Expression In Colon Cancer Cells Research Articles

Upregulation of Immune checkpoint PD-L1 in Colon cancer cell lines and activation of T cells by Leuconostoc mesenteroides

Globally colorectal cancer ranks as the third most widespread disease and the third leading cause of cancer-associated mortality. Immunotherapy treatments like PD-L1 blockade have been used to inhibit the PD-L1 legend, which boosts the activity of cytotoxic T lymphocytes. Recently, studies suggest that some probiotics could potentially enhance the effectiveness of immunotherapy treatments for cancer patients. We found that in Caco-2 and HT-29 cells, the live Leuconostoc mesenteroides treatment resulted an increase in the PD-L1 expression and this treatment stimulated interferon-gamma (IFN-γ) production in Jurkat T-cells. Due to the well-established ability of IFN-γ to enhance PD-L1 expression, the combination of IFN-γ and L. mesenteroides was used in colon cancer cell lines and a resulting remarkable increase of over tenfold in PD-L1 expression was obtained. Interestingly, when L. mesenteroides and IFN-γ are present, the blockage of PD-L1 using PD-L1 antibodies not only improved the viability of Jurkat T-cells but also significantly boosted the levels of IFN-γ and IL-2, the T-cells activation marker cytokines. In addition to upregulating PD-L1, L. mesenteroides also activated Toll-like receptors (TLRs) and NOD-like receptors (NODs) pathways, specifically through TLR2 and NOD2, while also exerting a suppressive effect on autophagy in colon cancer cell lines. In conclusion, our findings demonstrate a significant upregulation of PD-L1 expression in colon cancer cells upon co-culturing with L. mesenteroides. Moreover, the presence of PD-L1 antibodies during co-culturing activates Jurkat T cells. The observed enhancement in PD-L1 expression may be attributed to the inhibition of the Autophagy pathway or activation of the hippo pathway.Graphical abstract textThe administration of Live Lactobacillus mesenteroides on colon cancer cells leads to the elevation of PD-L1, with a further increase observed in the presence of IFN-γ. Co-cultivation of Live L. mesenteroides with colon cancer cells in conjunction with anti-PD-L1 blockade antibody results in the enhanced viability of T cells.Key PointsCo-culturing L. mesenteroides increases PD-L1 gene and protein transaction in colon cancer.L. mesenteroides existing enhances T cells viability and activity.GPCR41/42 is a possible link between L. mesenteroides, YAP-1 and PD-L1.

MicroRNA 429 regulates MMPs expression by modulating TIMP2 expression in colon cancer cells and inflammatory colitis.

In a previous study, we found that the expression of microRNA 429 (MIR429) was decreased in dextran sodium sulfate (DSS)-induced mouse colitis tissues. In this study, we aimed to investigate the interaction of MIR429 with TIMP metallopeptidase inhibitor 2 (TIMP2), one of its candidate target genes, in human colorectal cancer (CRC) cells and DSS-induced mouse colitis tissues. A luciferase reporter system was used to confirm the effect of MIR429 on TIMP2 expression. The expression levels of MIR429 and target genes in cells or tissues were evaluated through quantitative RT-PCR, western blotting, or immunohistochemistry. We found that the expression level of MIR429 was downregulated in human CRC tissues, and also showed that TIMP2 is a direct target gene of MIR429 in CRC cell lines. Furthermore, MIR429 regulate TIMP2-mediated matrix metallopeptidases (MMPs) expression in CRC cells. We also generated cell lines stably expressing MIR429 in CRC cell lines and showed that MIR429 regulates the expression of MMPs by mediating TIMP2 expression. In addition to human CRC tissues, we found that TIMP2 was highly expressed in mouse colitis tissues and human ulcerative colitis (UC) tissues. Our findings suggest that the expression of endogenous MIR429 was reduced in human CRC tissues and colitis, leading to upregulation of its target gene TIMP2. The upregulation of TIMP2 by decreased MIR429 expression in CRC tissues and inflamed tissues suggests that it may affect extracellular matrix (ECM) remodeling through downregulation of MMPs. Therefore, MIR429 may have therapeutic value for human CRC and colitis.

GSK0660 enhances antitumor immunotherapy by reducing PD-L1 expression

Blockade of PD-1/PD-L1 immune checkpoint is wildly used for multiple types of cancer treatment, while the low response rate for patients is still completely unknown. As nuclear hormone receptor, PPARδ (peroxisome-proliferator-activated receptor) regulates cell proliferation, inflammation, and tumor progression, while the effect of PPARδ on tumor immune escape is still unclear. Here we found that PPARδ antagonist GSK0660 significantly reduced colon cancer cell PD-L1 protein and gene expression. Luciferase analysis showed that GSK0660 decreased PD-L1 gene transcription activity. Moreover, reduced PD-L1 expression in colon cancer cells led to increased T cell activity. Further analysis showed that GSK0660 decreased PD-L1 expression in a PPARδ dependent manner. Implanted tumor model analysis showed that GSK0660 inhibited tumor immune escape and the combined PD-1 antibody with GSK0660 effectively enhanced colorectal cancer immunotherapy. These findings suggest that GSK0660 treatment could be an effective strategy for cancer immunotherapy.

Abstract 5419: Claudin-7 mediates Rab25-induced suppression of colon cancer cell invasion

Abstract Ras-related protein 25 (Rab25) is a member of small GTPase, and growing evidence demonstrates the context-dependent roles of Rab25 in cancer progression. While Rab25 aggravates cancer cell invasion in ER-positive breast, ovarian, and gastric cancers, Rab25 is under-expressed and suppresses colon and triple-negative breast cancer progression. Claudins are major components of tight junctions, and disruption of claudins is implicated in tumorigenesis. Among 27 known members of the claudin family, claudin-7 is suggested to suppress colon cancer progression. Previously, we identified that Rab25 induces endothelial-mesenchymal transition (EMT) and invasiveness of several types of cancer cells through activating the β1 integrin/epidermal growth factor receptor (EGFR)/Snail signaling cascades. However, little is known about the underlying mechanism of Rab25-induced tumor suppression. In the present study, we identify the critical role of claudin-7 in Rab25-induced suppression of colon cancer invasion. 3D Matrigel and modified Boden chamber were used to assess the invasiveness of colon cancer cells. Wound-healing assay was used to analyze colon cancer cell migration. 3D Matrigel was used to analyze tumor growth. Immunoblotting was used to assess the protein expression. Rab25 induced claudin-7 expression in colon cancer cells through protein stabilization. In addition, the enforced expression of claudin-7 not only reduced the EGFR/Ras signaling activity and Snail expression but also attenuated colon cancer cell invasion. However, silencing of claudin-7 expression rescued the tumor suppressive role of Rab25, thereby increasing colon cancer cell invasiveness. Collectively, our data demonstrate for the first time that Rab25 reduces the EGFR/Ras/Snail signaling activity and inhibits colon cancer cell invasion by upregulating claudin-7 expression. Citation Format: Su Jin Cho, Bo Young Jeong, Se-Hee Yoon, Chang Gyo Park, Hoi Young Lee. Claudin-7 mediates Rab25-induced suppression of colon cancer cell invasion [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 5419.

Abstract 3022: GPR109A activation limits cell growth by inducing p53 and its targets in human colon cancer cells

Abstract Chemical carcinogens or xenobiotics present in the diet play a significant role in colon cancer (CC) formation. p53, a tumor suppressor and multi-functional protein, provides an important barrier against the initiation and maintenance of CC. p53 mutation or inactivation is observed in about 60% of CCs and about 90% of all cancers. Therefore, therapeutic avenues that will rejuvenate wild-type p53 or target mutant p53 should have a significant impact on the prevention and treatment of CC. GPR109A, a G-protein coupled receptor for the FDA-approved lipid-lowering drug Niacin (NA), bacterial metabolite Butyrate (BTR), and ketone body β-hydroxybutyrate (BHB), functions as a novel tumor suppressor in the colon by providing the molecular link between colonic bacteria and p53 function. GPR109A activation limits cAMP by inhibiting adenylate cyclase (AC), which induces RAS and its downstream growth-promoting signaling. cAMP also enhances the binding of MDM2 to p53 and induces the proteasomal degradation of p53. p53 is an NAD+-dependent molecule and deficiency of NA, the precursor of NAD+, impairs p53 function. We tested the role of GPR109A and its agonists (NA, BTR, and BHB) in normal immortalized colon epithelial cell lines (CCD33 and CCD81) and various CC lines such as HCT116-p53+/+, HCT116-p53−/−, DLD1 (p53 point mutation), VACO4A (p53 insertion mutation), and SW498 (p53 deletion) and measured cell proliferation, cell cycle, apoptosis, and analyzed the expression of GPR109A and p53 (total, phosphorylated, and acetylated), MDM2, p21, Bax, and Puma in the presence and absence of GPR109109 agonists. We found that GPR109A expression is significantly reduced in CC cell lines, especially in p53 mutant cell lines, when compared to normal immortalized colonic epithelial cell lines. Treatment of CC cell lines with GPR109A agonists-induced apoptosis in wild-type p53 expressing CC by activating p53 and its targets. However, some lesser extent in p53 mutant or p53 null cell lines. Lentiviral-mediated GPR109A expression in CC cell lines itself induced apoptosis in both wild-type and mutant p53 expressing CC cell lines and GPR109A agonists treatment induced further. Interestingly, GPR109A agonists BTR and BHB significantly increased apoptosis in p53 mutant and p53 null CC cell lines in a p53-independent manner. Overall, our results provide evidence that GPR109A activation can be used as an effective therapeutic strategy for the treatment of CC. Citation Format: Muthusamy Thangaraju, Nanditi N. Thangaraju, Lauren Gilstrap, Madison Chimenti, Tulshi Patel, Sathish Sivaprakasam, Nikhil Patel, Santhakumar Manicassamy, Puttur D. Prasad, Vadivel Ganapathy. GPR109A activation limits cell growth by inducing p53 and its targets in human colon cancer cells [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 3022.

Study of Therapeutic Mechanisms of Bupi Yichang Formula against Colon Cancer Based on Network Pharmacology, Machine Learning, and Experimental Verification.

Bupi Yichang formula (BPYCF) has shown the anti-cancer potential; however, its effects on colon cancer and the mechanisms remain unknown. This study intended to explore the effects of BPYC on colon cancer and its underlying mechanisms. BPYCF-related and colon cancer-related targets were acquired from public databases, followed by differentially expressed genes (DEG) identification. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed using clusterProfiler. A protein-protein interaction (PPI) network was constructed using STRING database. CytoHubba and MCODE to screen the hub targets. A diagnostic model was built using random forest algorithm. Molecular docking was conducted using PyMOL and AutoDock. High-performance liquid chromatograph-mass spectrometry (HPLC-MS) analysis and in vitro validation were performed. Forty-six overlapping targets of BPYCF-related, colon cancer-related targets, and DEGs were obtained. GO and KEGG analyses showed that the targets were mainly enriched in response to lipopolysaccharide, neuronal cell body, protein serine/threonine/tyrosine, as well as C-type lectin receptor, NOD-like receptor, and TNF signaling pathways. Five targets were identified as the pivotal targets, among which, NOS3, CASP8, RIPK3, and TNFRSF10B were stably docked with the core active component, naringenin. Naringenin was also identified from the BPYCF sample through HPLC-MS analysis. In vitro experiments showed that BPYCF inhibited cell viability, reduced NOS3 expression, and elevated CASP8, RIPK3, and TNFRSF10B expression in colon cancer cells. BPYCF might treat colon cancer mainly by regulating NOS3, CASP8, RIPK3, and TN-FRSF10B. This study first revealed the therapeutic effects and mechanisms of BPYCF against colon cancer, paving the path for the development of targeted therapeutic strategies for this cancer in the clinic.

SIX4 Controls Anti-PD-1 Efficacy by Regulating STING Expression.

Our studies demonstrate that SIX4 is an important regulator of STING expression, providing a genetic marker or a therapeutic target to predict or enhance immune checkpoint blockade therapy responses in colon cancer.

An Increase in Mucin2 Expression Is Required for Colon Cancer Progression Mediated by L1.

An induction in the expression of the cell adhesion receptor L1, a Wnt target gene, is a characteristic feature of Wnt/β-catenin activation in colon cancer cells at later stages of the disease. We investigated the proteins secreted following L1 expression in colon cancer cells and identified Mucin2 among the most abundant secreted proteins. We found that suppressing Mucin2 expression in L1-expressing colon cancer cells inhibits cell proliferation, motility, tumorigenesis, and liver metastasis. We detected several signaling pathways involved in Mucin2 induction in L1-expressing cells. In human colon cancer tissue, Mucin2 expression was significantly reduced or lost in the adenocarcinoma tissue, while in the mucinous subtype of colon cancer tissue, Mucin2 expression was increased. An increased signature of L1/Mucin2 expression reduced the survival rate of human colon cancer patients. Thus, induction of Mucin2 expression by L1 is required during mucinous colon cancer progression and can serve as a marker for diagnosis and a target for therapy.

Transcription activation of SPINK4 by ELF-1 augments progression of colon cancer by regulating biological behaviors

BackgroundSPINK4 was highly expressed in colorectal cancer and resulted in worse prognosis of colorectal cancer patients. However, the expression and function of SPINK4 in colon cancer have not been revealed. MethodsAnalysis from GEPIA website showed the expression and function of SPINK4 in colon cancer samples. Colon cancer cell lines were applied to detect the biological function of SPINK4. Functionally, the transcriptional factor of SPINK4 has been predicted and verified. Finally, the associations between transcriptional factor and SPINK4 have been confirmed. ResultsSPINK4 expression was obviously increased in colon cancer samples. HCT-116 and DXH-1 cells in si-SPINK4–1 or si-SPINK4–2 group displayed an obvious reduction in its proliferation, cell cycle, invasion and migration compared to those in the si-control group. Moreover, transcriptional factor ELF-1 bound to the promoter of SPINK4 and affected its expression in colon cancer cells. High ELF-1 expression was presented in colon cancer samples and resulted in worse prognosis of colon cancer patients. Additionally, si-SPINK4 antagonized the function of ELF-1 overexpression in modulating colon cancer cell proliferation, cell cycle and mobility. ConclusionsOur findings afforded a theoretical basis for further research on the treatment of colon cancer based on the control of ELF-1/SPINK4 expression.

The tumor promoter cysteinyl leukotriene receptor 1 regulates PD-L1 expression in colon cancer cells via the Wnt/β-catenin signaling axis

Immunotherapy targeting programmed death-ligand 1 (PD-L1) or PD-1 in solid tumors has been shown to be clinically beneficial. However, in colorectal cancer (CRC), only a subset of patients benefit from PD-1/PD-L1 treatment. Previously, we showed that high cysteinyl leukotriene receptor 1 (CysLT1R) levels are associated with poor prognosis in CRC patients. Recently, we have revealed the role of the tumor promoter CysLT1R in drug resistance and stemness in colon cancer (CC) cells. Here, we show the role of the CysLT1R/Wnt/β-catenin signaling axis in the regulation of PD-L1 using both in vitro and in vivo preclinical model systems. Interestingly, we found that both endogenous and IFNγ-induced PD-L1 expression in CC cells is mediated through upregulation of CysLT1R, which enhances Wnt/β-catenin signaling. Therapeutic targeting of CysLT1R with its antagonist montelukast (Mo), as well as CRISPR/Cas9-mediated or doxycycline-inducible functional absence of CysLT1R, negatively regulated PD-L1 expression in CC cells. Interestingly, an anti-PD-L1 neutralizing antibody exhibited stronger effects together with the CysLT1R antagonist in cells (Apcmut or CTNNB1mut) with either endogenous or IFNγ-induced PD-L1 expression. Additionally, mice treated with Mo showed depletion of PD-L1 mRNA and protein. Moreover, in CC cells with combined treatment of a Wnt inhibitor and an anti-PD-L1 antibody was effective only in β-catenin-dependent (APCmut) context. Finally, analysis of public dataset showed positive correlations between the PD-L1 and CysLT1R mRNA levels. These results elucidate a previously underappreciated CysLT1R/Wnt/β-catenin signaling pathway in the context of PD-L1 inhibition in CC, which might be considered for improving the efficacy of anti-PD-L1 therapy in CC patients.7T-oMpUEusLw1sD21V8GVPVideo

Abstract 3916: role of her3 v104l mutation on tumor growth and her3 stabilization

Abstract We aimed to determine if naturally occurring HER3 mutations could drive oncogenic activity of HER2+ HCC1569 cells in which endogenous HER3 was knocked out using CRISPR-Cas9 technology. A series of HER3 mutations found in breast cancer patients (F94L, V104L, G284R, D297Y, T355I, and E928G) were introduced via lentiviral transduction and stable cell lines were generated in HER3 knock out (KO) HCC1569 cells and were maintained in 0.5 mg/mL puromycin. Our data indicated that cells stably transduced with HER3V104L mutation had significantly higher cell growth with higher p-HER3 expression versus wild-type (wt) and empty-vector (EV) HER3. We found that HCC1569HER3KO cells with WT and V104L were sensitive to increasing concentration of neratinib (0.1-0.5 µM) as indicated by crystal violet assay. Next we examined if V104L mutation rendered resistance to another FDA- approved irreversible HER2 tyrosine kinase inhibitor, tucatinib. Our results showed that V104L cells were sensitive to higher concentration of tucatinib compared to neratinib. In other experiments, we used COS7 cells to examine the signaling pathways of HER3V104L mutation. Our western blot data demonstrated that transient transfection of COS7 cells with HER3V104L mutant significantly upregulated p-HER3 and p-HER2 expression versus WT HER3 in a NRG- dependent manner. In addition, we found that the V104L mutation stabilizes HER3 protein expression independent of ligand stimulation. Our western blot data showed that V104L induced HER3 stabilization is independent on HER3 binding partners HER1, HER2 and HER4. Our cycloheximide chase data indicated that V104L mutation stabilizes HER3 expression in COS7 cells as well as low HER3 expressing colon cancer cell line, SNUC5. Our western blot data indicates that V104L mutation activates ERK signaling in NRG dependent manner over AKT pathway in COS7 cells. In addition, we are using various PDXs with different HER3 mutations (V104M and E928D) to determine the role of these driver HER3 mutations in cancer and how this can be targeted in the clinic using HER targeted therapy. Citation Format: Rosalin Mishra, Mary Kate Kilroy, Wasim Feroz, Hima Patel, Samar Alanazi, Joan T. Garrett. role of her3 v104l mutation on tumor growth and her3 stabilization. [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 3916.

COLITIS-ASSOCIATED COLON CANCER: EXPLORING THE DCLK1-S-MMP13-P53 AXIS

Chronic intestinal inflammation in patients with inflammatory bowel diseases (IBD) significantly increases the risk of colorectal cancer (CRC). Tuft cells in the intestine marked by doublecortin-like kinase-1 (DCLK1), are long-lived chemosensory cells with newly identified roles in colitis as epithelial ablation of DCLK1 worsens disease outcome. DCLK1 exists in long and short isoforms (DCLK1-L and DCLK1-S). Hypermethylation of DCLK1 alpha promoter encoding DCLK1-L particularly in CRC, allows switching to the DCLK1-S isoform that confers a more invasive tumor phenotype. In an infectious colitis model, we recently discovered that DCLK1-S is predominantly expressed in Ly6G+;MHCII- neutrophils that coincides with elevated levels of CXCL1/KC and MMP13 expression in the colon. In an unbiased molecular docking study, DCLK1’s kinase domain was found to interact with the catalytic domain of MMP13. MMP13 plays a crucial role in the modulation of extracellular matrix (ECM) degradation and cell-matrix interactions and wild type and mutant p53 differentially regulate MMP13 gene expression. Mutations in TP53 (tumor suppressor p53) are crucial for neoplasia initiation in IBD patients and are observed in 50-85% of colitis-associated colon cancers (CACCs). We further observed that p53 mutation status dictates DCLK1-S expression in colon cancer cells and in mice mutant for p53, expression of DCLK1-S and MMP13 correlated with neutrophilic inflammation and crypt hyperplasia. We will expand on these novel discoveries by employing high fidelity proteomic and phosphoproteomic studies and decipher how DCLK1-S-MMP13-p53 axes stimulate fibrosis, tumorigenesis, and tumor progression. These studies are expected to lead to therapeutic avenues to target CACCs.

COLITIS-ASSOCIATED COLON CANCER: EXPLORING THE DCLK1-S-MMP13-P53 AXIS

Abstract Chronic intestinal inflammation in patients with inflammatory bowel diseases (IBD) significantly increases the risk of colorectal cancer (CRC). Tuft cells in the intestine marked by doublecortin-like kinase-1 (DCLK1), are long-lived chemosensory cells with newly identified roles in colitis as epithelial ablation of DCLK1 worsens disease outcome. DCLK1 exists in long and short isoforms (DCLK1-L and DCLK1-S). Hypermethylation of DCLK1 alpha promoter encoding DCLK1-L particularly in CRC, allows switching to the DCLK1-S isoform that confers a more invasive tumor phenotype. In an infectious colitis model, we recently discovered that DCLK1-S is predominantly expressed in Ly6G+;MHCII- neutrophils that coincides with elevated levels of CXCL1/KC and MMP13 expression in the colon. In an unbiased molecular docking study, DCLK1’s kinase domain was found to interact with the catalytic domain of MMP13. MMP13 plays a crucial role in the modulation of extracellular matrix (ECM) degradation and cell-matrix interactions and wild type and mutant p53 differentially regulate MMP13 gene expression. Mutations in TP53 (tumor suppressor p53) are crucial for neoplasia initiation in IBD patients and are observed in 50-85% of colitis-associated colon cancers (CACCs). We further observed that p53 mutation status dictates DCLK1-S expression in colon cancer cells and in mice mutant for p53, expression of DCLK1-S and MMP13 correlated with neutrophilic inflammation and crypt hyperplasia. We will expand on these novel discoveries by employing high fidelity proteomic and phosphoproteomic studies and decipher how DCLK1-S-MMP13-p53 axes stimulate fibrosis, tumorigenesis, and tumor progression. These studies are expected to lead to therapeutic avenues to target CACCs.

The Effect of Different Anesthetic Techniques on Proliferation, Apoptosis, and Gene Expression in Colon Cancer Cells: A Pilot In Vitro Study.

Colorectal cancer is highly common and causes high mortality rates. Treatment for colorectal cancer is multidisciplinary, but in most cases the main option remains surgery. Intriguingly, in recent years, a number of studies have shown that a patient's postoperative outcome may be influenced by certain anesthetic drugs. Our main objective was to compare the effect of propofol-total intravenous anesthesia (TIVA) with sevoflurane anesthesia and to investigate the potential role of intravenous lidocaine on colon cancer cell functions. We tested the effects of serum from colorectal cancer patients undergoing TIVA vs. sevoflurane anesthesia with or without lidocaine on HCT 116 cell lines; on proliferation, apoptosis, migration, and cell cycles; and on cancer-related gene expressions. 60 patients who were scheduled for colorectal cancer surgery were randomized into four different groups (two groups with TIVA and two groups with sevoflurane anesthesia with or without intravenous lidocaine). Blood samples were collected at the start and at the end of surgery. HCT 116 cells were exposed to the patients' serum. 15 patients were included in each of the study groups. We did not find any significant difference on cell viability or apoptosis between the study groups. However, there was an increased apoptosis in propofol groups, but this result was not statistically significant. A significant increase in the expression profile of the TP53 gene in the propofol group was registered (p = 0.029), while in the other study groups, no significant differences were reported. BCL2 and CASP3 expressions increased in the sevoflurane-lidocaine group without statistical significance. In our study, serum from patients receiving different anesthetic techniques did not significantly influence the apoptosis, migration, and cell cycle of HCT-116 colorectal carcinoma cells. Viability was also not significantly influenced by the anesthetic technique, except the sevoflurane-lidocaine group where it was increased. The gene expression of TP53 was significantly increased in the propofol group, which is consistent with the results of similar in vitro studies and may be one of the mechanisms by which anesthetic agents may influence the biology of cancer cells. Further studies that investigate the effects of propofol and lidocaine in different plasma concentrations on different colon cancer cell lines and assess the impacts of these findings on the clinical outcome are much needed.

Nrf3 alleviates oxidative stress and promotes the survival of colon cancer cells by activating AKT/BCL-2 signal pathway.

Oxidative stress is closely linked to tumor initiation and development, conferring a survival advantage to cancer cells. Therefore, understanding cancer cells' antioxidant molecular mechanisms is crucial to cancer therapy. In this study, we discovered that H2O2-induced oxidative stress increased Nrf3 expression in colon cancer cells. Overexpression of Nrf3 decreased H2O2-mediated cytotoxicity and apoptosis. Furthermore, Nrf3 reduced reactive oxygen species levels and malondialdehyde concentrations after H2O2 treatment. Mechanistically, H2O2-mediated cell apoptosis involves multiple signaling proteins, including Akt, bcl-2, JNK, and p38. An increase in Nrf3 expression in colon cancer cells treated with H2O2 partly reversed Akt/Bcl-2 inhibition, whereas it decreased activation of p38 and JNK. In addition, we found that increasing Nrf3 decreased stress-associated chemical-induced cell death, resulting in drug resistance. According to these results, Nrf3 is critical for drug resistance and oxidant adaptation.

Multi-omics Analysis of the Role of PHGDH in Colon Cancer.

Objectives: Serine metabolism is essential for tumor cells. Endogenous serine arises from de novo synthesis pathways. As the rate-limiting enzyme of this pathway, PHGDH is highly expressed in a variety of tumors including colon cancer. Therefore, targeted inhibition of PHGDH is an important strategy for anti-tumor therapy research. However, the specific gene expression and metabolic pathways regulated by PHGDH in colon cancer are still unclear. Our study was aimed to clarified the role of PHGDH in serine metabolism in colon cancer to provide new knowledge for in-depth understanding of serine metabolism and PHGDH function in colon cancer. Methods: In this study, we analyzed the gene expression and metabolic remodeling process of colon cancer cells (SW620) after targeted inhibition of PHGDH by gene transcriptomics and metabolomics. LC-MS analysis was performed in 293T cells to PHGDH gene transcription and protein post-translational modification under depriving exogenous serine. Results: We found that amino acid transporters, amino acid metabolism, lipid synthesis related pathways compensation and other processes are involved in the response process after PHGDH inhibition. And ATF4 mediated the transcriptional expression of PHGDH under exogenous serine deficiency conditions. While LC-MS analysis of post-translational modification revealed that PHGDH produced changes in acetylation sites after serine deprivation that the K289 site was lost, and a new acetylation site K21was produced. Conclusion: Our study performed transcriptomic and metabolomic analysis by inhibiting PHGDH, thus clarifying the role of PHGDH in gene transcription and metabolism in colon cancer cells. The mechanism of high PHGDH expression in colon cancer cells and the acetylation modification that occurs in PHGDH protein were also clarified by serine deprivation. In our study, the role of PHGDH in serine metabolism in colon cancer was clarified by multi-omics analysis to provide new knowledge for in-depth understanding of serine metabolism and PHGDH function in colon cancer.

Expression of nucleus accumbens-1 in colon cancer negatively modulates antitumor immunity.

Nucleus accumbens-1 (NAC-1) is highly expressed in a variety of tumors, including colon cancer, and is closely associated with tumor recurrence, metastasis, and invasion. To determine whether and how NAC-1 affects antitumor immunity in colon cancer. NAC-1-siRNA was transfected into RKO colon cancer cells to knock down NAC expression; tumor cells with or without knockdown of NAC-1 were treated with CD8+ T cells to test their cytocidal effect. The level of the immune checkpoint programmed death receptor-1 ligand (PD-L1) in colon cancer cells with or without knockdown of NAC-1 was analyzed using Quantitative real-time polymerase chain reaction and Western blotting. A double luciferase reporter assay was used to examine the effects of NAC-1 on the transcription of PD-L1. Mice bearing MC-38-OVA colon cancer cells expressing NAC-shRNA or control-shRNA were treated with OT-I mouse CD8+ T cells to determine the tumor response to immunotherapy. Immune cells in the tumor tissues were analyzed using flow cytometry. NAC-1, PD-L1 and CD8+ T cells in colon cancer specimens from patients were examined using immunohistochemistry staining. Knockdown of NAC-1 expression in colon cancer cells significantly enhanced the cytocidal effect of CD8+ T cells in cell culture experiments. The sensitizing effect of NAC-1 knockdown on the antitumor action of cytotoxic CD8+ T cells was recapitulated in a colon cancer xenograft animal model. Furthermore, knockdown of NAC-1 in colon cancer cells decreased the expression of PD-L1 at both the mRNA and protein levels, and this effect could be rescued by transfection of an RNAi-resistant NAC-1 expression plasmid. In a reporter gene assay, transient expression of NAC-1 in colon cancer cells increased the promoter activity of PD-L1, indicating that NAC-1 regulates PD-L1 expression at the transcriptional level. In addition, depletion of tumoral NAC-1 increased the number of CD8+ T cells but decreased the number of suppressive myeloid-derived suppressor cells and regulatory T cells. Tumor expression of NAC-1 is a negative determinant of immunotherapy.

EMR1/ADGRE1 Expression in Cancer Cells Upregulated by Tumor-Associated Macrophages Is Related to Poor Prognosis in Colorectal Cancer.

EMR1, a member of the adhesion G protein-coupled receptor family (ADGRE1), is a macrophage marker that is abnormally expressed in cancer cells. However, its clinical significance in colorectal cancer (CRC) is not well-known. In this investigation, EMR1 expression in tumor cells (EMR1-TC) was found in 91 (22.8%) of the 399 CRC samples tested by immunohistochemical staining and showed a significant relationship with lymph node metastasis. Furthermore, EMR1-TC was significantly associated with CD68+ CD163+ tumor-associated macrophages (TAMs), and CRC with a high combined EMR1-TC+CD68+CD163+ score showed worse recurrence-free survival prognosis. In an in vitro co-culture assay of colon cancer cells with myeloid cells, we found that EMR1 expression significantly upregulated in cancer cells was induced by macrophages. In addition, there was increased expression of M2 markers (CD163 and interleukin-6 & 10) in myeloid portion, while that of M1 markers (CD86 and iNOS) remained unchanged. Accordingly, upon treatment with M2 macrophage polarization inhibitors (O-ATP, trametinib, bardoxolone methyl), EMR1 expression reduced significantly, along with M2 markers (CD163 and interleukin-6 & 10). In conclusion, EMR1-TC was a high-risk factor for lymph node metastasis and correlated with poor recurrence free survival, particularly in patients with TAM-rich CRC. Furthermore, EMR1 expression in colon cancer cells may be related to M2 macrophage polarization and vice versa.

ZDHHC9 promotes colon tumor growth by inhibiting effector T cells.

Zinc finger DHHC-type palmitoyltransferase 9 (ZDHHC9) has been reported to play an important role in the occurrence and development of several types of cancer. However, its effects on colon cancer growth remain unclear. Using Gene Expression Profiling Interactive Analysis and Tumor Immune Estimation Resource, data obtained from The Cancer Genome Atlas were analyzed, and the results showed that ZDHHC9 was highly expressed in colon cancer and that patients with higher ZDHHC9 expression levels had a worse prognosis. Inhibition of ZDHHC9 expression promoted the proliferation of colon cancer cells in vitro but decreased their growth in vivo. Additionally, inhibition of ZDHHC9 expression in cancer cells enhanced CD8+ T cell-mediated cytotoxicity in vitro and increased CD8+ T infiltration and activation in vivo. Furthermore, ZDHHC9 promoted IFN-γ-induced JAK/STAT1 activation and upregulated programmed death-ligand 1 (PD-L1) expression in colon cancer cells. In conclusion, the present findings showed that ZDHHC9 promoted colon cancer growth by upregulating the expression of PD-L1 and inhibiting the function of CD8+ T cells.

Metabolomic Signatures in Doxorubicin-Induced Metabolites Characterization, Metabolic Inhibition, and Signaling Pathway Mechanisms in Colon Cancer HCT116 Cells.

Doxorubicin (DOX) is a chemotherapeutic agent is used for various cancer cells. To characterize the chemical structural components and metabolic inhibition, we applied a DOX to HCT116 colon cancer cells using an independent metabolites profiling approach. Chemical metabolomics has been involved in the new drug delivery systems. Metabolomics profiling of DOX-applied HCT116 colon cancer cellular metabolisms is rare. We used 1H nuclear magnetic resonance (NMR) spectroscopy in this study to clarify how DOX exposure affected HCT116 colon cancer cells. Metabolomics profiling in HCT116 cells detects 50 metabolites. Tracking metabolites can reveal pathway activities. HCT116 colon cancer cells were evenly treated with different concentrations of DOX for 24 h. The endogenous metabolites were identified by comparison with healthy cells. We found that acetate, glucose, glutamate, glutamine, sn-glycero-3-phosphocholine, valine, methionine, and isoleucine were increased. Metabolic expression of alanine, choline, fumarate, taurine, o-phosphocholine, inosine, lysine, and phenylalanine was decreased in HCT116 cancer cells. The metabolic phenotypic expression is markedly altered during a high dose of DOX. It is the first time that there is a metabolite pool and phenotypic expression in colon cancer cells. Targeting the DOX-metabolite axis may be a novel strategy for improving the curative effect of DOX-based therapy for colon cancer cells. These methods facilitate the routine metabolomic analysis of cancer cells.