+ Cytotoxic T Lymphocyte Cytotoxicity.

Myelosuppression is a major adverse effect of 5-fluorouracil (5-FU) chemotherapy. However, recent findings indicate that 5-FU selectively suppresses myeloid-derived suppressor cells (MDSCs), to enhance antitumor immunity in tumor-bearing mice. 5-FU-mediated myelosuppression may thus have a beneficial effect for cancer patients. The molecular mechanism underlying 5-FU's suppression of MDSCs is currently unknown. We aimed at testing the hypothesis that 5-FU suppresses MDSCs through enhancing MDSC sensitivity to Fas-mediated apoptosis. We observed that, although FasL is highly expressed in T cells, Fas is weakly expressed in myeloid cells in human colon carcinoma, indicating that downregulation of Fas is a mechanism underlying myeloid cell survival and accumulation in human colon cancer. 5-FU treatment upregulated expression of both p53 and Fas, and knocking down p53 diminished 5-FU-induced Fas expression in MDSC-like cells, in vitro. 5-FU treatment also increased MDSC-like cell sensitivity to FasL-induced apoptosis in vitro. Furthermore, we determined that 5-FU therapy increased expression of Fas on MDSCs, suppressed MDSC accumulation, and increased CTL tumor infiltration in colon tumor-bearing mice. In human colorectal cancer patients, 5-FU chemotherapy decreased MDSC accumulation and increased CTL level. Our findings determine that 5-FU chemotherapy activates the p53-Fas pathway, to suppress MDSC accumulation, to increase CTL tumor infiltration.

Sphingosine 1-phosphate lyase facilitates cancer progression through converting sphingolipids to glycerophospholipids.

BackgroundIn addition to potent agonist properties for sphingosine 1‐phosphate (S1P) receptors, intracellularly, S1P is an intermediate in metabolic conversion pathway from sphingolipids to glycerolysophospholipids (glyceroLPLs). We hypothesized that this S1P metabolism and its products might possess some novel roles in the pathogenesis of cancer, where S1P lyase (SPL) is a key enzyme.MethodsThe mRNA levels of sphingolipid‐related and other cancer‐related factors were measured in human hepatocellular carcinoma (HCC), colorectal cancer, and esophageal cancer patients’ tumours and in their adjacent non‐tumour tissues. Phospholipids (PL) and glyceroLPLs were measured by using liquid chromatography‐tandem mass spectrometry (LC‐MS/MS). In‐vitro experiments were performed in Colon 26 cell line with modulation of the SPL and GPR55 expressions. Xenograft model was used for determination of the cancer progression and for pharmacological influence.ResultsBesides high SPL levels in human HCC and colon cancer, SPL levels were specifically and positively linked with levels of glyceroLPLs, including lysophosphatidylinositol (LPI). Overexpression of SPL in Colon 26 cells resulted in elevated levels of LPI and lysophosphatidylglycerol (LPG), which are agonists of GPR55. SPL overexpression‐enhanced cell proliferation was inhibited by GPR55 silencing. Conversely, inhibition of SPL led to the opposite outcome and reversed by adding LPI, LPG, and metabolites generated during S1P degradation, which is regulated by SPL. The xenograft model results suggested the contribution of SPL and glyceroLPLs to tumour progression depending on levels of SPL and GPR55. Moreover, the pharmacological inhibition of SPL prevented the progression of cancer. The underlying mechanisms for the SPL‐mediated cancer progression are the activation of p38 and mitochondrial function through the LPI, LPG‐GPR55 axis and the suppression of autophagy in a GPR55‐independent manner.ConclusionA new metabolic pathway has been proposed here in HCC and colon cancer, SPL converts S1P to glyceroLPLs, mainly to LPI and LPG, and facilitates cancer development.

JNK pathway plays a critical role for expansion of human colorectal cancer in the context of BRG1 suppression.

Tumor stem cells (TSCs), capable of self‐renewal and continuous production of progeny cells, could be potential therapeutic targets. We have recently reported that chromatin remodeling regulator Brg1 is required for maintenance of murine intestinal TSCs and stemness feature of human colorectal cancer (CRC) cells by inhibiting apoptosis. However, it is still unclear how BRG1 suppression changes the underlying intracellular mechanisms of human CRC cells. We found that Brg1 suppression resulted in upregulation of the JNK signaling pathway in human CRC cells and murine intestinal TSCs. Simultaneous suppression of BRG1 and the JNK pathway, either by pharmacological inhibition or silencing of c‐JUN, resulted in even stronger inhibition of the expansion of human CRC cells compared to Brg1 suppression alone. Consistently, high c‐JUN expression correlated with worse prognosis for survival in human CRC patients with low BRG1 expression. Therefore, the JNK pathway plays a critical role for expansion and stemness of human CRC cells in the context of BRG1 suppression, and thus a combined blockade of BRG1 and the JNK pathway could be a novel therapeutic approach against human CRC.

TRF2 cooperates with CTCF for controlling the oncomiR-193b-3p in colorectal cancer

The Telomeric Repeat binding Factor 2 (TRF2), a key protein involved in telomere integrity, is over-expressed in several human cancers and promotes tumor formation and progression. Recently, TRF2 has been also found outside telomeres where it can affect gene expression. Here we provide evidence that TRF2 is able to modulate the expression of microRNAs (miRNAs), small non-coding RNAs altered in human tumors. Among the miRNAs regulated by TRF2, we focused on miR-193b-3p, an oncomiRNA that positively correlates with TRF2 expression in human colorectal cancer patients from The Cancer Genome Atlas dataset. At the mechanistic level, the control of miR-193b-3p expression requires the cooperative activity between TRF2 and the chromatin organization factor CTCF. We found that CTCF physically interacts with TRF2, thus driving the proper positioning of TRF2 on a binding site located upstream the miR-193b-3p host-gene. The binding of TRF2 on the identified region is necessary for promoting the expression of miR-193b3p which, in turn, inhibits the translation of the onco-suppressive methyltransferase SUV39H1 and promotes tumor cell proliferation. The translational relevance of the oncogenic properties of miR-193b-3p was confirmed in patients, in whom the association between TRF2 and miR-193b-3p has a prognostic value.

Restoring FAS Expression via Lipid-Encapsulated FAS DNA Nanoparticle Delivery Is Sufficient to Suppress Colon Tumor Growth In Vivo.

Simple SummaryA key feature of human colorectal tumor is loss of FAS expression. FAS is the death receptor for FASL of activated T cells. Loss of FAS expression therefore may promote tumor cell immune escape. We aimed at determining whether restoring FAS expression is sufficient to suppress colorectal tumor growth. Mouse and human FAS cDNA was synthesized and encapsulated into cationic lipid nanoparticle DOTAP-Cholesterol to formulate DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Restoring FAS expression in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS+ tumor cells in vitro and suppressed colon-tumor growth and progression in tumor-bearing mice in vivo. Restoring FAS expression induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells in vitro. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. Tumor-selective delivery of FAS DNA nanoparticle is potentially an effective therapy for human colorectal cancer.A hallmark of human colorectal cancer is lost expression of FAS, the death receptor for FASL of cytotoxic T lymphocytes (CTLs). However, it is unknown whether restoring FAS expression alone is sufficient to suppress csolorectal-cancer development. The FAS promoter is hypermethylated and inversely correlated with FAS mRNA level in human colorectal carcinomas. Analysis of single-cell RNA-Seq datasets revealed that FAS is highly expressed in epithelial cells and immune cells but down-regulated in colon-tumor cells in human colorectal-cancer patients. Codon usage-optimized mouse and human FAS cDNA was designed, synthesized, and encapsulated into cationic lipid to formulate nanoparticle DOTAP-Chol-mFAS and DOTAP-Chol-hFAS, respectively. Overexpression of codon usage-optimized FAS in metastatic mouse colon-tumor cells enabled FASL-induced elimination of FAS+ tumor cells in vitro, suppressed colon tumor growth, and increased the survival of tumor-bearing mice in vivo. Overexpression of codon-optimized FAS-induced FAS receptor auto-oligomerization and tumor cell auto-apoptosis in metastatic human colon-tumor cells. DOTAP-Chol-hFAS therapy is also sufficient to suppress metastatic human colon tumor xenograft growth in athymic mice. DOTAP-Chol-mFAS therapy exhibited no significant liver toxicity. Our data determined that tumor-selective delivery of FAS DNA nanoparticles is sufficient for suppression of human colon tumor growth in vivo.

HOXC10 Promotes Metastasis in Colorectal Cancer by Recruiting Myeloid-derived Suppressor Cells.

Background: Since metastasis is the primary cause of death in human colorectal cancer (CRC) patients, the exact mechanism underlying CRC metastasis remains unclear. Here, we provide evidence for a unique function of HomeoboxC10 (HOXC10) in driving CRC metastasis, as well as treatment options for these subpopulation patients.Methods: Immunohistochemistry detected the expression of HOXC10 in the human CRC cohort. The function of HOXC10 in CRC metastasis was investigated using the cecum orthotopic model.Results: In CRC patients, elevated expression of HOXC10 expression was linked to lymph node metastases, distant metastasis, worse tumor differentiation, higher AJCC stage, and poor prognosis. HOXC10 is also an independent predictive predictor for CRC patients (P<0.001). HOXC10 overexpression increased the metastasis ability of MC38 cells and promoted the infiltration of MDSCs by upregulating CXCL5 at the same time. The CXCR2 inhibitor can reduce the rate of metastasis in MC38 cells by reducing MDSCs infiltration. SB225002, a CXCR2 inhibitor, and anti-programmed death-ligand 1 (anti-PD-L1) can significantly prevent CRC metastasis.Conclusions: HOXC10 overexpression upregulated CXCL5, which promoted MDSCs infiltration. Interrupting this loop might be a potential therapy option for HOXC10-induced CRC metastasis.

A Novel Mechanism of Endoplasmic Reticulum Stress- and c-Myc-Degradation-Mediated Therapeutic Benefits of Antineurokinin-1 Receptor Drugs in Colorectal Cancer.

The neurokinin‐1 receptor (NK‐1R) antagonists are approved as treatment for chemotherapy‐associated nausea and vomiting in cancer patients. The emerging role of the substance P‐NK‐1R system in oncogenesis raises the possibility of repurposing well‐tolerated NK‐1R antagonists for cancer treatment. This study reports that human colorectal cancer (CRC) patients with high NK‐1R expression have poor survival, and NK‐1R antagonists SR140333 and aprepitant induce apoptotic cell death in CRC cells and inhibit CRC xenograft growth. This cytotoxicity induced by treatment with NK‐1R antagonists is mediated by induction of endoplasmic reticulum (ER) stress. ER stress triggers calcium release, resulting in the suppression of prosurvival extracellular signal‐regulated kinase (ERK)‐c‐Myc signaling. Along with ER calcium release, one ER stress pathway mediated by protein kinase RNA‐like ER kinase (PERK) is specifically activated, leading to increased expression of proapoptotic C/EBP‐homologous protein (CHOP). Moreover, NK‐1R antagonists enhance the efficacy of chemotherapy by increasing the sensitivity and overcoming resistance to 5‐fluorouracil in CRC cells through the induction of sustained ER stress and the consequent suppression of ERK‐c‐Myc signaling both in vitro and in vivo. Collectively, the findings provide novel mechanistic insights into the efficacy of NK‐1R antagonists either as a single agent or in combination with chemotherapy for cancer treatment.

Brg1 is required to maintain colorectal cancer stem cells.

Tumor cells capable of self-renewal and continuous production of progeny cells are called tumor stem cells (TSCs) and are considered to be potential therapeutic targets. However, the mechanisms underlying the survival and function of TSCs are not fully understood. We previously reported that chromatin remodeling regulator Brg1 is essential for intestinal stem cells in mice and Dclk1 is an intestinal TSC marker. In this study, we investigated the role of Brg1 in Dclk1+ intestinal tumor cells for the maintenance of intestinal tumors in mice. Specific ablation of Brg1 in Dclk1+ intestinal tumor cells reduced intestinal tumors in ApcMin mice, and continuous ablation of Brg1 maintained the reduction of intestinal tumors. Lineage tracing in the context of Brg1 ablation in Dclk1+ intestinal tumor cells revealed that Brg1-null Dclk1+ intestinal tumor cells did not give rise to their descendent tumor cells, indicating that Brg1 is essential for the self-renewal of Dclk1+ intestinal tumor cells. Five days after Brg1 ablation, we observed increased apoptosis in Dclk1+ tumor cells. Furthermore, Brg1 was crucial for the stemness of intestinal tumor cells in a spheroid culture system. BRG1 knockdown also impaired cell proliferation and increased apoptosis in human colorectal cancer (CRC) cells. Microarray analysis revealed that apoptosis-related genes were upregulated and stem cell-related genes were downregulated in human CRC cells by BRG1 suppression. Consistently, high BRG1 expression correlated with poor disease-specific survival in human CRC patients. These data indicate that Brg1 plays a crucial role in intestinal TSCs in mice by inhibiting apoptosis and is critical for cell survival and stem cell features in human CRC cells. Thus, BRG1 represents a new therapeutic target for human CRC. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Antitumor Activities of Immune Costimulatory Molecules of TNF Superfamily in Colorectal and Lung Cancers

Cancer immunotherapy has emerged as a promising treatment that utilizes the innate or adaptive immunity to generate a robust killing of malignant cells. However, only a limited number of cancer patients showed responsiveness to immunotherapies such as checkpoint inhibitors, suggesting the need for potent alternative strategies. In the present study, we explored the therapeutic potentials of costimulatory molecules including TNF superfamily member 4 (TNFSF4), member 9 (TNFSF9), and member 18 (TNFSF18). In tumor samples from human colorectal and lung cancer patients, expression of these factors positively correlated with lymphocyte infiltration and expression of several immune effector genes. In syngeneic mouse tumor models, overexpression of the TNF superfamily costimulatory factors in murine colorectal or lung cancer cells significantly suppressed tumor progression. Especially, TNFSF9 and 18 showed stronger antitumor effects than TNFSF4. Together, our study demonstrated the great potential of cancer immunotherapy targeting these immune costimulatory molecules.

FBXL6 degrades phosphorylated p53 to promote tumor growth.

The ubiquitin-proteasome system regulates many distinct biological processes. Its dysregulation causes various diseases, including but not limited to cancer. In this study, based on the analysis of gene expression in several colorectal cancer (CRC) datasets, we show that FBXL6, a poorly-characterized F-box protein, is amplified, over-expressed, and highly correlated with poor prognosis in human CRC patients. Mechanistically, FBXL6 targets phospho-p53 (S315) to mediate its polyubiquitination and proteasomal degradation, thereby inhibiting p53 signaling. FBXL6 depletion inhibits proliferation of p53 wild-type (WT) CRC cells by inducing cell cycle arrest and apoptosis. Furthermore, p53 transcriptionally suppresses FBXL6 expression by binding its core promoter region. Taken together, these results identify the feed-forward loop of FBXL6-p53 as a potential therapeutic target for CRC treatments.

The lipid peroxidation product EKODE exacerbates colonic inflammation and colon tumorigenesis

Oxidative stress is emerging as an important contributor to the pathogenesis of colorectal cancer (CRC), however, the molecular mechanisms by which the disturbed redox balance regulates CRC development remain undefined. Using a liquid chromatography–tandem mass spectrometry-based lipidomics, we found that epoxyketooctadecenoic acid (EKODE), which is a lipid peroxidation product, was among the most dramatically increased lipid molecules in the colon of azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced CRC mice. This is, at least in part, due to increased oxidative stress in colon tumors, as assessed by analyzing gene expression of oxidative markers in AOM/DSS-induced CRC mice and human CRC patients in the Cancer Genome Atlas (TCGA) database. Systemic, short-time treatment with low-dose EKODE increased the severity of DSS-induced colitis, caused intestinal barrier dysfunction and enhanced lipopolysaccharide (LPS)/bacterial translocation, and exacerbates the development of AOM/DSS-induced CRC in mice. Furthermore, treatment with EKODE, at nM doses, induced inflammatory responses via JNK-dependent mechanisms in both colon cancer cells and macrophage cells. Overall, these results demonstrate that the lipid peroxidation product EKODE is an important mediator of colonic inflammation and colon tumorigenesis, providing a novel mechanistic linkage between oxidative stress and CRC development.

Autocrine IL6 activates the STAT3-DNMT axis to silence the TNFa-RIP1 necroptosis pathway to sustain myeloid-derived suppressor cell survival and accumulation

Abstract Accumulation of myeloid-derived suppressor cells (MDSCs) is a hallmark of cancer. However, the underlying mechanism of MDSC accumulation in the tumor microenvironment (TME) remain incompletely understood. We report that MDSC accumulation is regulated by the TNFα-RIP1-mediated necroptosis. We determined that inhibition of DNMTs with Decitabine (DAC) abolished MDSC accumulation and increased activation of antigen-specific cytotoxic T lymphocytes (CTLs) in tumor-bearing mice. DAC-induced decrease of MDSC accumulation is correlated with increased IRF8 expression in MDSCs. However, DAC also abolished MDSC-like cell accumulation in IRF8 KO mice, indicating that DNA methylation does not regulate MDSC lineage differentiation but mediates MDSC accumulation at post differentiation stage. We determined that DAC decreased MDSC accumulation through increasing cell death and identified RIP1-dependent necroptosis as target of DNA methylation in MDSCs. Genome-wide DNA bisulfite sequencing revealed that the Tnf promoter is hypermethylated in tumor-induced MDSCs in vivo. Consequently, DAC dramatically increased TNFα level in MDSCs and neutralizing TNFα significantly decreased MDSC cell death. Furthermore, recombinant TNFα induced MDSC cell death in a does- and RIP1-dependent manner. IL6 which is expressed in MDSCs in tumor-bearing mice and human colorectal cancer patients. Our data shows that the autocrine IL6 activates the STAT3-DNMT axis to epigenetically silence the TNFα-RIP1 necroptosis pathway to sustain MDSC survival and accumulation in cancer. Targeting the TNFα-RIP1 necroptosis is potentially an effective approach to supress MDSCs to activate tumor-reactive CTLs in the TME.