GCSH promotes colorectal cancer progression by inhibiting Cuproptosis through the PI3K/AKT-FDX1 axis.

444 Background: Wnt signaling is well known for its role in colorectal cancer (CRC) formation through transcriptional activities of nuclear β-catenin. Although activation of Wnt signaling depends on specific Wnt/Frizzled receptors (FZD) combinations, the specificity of the interaction and the role of FZD in that particular interaction are still unknown. Among the 10 Wnt receptors of the FZD protein family, FZD-3 is involved in neurodevelopmental abnormalities and gastric cancer carcinogenesis. However, the expression of FZD-3 in CRC is not clear. Therefore in this study, we examined the expression of FZD-3 in CRC cell lines and CRC patient tissues with various pathological stages. The information obtained will be important for us to understand the role of FZD-3 in the development of CRC. Methods: FZD-3 mRNA expression was studied in CRC metastatic SW620, primary SW480 and normal CCD18co cell lines using quantitative real-time polymerase chain reaction with primers and a Taqman minor grove binder probe (Applied Biosystems, Foster City, USA). Moreover, paraffin-embedded specimens of 40 CRC patient tissues, 25 colorectal adenoma (CA) tissues were retrieved from the Department of Pathology, Queen Elizabeth Hospital, Hong Kong Special Administrative Region for FZD-3 immunostaining using an anti-FZD-3 antibody (Catalog no: MAB1001, R&D systems Inc., Minneapolis, USA) in an automatic Ventana Benchmark XT immunostainer (Ventana Medical Systems Inc., Tucson, USA). Results: FZD-3 mRNA was up-regulated in metastatic SW620 cell line (fold-change: 622) and in primary SW480 cell line (fold-change: 820) when compared to that in normal CCD18co cell line. Furthermore, immunostaining showed that FZD-3 protein was expressed in 100% (40/40) of CRC specimens and 84% (21/25) of CA specimens. Detailed analysis showed that FZD-3 protein was significantly up-regulated in CRC, CA when compared to their adjacent normal colorectal epithelial tissues (p < 0.0005, Wilcoxon matched pairs test). Conclusions: This study provided evidence that FZD-3 is involved in CRC carcinogenesis and it is a potential therapeutic target in CRC. No significant financial relationships to disclose.

The aim of the present study was to investigate the effect of the actin‑binding protein Girdin on the proliferation, invasion and migration of colorectal cancer (CRC) cells. Cultured CRC cells (LoVo cell line) were transfected by Girdin‑specific and control shRNA constructs and analyzed for proliferation, invasion and migration by the MTT, Transwell and wound‑healing assays, respectively. The activation of the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway and expression of proinflammatory cytokines was examined by western blotting and ELISA assay, respectively. The effect of Girdin silencing on CRC growth was also evaluated in a xenograft model using nude mice, which were subcutaneously injected with Girdin‑deficient and negative control LoVo cells and analyzed for tumor volume and weight. Transfection of LoVo cells with Girdin‑specific shRNA inhibited Girdin mRNA expression to 27.5% and protein expression to 36.7% when compared with expression levels in the control cells (P<0.001) and significantly demonstrated suppression of LoVo cell proliferation (P<0.05), invasion (P<0.01) and migration (P<0.01). Furthermore, Girdin silencing downregulated the phosphorylation of the signaling proteins JAK (by 42%, P<0.001) and STAT3 (by 34%, P<0.01) and the content of IFN (by 28%, P<0.001) and IL‑6 (by 44%, P<0.001) compared to the control. Notably, inhibition of Girdin expression effectively suppressed tumorigenicity of LoVo cells invivo as evidenced by the reduced volume (P<0.05) and weight (P<0.05) of the tumors derived from Girdin shRNA‑transfected LoVo cells compared to those from the control cells. In conclusion, the silencing of Girdin expression inhibited the malignant behavior of CRC cells via the downregulation of the JAK/STAT signaling pathway, indicating Girdin as a potential therapeutic target in CRC. In the present study, we revealed, for the first time, that the malignant behavior of CRC cells depended on the expression of an actin‑binding protein, Girdin. Silencing of Girdin expression by specific shRNA suppressed the proliferation, invasion, and migration of CRC cells through the decrease in proinflammatory cytokines IFN and IL‑6 and the downregulation of the JAK/STAT signaling pathway. Our findings indicated that Girdin expression may be a potential novel therapeutic target in CRC.

Comprehensive RNA Sequencing in Adenoma-Cancer Transition Identified Predictive Biomarkers and Therapeutic Targets of Human CRC

Fucosylation alteration is involved in several steps of human cancer pathogenesis. Dysregulated long non-coding RNA (lncRNA) often leads to malignancy in colorectal cancer (CRC).Differential levels of LEF1-AS1, LEF1 and FUT8 are analyzed by qRT-PCR and western blot. Chip, RIP, EMSA and luciferase reporter assay confirm the direct interaction among LEF1-AS1, MLL1, H3K4me3, LEF1 and FUT8. Functionally, CRC cell proliferation, migration and invasion are analyzed by CCK8 assay, colony formation assay, transwell assay and flow cytometry. The xenografts nude mice models, lung metastasis and liver metastasis are established to determine the effect of LEF1-AS1/LEF1/FUT8 axis on CRC progression in vivo.Here, we identify that LEF1-AS1 and LEF1 are higher in CRC tissues than that in adjacent tissues, as well as upregulated in CRC cell lines than that in normal colorectal cells. Altered levels of LEF1-AS1 modulate LEF1 expression, while altered LEF1 could not regulate LEF1-AS1. LEF1-AS1 recruits MLL1 to the promoter region of LEF1, induces H3K4me3 methylation modification and mediates LEF1 transcription. Furthermore, α1-6 fucosyltransferase FUT8 is overexpressed in CRC tissues and positively correlated to LEF1. FUT8 is a direct target of transcription factor LEF1, which regulates FUT8 level. Altered FUT8 also regulates the core fucosylation of CRC cells, and LEF1-AS1 mediates FUT8 level through activation of Wnt/β-catenin/LEF1 pathway, thereby resulting in β-catenin nuclear translocation. In addition, LEF1-AS1 mediates the proliferation, migration and invasion of CRC cells in vitro. LEF1-AS1 silence hinders the tumorigenesis, liver and lung metastasis of SW620 cells in vivo, while overexpressed FUT8 abolishes the suppressive impact of LEF1-AS1 repression on the biological behavior of SW620 cells.Our studies uncovered a novel mechanism for constitutive LEF1-AS1/LEF1/FUT8 axis in CRC progression by regulating α1, 6-fucosylation via Wnt/β-catenin pathway, and consequently, as a potential therapeutic target in CRC.

High mobility group box 1 (HMGB1) has been implicated in the development of various cancers, but its role in colorectal cancer (CRC) remains poorly understood. This study investigated the role of HMGB1 in CRC progression, particularly through its interaction with DEAD-box helicase 3 (DDX3), which, as demonstrated by our previous research, regulates CRC via the MAPK pathway. We analysed HMGB1 expression in CRC using public databases and tissue microarrays and detected significantly higher expression in CRC tissues than in normal tissues, which was associated with poor prognosis. HMGB1 expression was knocked down in the SW480 and HCT116 cell lines using siRNA and lentiviral vectors, and this knockdown inhibited CRC cell proliferation, migration, invasion, and adhesion, as confirmed by both in vitro and in vivo experiments. Molecular analyses revealed reduced phosphorylation of Erk1/2, c-Jun, and Elk1, along with decreased β-catenin and Snail expression and increased E-cadherin expression. Coimmunoprecipitation assay results further confirmed the interaction between HMGB1 and DDX3. These findings suggest that HMGB1 is an oncogene in CRC that promotes tumour progression through the MAPK pathway by downregulating DDX3. These findings highlight HMGB1 as a potential therapeutic target in CRC.

While it is known that miR-203 is frequently downregulated in many types of human cancer, little is known regarding its expression and functional role in colorectal cancer (CRC). In this study, we aimed to investigate the expression and the potential mechanisms of miR-203 in colorectal cancer. MiR-203 was significantly downregulated in CRC tissues compared with matched normal adjacent tissues. Our clinical data show that decreased miR-203 was associated with an advanced clinical tumor-node-metastasis stage, lymph node metastasis, and poor survival in CRC patients. Furthermore, externally induced expression of miR-203 significantly inhibited CRC cell proliferation and invasion in vitro and in vivo. Mechanistically, we identified EIF5A2 as a direct and functional target of miR-203. The levels of miR-203 were inversely correlated with levels of the EIF5A2 in the CRC tissues. Restoration of EIF5A2 in the miR-203-overexpressing CRC cells reversed the suppressive effects of miR-203. Our results demonstrate that miR-203 serves as a tumor suppressor gene and may be useful as a new potential therapeutic target in CRC.

ADAMTSL2 facilitates ACLY-mediated lipid metabolism in colorectal cancer by activating Notch signaling pathway.

Colorectal cancer (CRC) accounts for over 600 000 deaths annually worldwide. The current study aims to evaluate the value of proto‐oncogene PIM1 as a therapeutic target in CRC and investigate the anticancer activity of hispidulin, a naturally occurring phenolic flavonoid compound, against CRC. Immunohistochemistry analysis showed that PIM1 was upregulated in CRC tissue. The role of PIM1 as an oncogene was evidenced by the fact that PIM1 knockdown inhibits cell growth, induces apoptosis, and suppresses invasion. Our results showed that hispidulin exerts antitumor activity in CRC through inhibiting the expression of PIM1. Moreover, our findings revealed that hispidulin downregulated the expression of PIM1 by inhibiting JAK2/STAT3 signaling by generating reactive oxygen species. Furthermore, our in vivo studies showed that hispidulin can significantly inhibit tumor growth and metastasis in CRC. Collectively, our results provide an experimental basis for trialing hispidulin in CRC treatment. PIM1 can be considered a potential therapeutic target in CRC.

Background: Colorectal cancer (CRC) persists as one of the most lethal malignancies worldwide, with therapeutic resistance representing a significant obstacle in clinical management. Ferroptosis, a form of programmed cell death triggered by iron accumulation and lipid peroxidation, has recently emerged as a promising target for cancer therapy. Although low-density lipoprotein receptor-related protein 8 (LRP8) has been implicated in oncogenic processes across cancer types, its involvement in CRC progression and ferroptosis regulation has not been fully elucidated. Methods: This study utilized an integrative multi-omics approach, incorporating transcriptomic profiling across the colorectal carcinogenesis spectrum (normal mucosa, adenoma, carcinoma; n=5 each) and proteomic analysis via 4D-DIA mass spectrometry. LRP8 expression patterns were examined in 40 paired CRC and adjacent normal tissues and a tissue microarray comprising 94 cases. Functional investigations were conducted in CRC cell lines following LRP8 knockdown or overexpression. Xenograft models were employed for in vivo validation. Mechanistic insights were gained through co-immunoprecipitation, redox assays, and transmission electron microscopy. Results: Transcriptomic data revealed a stepwise increase in LRP8 expression during CRC development. Clinical analyses demonstrated that elevated LRP8 levels correlated significantly with advanced tumour stage, lymphatic metastasis, and poorer patient prognosis. Functional assays indicated that LRP8 enhances oncogenic behaviors by interacting with SLC3A2. Reintroducing SLC3A2 in LRP8-depleted cells restored glutathione peroxidase 4 (GPX4) expression and mitigated oxidative stress, thereby rescuing ferroptosis resistance. In vivo, silencing LRP8 inhibited tumour growth and induced ferroptosis-associated alterations, including disrupted iron homeostasis and increased lipid peroxidation. Conclusion: LRP8 facilitates CRC progression by antagonizing ferroptosis via modulation of the SLC3A2/GPX4 signalling axis. These findings highlight LRP8 as a previously unrecognized regulator of ferroptotic vulnerability and a potential therapeutic target in CRC.

Background: The prognostic value of many circulating cytokines in metastatic colorectal cancer (CRC) patients is undetermined. By using high through-put approach, this study was to evaluate the prognostic values of circulating cytokines in patients with metastatic CRC and identify potential therapeutic targets. Methods: Thirty-nine cytokines were simultaneously analyzed in serum samples from 99 patients with metastatic CRC by using multiplex bead-based Luminex technology. Three CRC cell lines, together with three more aggressive lines derived from them, were used to validate three potential therapeutic targets by applying neutralizing antibodies. Results: Elevated levels of 17 cytokines were found to be significant risk factors for the overall survival (OS) of patients. Only macrophage-derived chemokine (MDC) positively correlated with better prognosis. Interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1), MCP-3 and MDC were significant independent predictor of OS in multivariate analyses. The risk scoring system developed by combining IL-8, MCP-1, MCP-3, and MDC, was more effective than each individual factor to predictor the OS. IL-8 was secreted by three CRC cell lines, with higher expression observed in more-aggressive cells. Blocking of IL-8 with a neutralizing antibody inhibited the invasiveness of CRC cells. MCP-1 and MCP-3 were secreted by SW480 cell line, with higher expression in more-aggressive cell. Neutralizing antibodies against MCP-1 or MCP-3 significantly inhibited the cellular invasion. Conclusions: The serum levels of 18 cytokines correlated with the prognosis of metastatic CRC patinets. Among them, IL-8, MCP-1, MCP-3 and MDC were independent prognostic factors for OS. IL-8, MCP-1, and MCP-3 are potential therapeutic targets in CRC. Citation Format: Zhi-Yuan Chen, Wen-Zhuo He, Li-Xia Peng, Rong-Ping Guo, Liang-Ping Xia, Chao-Nan Qian. Serum cytokine profiling in metastatic colorectal cancer patients: IL-8, MCP-1, and MCP-3 are theranostic targets. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1225. doi:10.1158/1538-7445.AM2013-1225

Colorectal cancer (CRC) is the most common gastrointestinal tumor worldwide, which is a severe malignant disease that threatens mankind. Cathepsin G (CTSG) has been reported to be associated with tumorigenesis, whereas its role in CRC is still unclear. This investigation aims to determine the function of CTSG in CRC. Our results indicated that CTSG was inhibited in CRC tissues, and patients with CTSG low expression have poor overall survival. Functional experiments revealed that CTSG overexpression suppressed CRC cell progression in vitro and in vivo, whereas CTSG suppression supports CRC development cells in vitro and in vivo. Mechanistically, CTSG overexpression suppressed Akt/mTOR signaling mechanism and elevated apoptotic-associated markers, and CTSG silencing activated Akt/mTOR signaling mechanisms and inhibited apoptotic-associated markers. Furthermore, the Akt suppression signaling pathway by MK2206 abolishes CTSG-silenced expression-induced cell viability and Bcl2 up-regulation in vitro and in vivo. Altogether, these outcomes demonstrate that CTSG may act as a tumor suppressor gene via Akt/mTOR/Bcl2-mediated anti-apoptotic signaling inactivation, and CTSG represents a potential therapeutic target in CRC.

Colorectal cancer (CRC) persists as one of the most lethal malignancies worldwide, with therapeutic resistance representing a significant obstacle in clinical management. Ferroptosis, a form of programmed cell death triggered by iron accumulation and lipid peroxidation, has recently emerged as a promising target for cancer therapy. Although low-density lipoprotein receptor-related protein 8 (LRP8) has been implicated in oncogenic processes across cancer types, its involvement in CRC progression and ferroptosis regulation has not been fully elucidated. This study utilized an integrative multi-omics approach, incorporating transcriptomic profiling across the colorectal carcinogenesis spectrum (normal mucosa, adenoma, carcinoma; n = 5 each) and proteomic analysis via 4D-DIA mass spectrometry. LRP8 expression patterns were examined in 40 paired CRC and adjacent normal tissues and a tissue microarray comprising 94 cases. Functional investigations were conducted in CRC cell lines following LRP8 knockdown or overexpression. Xenograft models were employed for in vivo validation. Mechanistic insights were gained through co-immunoprecipitation, redox assays, and transmission electron microscopy. Transcriptomic data revealed a stepwise increase in LRP8 expression during CRC development. Clinical analyses demonstrated that elevated LRP8 levels correlated significantly with advanced tumour stage, lymphatic metastasis, and poorer patient prognosis. Functional assays indicated that LRP8 enhances oncogenic behaviors by interacting with SLC3A2. Reintroducing SLC3A2 in LRP8-depleted cells restored glutathione peroxidase 4 (GPX4) expression and mitigated oxidative stress, thereby rescuing ferroptosis resistance. In vivo, silencing LRP8 inhibited tumour growth and induced ferroptosis-associated alterations, including disrupted iron homeostasis and increased lipid peroxidation. LRP8 facilitates CRC progression by antagonizing ferroptosis via modulation of the SLC3A2/GPX4 signalling axis. These findings highlight LRP8 as a previously unrecognized regulator of ferroptotic vulnerability and a potential therapeutic target in CRC.

This study was designed to develop novel and better reliable serum prognostic biomarkers for colorectal cancer (CRC). A 50 sample set including CRC, adenoma and healthy control sera was used to identify the serum proteins involved in CRC carcinogenesis using serum proteomic approach. Alpha-2-glycoprotein 1, zinc-binding (AZGP1), pigment epithelium derived factor (PEDF) and peroxiredoxin 2 (PRDX2) were selected as good candidates. Two independent cohorts of 868 individuals were enrolled. The expression of selected proteins in serum from cohort 1 (n = 534) was quantified with enzyme-linked immunosorbent assays. CRC sera of this cohort (n = 405) were assigned to training and test sets, which were used to identify and verify the prognostic markers. The prognostic values of identified proteins were further validated in cohort 2 (n = 334) using quantitative reverse transcription PCR and immunohistochemical staining. Our data showed that the elevated AZGP1 and decreased PEDF and PRDX2 expressions in CRC serum and tissues were correlated with liver metastases. In the training set, higher AZGP1 and lower PEDF levels in sera were significantly associated with a poorer overall survival (OS), higher AZGP1 was also associated with a poorer disease-free survival (DFS). This association was verified in the testing set and further validated in patients in cohort 2. Patients with lower PEDF or PRDX2 levels in their CRC tissues had a significantly poorer DFS or OS than patients with high levels of these proteins in cohort 2. Univariate and multivariate analyses indicated that the prognostic performance of serum AZGP1 and PEDF was independent of other clinicopathological factors. We propose that they may serve as prognostic markers and potential therapeutic targets in CRC.

Bacterial extracellular vesicle ssRNA prevents colorectal cancer progression via Piezo1.

AIMTo compare the expression levels of interleukin (IL)-6 in colorectal cancer (CRC) tissues and adjacent non-cancerous tissues, and analyse the correlation of IL-6 expression with the clinicopathological parameters of CRC.METHODSFifty CRC tissue specimens and 50 matched adjacent mucosa specimens were collected. The expression of IL-6 in these clinical samples was examined by immunohistochemical staining. The correlation between IL-6 expression and clinicopathological parameters was assessed by statistical analysis.RESULTSIL-6 expression was significantly elevated in CRC tissues compared with noncancerous tissues (P < 0.001). IL-6 expression was positively correlated with tumour TNM stage (P < 0.001), but a negative correlation was detected between IL-6 expression and tumor histological differentiation in CRC (P < 0.05). Furthermore, IL-6 expression was associated with invasion depth and lymph node metastasis in CRC.CONCLUSIONIL-6 might be a useful marker for predicting a poor prognosis in patients with CRC and might be used as a potential therapeutic target in CRC.