Colorectal Cancer Tumor Growth Research Articles

ATP6V0A1-dependent cholesterol absorption in colorectal cancer cells triggers immunosuppressive signaling to inactivate memory CD8+ T cells

Obesity shapes anti-tumor immunity through lipid metabolism; however, the mechanisms underlying how colorectal cancer (CRC) cells utilize lipids to suppress anti-tumor immunity remain unclear. Here, we show that tumor cell-intrinsic ATP6V0A1 drives exogenous cholesterol-induced immunosuppression in CRC. ATP6V0A1 facilitates cholesterol absorption in CRC cells through RAB guanine nucleotide exchange factor 1 (RABGEF1)-dependent endosome maturation, leading to cholesterol accumulation within the endoplasmic reticulum and elevated production of 24-hydroxycholesterol (24-OHC). ATP6V0A1-induced 24-OHC upregulates TGF-β1 by activating the liver X receptor (LXR) signaling. Subsequently, the release of TGF-β1 into the tumor microenvironment by CRC cells activates the SMAD3 pathway in memory CD8+ T cells, ultimately suppressing their anti-tumor activities. Moreover, we identify daclatasvir, a clinically used anti-hepatitis C virus (HCV) drug, as an ATP6V0A1 inhibitor that can effectively enhance the memory CD8+ T cell activity and suppress tumor growth in CRC. These findings shed light on the potential for ATP6V0A1-targeted immunotherapy in CRC.

TRIP13 Activates Glycolysis to Promote Cell Stemness and Strengthen Doxorubicin Resistance of Colorectal Cancer Cells.

Chemotherapy resistance is one of the main causes of clinical chemotherapy failure. Current cancer research explores the drug resistance mechanism and new therapeutic targets. This work aims to elucidate the mechanism of thyroid hormone receptor interactor 13 (TRIP13) affecting doxorubicin (DOX) resistance in colorectal cancer (CRC). Bioinformatics analyses were employed to clarify TRIP13 expression in CRC tissues and predict the correlation of the TRIP13 enrichment pathway with glycolysis-related genes and stemness index mRNAsi. Quantitative real-time polymerase chain reaction and western blot were adopted to analyze the expression of TRIP13 and glycolysis- related genes. Cell Counting Kit-8 was utilized to determine the cell viability and IC50 value. Western blot was employed to measure the expression of stemness-related factors. Cell function assays were performed to detect cells' sphere-forming ability and glycolysis level. Animal models were constructed to determine the effects of TRIP13 expression on CRC tumor growth. TRIP13 was significantly overexpressed in CRC, concentrated in the glycolysis signaling pathway, and positively correlated with stemness index mRNAsi. High expression of TRIP13 facilitated DOX resistance in CRC. Further mechanistic studies revealed that overexpression of TRIP13 could promote cell stemness through glycolysis, which was also confirmed in animal experiments. TRIP13 was highly expressed in CRC, which enhanced the DOX resistance of CRC cells by activating glycolysis to promote cell stemness. These findings offer new insights into the pathogenesis of DOX resistance in CRC and suggest that TRIP13 may be a new target for reversing DOX resistance in CRC.

Death receptors 4/5 mediate tumour sensitivity to natural killer cell-mediated cytotoxicity in mismatch repair deficient colorectal cancer.

Identifying the target of natural killer (NK) cells in colorectal cancer (CRC) is critical for optimising the clinical use of NK cell-mediated immunotherapy. Mismatch repair deficiency (dMMR) is associated with high immune cell infiltration and MHC Class I defects. Whether dMMR CRC responses to NK cell therapy remains unclear. MLH1, DR4, and DR5 knockout cell lines were established using CRISPR-Cas9 system. NK92-MI or NK cell isolated from BABL/C mice were used as effector cells against tumour cells. Inflammatory cytokines secretion by CRC cells was assessed via cytokine analysis. NK-cell-deficient/proficient animal models were used to validate the NK cell sensitivity. We observed that dMMR CRC cells were more sensitive to NK cell-mediated cytotoxicity than were mismatch-repair-proficient (pMMR) CRC cells. In dMMR CRC, Death receptor (DR)4/5 was upregulated and mediated sensitivity to NK cell-mediated cytotoxicity. DR4/5-mediated secretion of interleukin -12 sustained NK cell viability in dMMR CRC. NK cell depletion induced dMMR CRC tumour growth, and NK cell transfer inhibited lung metastasis of dMMR CRC with DR4/5 expression in vivo. TP53 upregulated DR4/DR5 expression in dMMR CRC. dMMR associated with increased sensitivity to NK cell-mediated cytotoxicity in CRC. DR4/DR5 sensitise dMMR CRC to NK cell-mediated cytotoxicity.

METTL5 promotes cell proliferation, invasion, and migration by up-regulating Toll-like receptor 8 expression in colorectal cancer.

N6-methyladenosine (m6A) modification represents the predominant alteration found in eukaryotic messenger RNA and plays a crucial role in the progression of various tumors. However, despite its significance, the comprehensive investigation of METTL5, a key m6A methyltransferase, in colorectal cancer (CRC) remains limited. To investigate the role of METTL5 in CRC. We assessed METTL5 expression levels in clinical samples obtained from CRC patients as well as in CRC cell lines. To elucidate the downstream targets of METTL5, we performed RNA-sequencing analysis coupled with correlation analysis, leading us to identify Toll-like receptor 8 (TLR8) as a potential downstream target. In vitro functional assessments of METTL5 and TLR8 were conducted using CCK-8 assays, scratch assays, as well as assays measuring cell migration and invasion. Our findings reveal a pronounced upregulation of METTL5 expression in both CRC cells and tissues, which correlated significantly with an unfavorable prognosis. In vitro experiments unequivocally demonstrated the oncogenic role of METTL5, as evidenced by its promotion of CRC cell proliferation, invasion, and migration. Notably, we identified TLR8 as a downstream target of METTL5, and subsequent down-regulation of TLR8 led to a significant inhibition of CRC cell proliferation, invasion, and tumor growth. The heightened expression of METTL5 in CRC is strongly associated with clinicopathological features and a poor prognosis, thereby underscoring its potential utility as a critical marker for facilitating early diagnosis and prognostication in CRC.

Isthmin-1 promotes growth and progression of colorectal cancer through the interaction with EGFR and YBX-1

While previous studies have indicated the involvement of Isthmin 1 (ISM1), a secreted protein, in cancer development, the precise mechanisms have remained elusive. In this study, we unveiled that ISM1 is significantly overexpressed in both the blood and tissue samples of colorectal cancer (CRC) patients, correlating with their poor prognosis. Functional experiments demonstrated that enforced ISM1 expression significantly enhances CRC proliferation, migration, invasion and tumor growth. Notably, our investigation reveals an interaction of ISM1 with epidermal growth factor receptor (EGFR), a member of the receptor tyrosine kinase (RTK) family of CRC cells. The binding of ISM1 triggered EGFR activation and initiate downstream signaling pathways. Meanwhile, intracellular ISM1 interacted with Y-box binding protein 1 (YBX1), enhancing its transcriptional regulation on EGFR. Furthermore, our research uncovered the regulation of ISM1 expression by the hypoxia-inducible transcription factor HIF-1α in CRC cells. Mechanistically, we identified HIF-1α as a direct regulator of ISM1, binding to a hypoxia response element on its promoter. This novel mechanism illuminated potential therapeutic targets, offering insights into restraining HIF-1α/ISM1/EGFR-driven CRC progression and metastasis.

Abstract 5937: KS20226, a first-in-class IRP2 inhibitor, preferentially induces reprogramming of iron metabolism and suppresses tumor growth in colorectal cancer

Abstract Background: Iron homeostasis plays a role in redox activity, mitochondrial function, and cell cycle regulation. Specifically, the dysregulation of iron metabolism is implicated in malignant transformation and cancer progression. Iron regulatory protein 2 (IRP2) is crucial for maintaining balance within iron levels and primarily controls genes associated with iron utilization. Our discovery of KS20226, a first-in-class IRP2 inhibitor, reveals remarkable potential as an effective anti-cancer drug candidate. We have elucidated the mechanisms underlying the therapeutic effects of KS20226, thereby highlighting the feasibility of targeting IRP2 in colorectal cancer (CRC). Methods: The proliferation inhibitory activity of KS20226 was assessed the CCK-8 and 3D Spheroid models. To investigate mitochondrial function, we conducted measurement of oxidative phosphorylation (OCR) and glycoPER. Furthermore, gene expression analysis was performed through RNA sequencing (RNA-seq) using the Illumina HiSeq 2500 system platform (Macrogen Inc.). Differential expressed gene (DEG) analysis was carried out using the R package DESeq2, and pathway enrichment analysis was analyzed using GSEA (v_4.2.3.). For the observation of autophagic morphology, we utilized transmission electron microscopy (TEM), immunofluorescence staining targeting the autophagy marker LC3B, and immunoblotting. Results: KS20226 exhibited a remarkable growth inhibitory effect in 10 different CRC cell lines, with GI50 values ranging from 1 to 10 μM. KS20226 suppressed IRP2 expression and the corresponding occupancy of the iron-responsive elements of ferritin H and transferrin receptor 1, resulting in iron deprivation, and efficiently controlled CRC growth. Pharmacologic inhibition of IRP2 induced reprogramming of mitochondrial respiration, reducing reliance on oxidative phosphorylation. The observed mitochondrial dysfunction induced by KS20226 was correlated with pathway analysis, revealing a decrease in the gene-set for HALLMARK_OXIDATIVE_PHOSPHORYLATION (NES = -1.38, p-value = 0.008) and an increase in the gene-set for POSITIVE_REGULATION_OF_AUTOPHAGY (NES=1.43, p-value=0.016). Immunoblotting demonstrated that KS20226 activated the phosphorylation of AMPK, ULK, and Beclin1 cascade, leading to the autophagic cell death of the CRC cells. Conclusions: The reliance of cancer cells on iron metabolism to drive proliferation has led to the development of therapeutic strategies targeting the inhibition of IRP2. KS20226, a first-in-class IPR2 inhibitor, exhibits distinct therapeutic properties, such as perturbation of mitochondrial iron metabolism and induction of autophagic cell death. Our findings represent important progress toward exploiting IRP2-mediated abnormal iron metabolism in cancer, an unexplored area of research with substantial unmet needs. Citation Format: Jieon Hwang, Areum Park, Chinwoo Kim, Chang Gon Kim, Jaesung Kwak, Byungil Kim, Hyunjin Shin, Minhee Ku, Jaemoon Yang, Ayoung Baek, Jiwon Choi, Hocheol Lim, Kyoung Tai No, Xianghua Zhao, Uyeong Choi, Tae Il Kim, Kyu-Sung Jeong, Hyuk Lee, Sang Joon Shin. KS20226, a first-in-class IRP2 inhibitor, preferentially induces reprogramming of iron metabolism and suppresses tumor growth 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 5937.

Abstract 6832: Gender specific role of LMTK3 in neuronal-tumor microenvironment crosstalk in CRC

Abstract Background: Lemur tail kinase 3 (LMTK3) is a serine/threonine protein kinase with roles in multiple cellular pathways, including Wnt signaling, KIT modulation, and the estrogen receptor pathway; identifying it as a potential target in various cancers. LMTK3 is highly expressed within the central nervous system (CNS) and has been demonstrated to influence neuronal signaling. This study aimed to investigate the potential connection between LMTK3 and neuronal signaling pathways in colorectal cancer (CRC). Methods: Using the Cre/loxP system, we generated a C57BL/6 mouse strain that lacks the expression of LMTK3. 1 × 106 MC38 cells were subcutaneously implanted into 8 weeks old male and female wildtype (WT) and knockout (KO) mice. Tumor growth was measured until endpoint was reached. Tumors were then processed for RNA sequencing. Differentially expressed genes (DEGs) for each comparison group: WT Female vs WT Male, WT Female vs KO Female, WT Male vs KO Male, and KO Female vs KO Male were analyzed. Ingenuity pathway analysis (IPA) was done for identification of upstream regulators involved and affected cellular pathways. Expression levels of DEGs for glutamate receptor subunits were validated using qRT-PCR. Results: Both male and female LMTK3 KO mice showed highly significant (p<0.01) reduced tumor size when compared to WT mice of the same sex. Additionally, the KO female tumors were significantly (p<0.05) smaller when compared to the KO males. RNA sequencing revealed 761 DEGs in KO and WT female and 133 DEGs in KO female and male cohort comparisons (q<0.05). IPA results showed significant (p<0.01, Z-score←2) alterations in pathways including glutaminergic receptor signaling, neurovascular coupling, CREB signaling in neurons, and synaptic long-term depression in the WT vs KO female tumor cohort comparison. Within these pathways DEGs: CACNA1E, CACNA2DA, CACNG2, GRIA1, GRIA2, GRIN1, and GRIN2B, were downregulated in KO females. Upstream regulator analysis identified transcription regulators such as OTX2 and NEUROD1 as well as beta-estradiol to be significantly inhibited (p<0.01, Z-score←2) in KO females. qRT-PCR analysis showed significant (p<0.05) decreased expression in GRIA1, GRIN1, and GRIN2B as well as GRIN2A and GRIN2C in KO females when compared to WT females or KO males, validating RNA seq results. Conclusions: These results showed the involvement of LMTK3 in the gut-brain axis and its effect on CRC tumor growth. Downregulation of multiple neuronal pathways and genes suggest that LMTK3 may influence neuron signaling and neurotransmitter release to produce stimulatory or inhibitory effects within the tumor environment. The concurrent inhibition of beta-estradiol in LMTK3 KO tumors allude to possible sex differences the kinase’s effect on the observed tumor growth. Further studies are needed to confirm the mechanism of interaction of LMTK3 within these pathways. Citation Format: Lesly Torres-Gonzalez, Shivani Soni, Yan Yang, Goar Smbatyan, Jae Ho Lo, Pooja Mittal, Francesca Battaglin, Indrakant K. Singh, Priya Jayachandran, Sandra Algaze, Alexandra Wong, Karam Ashouri, Wu Zhang, Georgios Giamas, Joshua Millstein, Heinz-Josef Lenz. Gender specific role of LMTK3 in neuronal-tumor microenvironment crosstalk in CRC [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 6832.

Heart failure-induced microbial dysbiosis contributes to colonic tumour formation in mice.

Heart failure (HF) and cancer are the leading causes of death worldwide. Epidemiological studies revealed that HF patients are prone to develop cancer. Preclinical studies provided some insights into this connection, but the exact mechanisms remain elusive. In colorectal cancer (CRC), gut microbial dysbiosis is linked to cancer progression and recent studies have shown that HF patients display microbial dysbiosis. This current study focussed on the effects of HF-induced microbial dysbiosis on colonic tumour formation. C57BL/6J mice were subjected to myocardial infarction (MI), with sham surgery as control. After six weeks faeces were collected, processed for 16 s rRNA sequencing, and pooled for faecal microbiota transplantation. CRC tumour growth was provoked in germ-free mice by treating them with Azoxymethane/Dextran sodium sulphate. The CRC mice were transplanted with faeces from MI or sham mice. MI-induced HF resulted in microbial dysbiosis, characterized by a decreased α-diversity and microbial alterations on the genus level, several of which have been associated with CRC. We then performed faecal microbiota transplantation with faeces from HF mice in CRC mice, which resulted in a higher endoscopic disease score and an increase in the number of tumours in CRC mice. We demonstrated that MI-induced HF contributes to colonic tumour formation by altering the gut microbiota composition, providing a mechanistic explanation for the observed association between HF and increased risk for cancer. Targeting the microbiome may present as a tool to mitigate HF-associated co-morbidities, especially cancer.

KAT8-catalyzed lactylation promotes eEF1A2-mediated protein synthesis and colorectal carcinogenesis

Aberrant lysine lactylation (Kla) is associated with various diseases which are caused by excessive glycolysis metabolism. However, the regulatory molecules and downstream protein targets of Kla remain largely unclear. Here, we observed a global Kla abundance profile in colorectal cancer (CRC) that negatively correlates with prognosis. Among lactylated proteins detected in CRC, lactylation of eEF1A2K408 resulted in boosted translation elongation and enhanced protein synthesis which contributed to tumorigenesis. By screening eEF1A2 interacting proteins, we identified that KAT8, a lysine acetyltransferase that acted as a pan-Kla writer, was responsible for installing Kla on many protein substrates involving in diverse biological processes. Deletion of KAT8 inhibited CRC tumor growth, especially in a high-lactic tumor microenvironment. Therefore, the KAT8-eEF1A2 Kla axis is utilized to meet increased translational requirements for oncogenic adaptation. As a lactyltransferase, KAT8 may represent a potential therapeutic target for CRC.

The lncRNA TRG-AS1 promotes the growth of colorectal cancer cells through the regulation of P2RY10/GNA13

Background The lncRNA TRG-AS1 and its co-expressed gene P2RY10 are important for colorectal cancer (CRC) occurrence and development. The purpose of our research was to explore the roles of TRG-AS1 and P2RY10 in CRC progression. Methods The abundance of TRG-AS1 and P2RY10 in CRC cell lines (HT-29 and LoVo) and normal colon cells FHC was determined and difference between CRC cells and normal cells was compared. LoVo cells were transfected with si-TRG-AS1 and si-P2RY10 constructs. Subsequently, the viability, colony formation, and migration of the transfected cells were analyzed using cell counting kit-8, clonogenicity, and scratch-wound/Transwell® assays, respectively. Cells overexpressing GNA13 were used to further explore the relationship between TRG-AS1 and P2RY10 along with their downstream functions. Finally, nude mice were injected with different transfected cell types to observe tumor formation in vivo. Results TRG-AS1 and P2RY10 were significantly upregulated in HT-29 and LoVo compared to FHC cells. TRG-AS1 knockdown and P2RY10 silencing suppressed the viability, colony formation, and migration of LoVo cells. TRG-AS1 knockdown downregulated the expression of P2RY10, GNA12, and GNA13, while P2RY10 silencing downregulated the expression of TRG-AS1, GNA12, and GNA13. Additionally, GNA13 overexpression reversed the cell growth and gene expression changes in LoVo cells induced by TRG-AS1 knockdown or P2RY10 silencing. In vivo experiments revealed that CRC tumor growth was suppressed by TRG-AS1 knockdown and P2RY10 silencing. Conclusions TRG-AS1 knockdown repressed the growth of HT-29 and LoVo by regulating P2RY10 and GNA13 expression.

Itraconazole inhibits tumor growth via CEBPB-mediated glycolysis in colorectal cancer.

Advanced colorectal cancer (CRC) is characterized by a high recurrence and metastasis rate, which is the primary cause of patient mortality. Unfortunately, effective anti-cancer drugs for CRC are still lacking in clinical practice. We screened FDA-approved drugs by utilizing targeted organoid sequencing data and found that the antifungal drug itraconazole had a potential therapeutic effect on CRC tumors. However, the effect and mechanism of itraconazole on CRC tumors have not been investigated. A cell line-derived xenograft model in tumor-bearing mice was established and single-cell RNA sequencing was performed on tumor samples from four mice with or without itraconazole treatment. The proportion of cell populations and gene expression profiles was significantly different between the two groups. We found that itraconazole could inhibit tumor growth and glycolysis. We revealed that CEBPB was a new target for itraconazole, and that silencing CEBPB could repress CRC glycolysis and tumor growth by inhibiting ENO1 expression. Clinical analysis showed that CEBPB expression was obviously elevated in CRC patients, and was associated with poor survival. In summary, itraconazole treatment remodeled cell composition and gene expression profiles. Itraconazole inhibited cell glycolysis and tumor growth via the CEBPB-ENO1 axis. In this study, we illustrate a new energy metabolism mechanism for itraconazole on tumor growth in CRC that will provide a theoretical basis for CRC targeting/combination therapy.

Enterotoxigenic Bacteroides fragilis (ETBF) Enhances Colorectal Cancer Cell Proliferation and Metastasis Through HDAC3/miR-139-3p Pathway.

Enterotoxigenic Bacteroides fragilis (ETBF) is believed to promote the malignant process of colorectal cancer (CRC), but the underlying molecular mechanism still needs to be revealed. CRC cells (SW480 and HCT-116) were treated with ETBF strain. Cell proliferation, invasion and, migration were evaluated by cell counting kit 8 assay, EdU assay, colony formation assay, transwell assay, and wound healing assay. Protein expression was analyzed by western blot. MicroRNA (miR)-139-3p and histone deacetylase 3 (HDAC3) expression levels in tissues and cells were determined by qRT-PCR. Xenograft tumor model was conducted to evaluate the effect of miR-139-3p on CRC tumor growth. ETBF treatment could promote CRC cell proliferation, invasion and migration. MiR-139-3p expression was decreased by ETBF, and its overexpression reversed the effect of ETBF on CRC cell progression. HDAC3 negatively regulated miR-139-3p expression, and its overexpression facilitated CRC cell behaviors via reducing miR-139-3p expression. Moreover, HDAC3 expression was increased by ETBF, and its knockdown also abolished ETBF-mediated CRC cell progression. Additionally, miR-139-3p overexpression could reduce CRC tumor growth in vivo. ETBF aggravated CRC proliferation and metastasis via the regulation of HDAC3/miR-139-3p axis. The discovery of ETBF/HDAC3/miR-139-3p axis may provide a new direction for CRC treatment.

MAD2L2, a key regulator in ovarian cancer and promoting tumor progression

Ovarian cancer (OVCA), a prevalent gynecological malignancy, ranks as the fourth most common cancer among women. Mitotic Arrest Deficient 2 Like 2 (MAD2L2), a chromatin-binding protein and a component of DNA polymerase ζ, has been previously identified as an inhibitor of tumor growth in colorectal cancer. However, the roles of MAD2L2 in OVCA, including its expression, impact, and prognostic significance, remain unclear. We employed bioinformatics tools, Cox Regression analysis, and in vitro cell experiments to investigate its biological functions. Our findings reveal that MAD2L2 typically undergoes genomic alterations, such as amplifications and deep deletions. Moreover, we observed an overexpression of MAD2L2 mRNA in OVCA patients, correlating with reduced survival rates, particularly in those with Grade IV tumors. Furthermore, analysis of mRNA biofunctions indicated that MAD2L2 is predominantly localized in the organellar ribosome, engaging mainly in NADH dehydrogenase activity. This was deduced from the results of gene ontology enrichment analysis, which also identified its role as a structural constituent in mitochondrial translation elongation. These findings were corroborated by KEGG pathway analysis, further revealing MAD2L2’s involvement in tumor metabolism and the cell death process. Notably, MAD2L2 protein expression showed significant associations with various immune cells, including CD4+T cells, CD8+T cells, B cells, natural killer cells, and Myeloid dendritic cells. Additionally, elevated levels of MAD2L2 were found to enhance cell proliferation and migration in OVCA cells. The upregulation of MAD2L2 also appears to inhibit the ferroptosis process, coinciding with increased mTOR signaling activity in these cells. Our study identifies MAD2L2 as a novel regulator in ovarian tumor progression and offers new insights for treating OVCA.

Astragaloside IV regulates circ_0001615 and miR-873-5p/LASP1 axis to suppress colorectal cancer cell progression.

Astragaloside IV (AS-IV) has exhibited pivotal anti-cancer efficacy in multiple types of cancer, including colorectal cancer (CRC). Meanwhile, circular RNA (circRNA) circ_0001615 has been reported to be involved in the malignant development of CRC. Herein, this study is expected to figure out the interaction between circ_0001615 and AS-IV on CRC progression. The 50% inhibition concentration (IC50), proliferation, apoptosis, and migration were detected by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and wound healing assays. The expression of related proteins was examined by western blot. Circ_0001615, microRNA-873-5p (miR-873-5p), and LIM and SH3 protein 1 (LASP1) levels were detected by real-time quantitative polymerase chain reaction (RT-qPCR). The binding between miR-873-5p and circ_0001615, or LASP1, was predicted by Starbase, followed by verification by dual-luciferase reporter and RNA immunoprecipitation (RIP) assays. The biological role of circ_0001615 and AS-IV on CRC tumor growth was detected by the xenograft tumor model invivo. According to the IC50 of AS-IV in CRC cells, the 100 ng/mL AS-IV treatment for 24 h was chosen for the following research: Our data confirmed that AS-IV is a beneficial anti-cancer agent in CRC cells. Furthermore, circ_0001615 and LASP1 expression were increased, and miR-873-5p was decreased in CRC patients and cell lines, whereas their expression exhibited an opposite trend in AS-IV-treated cells. Functionally, applying AS-IV might act as a beneficial anti-cancer effect by downregulating circ_0001615 in CRC cells invitro. Mechanically, circ_0001615 serves as a sponge for miR-873-5p to affect LASP1 expression. In addition, AS-IV inhibited CRC cell growth invivo by modulating circ_0001615. Overall, AS-IV could mitigate CRC development, at least in part, through the circ_0001615/miR-873-5p/LASP1 axis. These findings support a theoretical basis for an in-depth study of the function of AS-IV and the clinical treatment of CRC.

Analyzing aberrant DNA methylation in colorectal cancer uncovered intangible heterogeneity of gene effects in the survival time of patients

Colorectal cancer (CRC) involves epigenetic alterations. Irregular gene-methylation alteration causes and advances CRC tumor growth. Detecting differentially methylated genes (DMGs) in CRC and patient survival time paves the way to early cancer detection and prognosis. However, CRC data including survival times are heterogeneous. Almost all studies tend to ignore the heterogeneity of DMG effects on survival. To this end, we utilized a sparse estimation method in the finite mixture of accelerated failure time (AFT) regression models to capture such heterogeneity. We analyzed a dataset of CRC and normal colon tissues and identified 3406 DMGs. Analysis of overlapped DMGs with several Gene Expression Omnibus datasets led to 917 hypo- and 654 hyper-methylated DMGs. CRC pathways were revealed via gene ontology enrichment. Hub genes were selected based on Protein–Protein-Interaction network including SEMA7A, GATA4, LHX2, SOST, and CTLA4, regulating the Wnt signaling pathway. The relationship between identified DMGs/hub genes and patient survival time uncovered a two-component mixture of AFT regression model. The genes NMNAT2, ZFP42, NPAS2, MYLK3, NUDT13, KIRREL3, and FKBP6 and hub genes SOST, NFATC1, and TLE4 were associated with survival time in the most aggressive form of the disease that can serve as potential diagnostic targets for early CRC detection.

STAM-binding Protein-like 1 Promotes Growth and Migration of Colorectal Cancer by NF-κB Pathway.

STAM-binding protein-like 1 (STAMBPL1) functions as a deubiquitinase to cleave Lys63 ubiquitin linkage, and is associated with cancer dissemination and progression. The role of STAMBPL1 in colorectal cancer (CRC) remains unclear. STAMBPL1 expression was determined by western blot and qRT-PCR. Cell proliferation was detected by colony formation and MTT assays, and apoptosis was assessed by flow cytometry. The metastasis was evaluated by transwell and wound healing assays. An animal xenograft experiment was used to investigate the effect of STAMBPL1 on tumor growth. The expression of STAMBPL1 was elevated in CRC cells. Knockdown of STAMBPL1 reduced cell viability of CRC and suppressed the proliferation, invasion, and migration. Apoptosis of CRC was induced by silence of STAMBPL1. Tumor growth of CRC was also suppressed by the silence of STAMBPL1. Knockdown of STAMBPL1 increased IκB and decreased phosphorylation of IκB to reduce p65 phosphorylation. Knockdown of STAMBPL1 inhibited cell growth and metastasis of CRC through inactivation of the NF-κB pathway.

Vasopressin Analog [V4Q5]dDAVP Exerts Cooperative Anticancer Effects in Combination With Low-Dose 5-Fluorouracil on Aggressive Colorectal Cancer Models.

Colorectal cancer (CRC) is a leading cause of cancer-associated mortality worldwide. Despite being an essential component of systemic chemotherapy for advanced CRC, 5-fluorouracil (5-FU) clinical use has severe limitations, such as high toxicity, low selectivity and drug resistance. [V4Q5]dDAVP (1-deamino-4-valine-5-glutamine-8-D-arginine vasopressin) is a peptide vasopressin analog and a selective agonist of the arginine vasopressin type 2 membrane receptor (AVPR2), expressed in microvascular and tumor tissue. This synthetic compound has well-proven antitumor and antimetastatic activity in different tumor types, including metastatic CRC. The objective of this work was to assess the potential combinational benefits in preclinical CRC models after [V4Q5]dDAVP addition to 5-FU. Effects on cellular viability, cell cycle progression, apoptosis and molecular mechanisms associated to [V4Q5]dDAVP treatment in combination with 5-FU were evaluated in murine CT-26 and human COLO-205 cell lines. In vivo, impact of dual therapy was explored on CRC tumor growth and metastatic spread. In CRC cells, [V4Q5]dDAVP (1 µM) addition to sub-IC50 5-FU concentrations resulted in the enhancement of cytostatic effects induced by chemotherapy. Reduction of cell viability after combined treatment was associated with cell cycle arrest in the G0/G1 phase, induction of apoptosis and increased gene expression of the cyclin-dependent kinase inhibitor p21 (CDKN1A) and the tumor suppressor p53 (TP53) in malignant cells, as assessed by flow cytometry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), and quantitative reverse transcription polymerase chain reaction (qRT-PCR), respectively. In vivo, intravenous administration of [V4Q5]dDAVP (0.3 µg/kg) in combination with safe low doses of 5-FU (50 or 80 mg/kg for CT-26 or COLO-205 tumor models, respectively) effectively abrogated CRC growth, reducing aggressiveness of primary lesions and increasing survival of tumor-bearing mice. In addition, concomitant administration of [V4Q5]dDAVP and 5-FU inhibited pulmonary metastasis formation by CT-26 cells in immunocompetent mice, especially reducing macrometastatic disease. [V4Q5]dDAVP seems to enhance the efficacy of 5-FU-based chemotherapy in CRC by modulating tumor progression, as well as metastatic dissemination, suggesting its potential role as a safe and cost-effective co-adjuvant agent for the management of advanced CRC.

Abstract B147: Preclinical evaluation of XPO1 inhibition with topoisomerase-I (topo-I) inhibition in colorectal cancer (CRC) cell lines and patient-derived xenograft (PDX) models

Abstract Background: Resistance to topo-I inhibitors is of significant importance in treating patients with metastatic CRC (mCRC). Selinexor and eltanexor are selective inhibitors of nuclear export, by binding to exportin 1 (XPO1, the primary nucleic export receptor, and is overexpressed in CRC). Eltanexor is an investigational XPO1 inhibitor, with similar mechanism and potency but improved tolerability in animal models. XPO1 inhibition results in nuclear localization of tumor suppressor proteins, oncoprotein mRNAs, and topoisomerases, and decreases DNA damage repair (DDR) response. Irinotecan is a topo-I inhibitor which induces DNA damage and is commonly used in mCRC. We hypothesized that XPO1 inhibition may improve response to topo-I inhibitor in mCRC by impairing the DDR response. Methods: The synergism of XPO1 inhibition (selinexor or eltanexor) with SN38 was assessed in CRC cell lines by CellTiter-Glo 2.0 and quantitated using Bliss Additivity Model using Synergy Finder 2.0. Each condition was plated either concurrent (both drugs for 72 hours) or sequential (SN38 for 24 hours then removed, followed by XPO1 inhibitor for 48 hours). Nude mice were implanted with one microsatellite stable CRC PDX model in the flanks followed by treatment with vehicle, XPO1 inhibitor, irinotecan (15 mg/kg intraperitoneal weekly), or combination. Tumor growth was measured by specific growth rate (SGR), within an IACUC-approved protocol. Tumor tissue was collected at day 7 for molecular analysis. Tissues were lysed and protein was run on 4%-12% SDS-PAGE Gel. p21 Waf/Cip and P-Histone H2A.X were used to determine DNA damage by western blot. P-Histone H2AX (pH2AX) was evaluated by immunofluorescence using primary conjugated P-Histone H2A.X antibody and DAPI for nuclear counter stain. Slides were imaged using Olympus iX83 microscope and analysis was performed using Olympus CellSens software. Results: In CRC cell lines, concurrent combination eltanexor and SN38 did not synergistically decrease viability but increased pH2AX compared to SN38 alone (~30-fold). However, sequential SN38 followed by eltanexor synergistically decreased viability at multiple dose levels; evaluation of pH2AX is ongoing. In PDX model CRC254, eltanexor (10 mg/kg and reduced to 7.5 mg/kg on day 8 to improve tolerability, orally 5 days per week) with irinotecan decreased SGR compared to irinotecan alone (SGR=0.007 vs 0.026, respectively, p=0.0003) and compared to eltanexor alone (SGR=0.029, p <0.0001). Combination treatment increased pH2AX and increased p21 expression compared to either single-agent treatment. Similar SGR results were observed with selinexor (5 mg/kg orally twice weekly). Conclusions: XPO1 inhibition and topo-I inhibition decreased CRC tumor growth in PDX models, presumably by increasing DNA double strand breaks. Cell line data suggests sequential dosing of topo-I inhibition followed by XPO1 inhibition may be effective. This work supports continued development of this combination in mCRC. Citation Format: Robert W Lentz, Adrian Dominguez, Stacey Bagby, Cameron Binns, Alexis Leal, Sunnie Kim, Sarah Lindsey Davis, Christopher H Lieu, Wells A Messersmith, Todd M Pitts. Preclinical evaluation of XPO1 inhibition with topoisomerase-I (topo-I) inhibition in colorectal cancer (CRC) cell lines and patient-derived xenograft (PDX) models [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 B147.

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transient Receptor Potential channels (TRP) in GtoPdb v.2023.3

Transcriptionally activates CCL28 expression to inhibit M2 polarization of macrophages and prevent immune escape in colorectal cancer cells

ObjectiveThis study aimed to investigate the potential molecular mechanism of SPDEF in immune evasion of colorectal cancer (CRC) and examine its impact on macrophage M2 polarization using the TCGA and GEO databases. MethodsBy combining TCGA and GEO databases, differential gene expression between CRC samples and standard tissue samples was analyzed to screen for immune-related genes (IRGs) associated with the prognosis of CRC patients. A predictive risk model was constructed based on 18 key IRGs, which were then validated using the GEO dataset. The relationship between transcription factors and IRGs was further explored to investigate their regulatory network in CRC. In vivo and in vitro experiments were carried out to validate these regulatory relationships and explore the function of SPDEF and CCL28 in CRC. ResultsTwelve key IRGs associated with clinical and pathological characteristics of CRC patients were identified. Among them, CCL28 significantly impacted macrophage infiltration in CRC cells and may be a critical factor in immune evasion. In both in vitro and in vivo experiments, overexpression of SPDEF upregulated CCL28 expression, thereby suppressing M2 polarization of macrophages and inhibiting CRC cell proliferation and tumor growth. Notably, interference with CCL28 could reverse the effect of SPDEF overexpression. ConclusionSPDEF can suppress immune evasion of CRC cells by activating CCL28, which is achieved through the modulation of M2 polarization of macrophages. This provides a new research direction and potential therapeutic target for immunotherapy in CRC.