Poor Prognosis In Colorectal Cancer Research Articles

Wnt/β-catenin signalling activates IMPDH2-mediated purine metabolism to facilitate oxaliplatin resistance by inhibiting caspase-dependent apoptosis in colorectal cancer

BackgroundOxaliplatin resistance usually leads to therapeutic failure and poor prognosis in colorectal cancer (CRC), while the underlying mechanisms are not yet fully understood. Metabolic reprogramming is strongly linked to drug resistance, however, the role and mechanism of metabolic reprogramming in oxaliplatin resistance remain unclear. Here, we aim to explore the functions and mechanisms of purine metabolism on the oxaliplatin-induced apoptosis of CRC.MethodsAn oxaliplatin-resistant CRC cell line was generated, and untargeted metabolomics analysis was conducted. The inosine 5ʹ-monophosphate dehydrogenase type II (IMPDH2) expression in CRC cell lines was determined by quantitative real-time polymerase chain reaction (qPCR) and western blotting analysis. The effects of IMPDH2 overexpression, knockdown and pharmacological inhibition on oxaliplatin resistance in CRC were assessed by flow cytometry analysis of cell apoptosis in vivo and in vitro.ResultsMetabolic analysis revealed that the levels of purine metabolites, especially guanosine monophosphate (GMP), were markedly elevated in oxaliplatin-resistant CRC cells. The accumulation of purine metabolites mainly arose from the upregulation of IMPDH2 expression. Gene set enrichment analysis (GSEA) indicated high IMPDH2 expression in CRC correlates with PURINE_METABOLISM and MULTIPLE-DRUG-RESISTANCE pathways. CRC cells with higher IMPDH2 expression were more resistant to oxaliplatin-induced apoptosis. Overexpression of IMPDH2 in CRC cells resulted in reduced cell death upon treatment with oxaliplatin, whereas knockdown of IMPDH2 led to increased sensitivity to oxaliplatin through influencing the activation of the Caspase 7/8/9 and PARP1 proteins on cell apoptosis. Targeted inhibition of IMPDH2 by mycophenolic acid (MPA) or mycophenolate mofetil (MMF) enhanced cell apoptosis in vitro and decreased in vivo tumour burden when combined with oxaliplatin treatment. Mechanistically, the Wnt/β-catenin signalling was hyperactivated in oxaliplatin-resistant CRC cells, and a reciprocal positive regulatory mechanism existed between Wnt/β-catenin and IMPDH2. Blocking the Wnt/β-catenin pathway could resensitize resistant cells to oxaliplatin, which could be restored by the addition of GMP.ConclusionsIMPDH2 is a predictive biomarker and therapeutic target for oxaliplatin resistance in CRC.

LncRNA PCAT6 is a predictor of poor prognosis of colorectal cancer.

The long non-coding RNA (lncRNA) prostate cancer-associated transcript 6 (PCAT6) has been studied in many cancers, yet its relationship with colorectal cancer (CRC) remains poorly defined. Here, we conducted an analysis of The Cancer Genome Atlas (TCGA) database to better clarify the role of PCAT6 in this cancer type. Wilcoxon rank-sum tests were utilized to assess relative levels of PCAT6 in CRC tumors and normal tissues, while logistic regression analyses were utilized to compare the relationships between PCAT6 levels and clinicopathological findings. Kaplan-Meier curves and Cox regression analyses were used to gauge correlations between PCAT6 and patient survival outcomes, while the biological roles of this lncRNA were investigated via a gene set enrichment analysis (GSEA) approach. The expression level of PCAT6 in CRC cell lines was detected by quantitative reverse transcription polymerase chain reaction (qRT-PCR). PCAT6 levels were significantly correlated with CRC patient lymph node metastasis (N) stage [odds ratio (OR) =1.8 for N1 & N2 vs. N0], lymphatic invasion [OR =1.9 for yes vs. no), distant metastasis (M stage) (OR =2.1 for M1 vs. M0), carcinoembryonic antigen (CEA) level (OR =1.9 for >5 vs. ≤5), perineural invasion (OR =1.9 for yes vs. no), pathologic stage (OR =1.9 for stage III/IV vs. stage I/II), and neoplasm type (OR =2.1 for rectal adenocarcinoma vs. colon adenocarcinoma) (all P<0.05). CRC patients expressing higher PCAT6 levels exhibited poorer survival outcomes than those expressing low levels of this lncRNA (P=0.017), and in univariate analyses, higher PCAT6 levels were linked to worse overall survival [hazard ratio (HR) =1.540; 95% confidence interval (CI): 1.079-2.199; P=0.017], with this relationship also being preserved in a multivariate analysis (HR =6.892; 95% CI: 1.713-27.727, P=0.007). GSEA revealed high PCAT6 expression to be linked to differential DNA methylation enrichment, with high PCAT6 levels being associated with changes in base excision repair, cellular senescence, G2/M DNA damage checkpoint, chromatin-modifying enzyme, and gene silencing by RNA activity. The high expression of lncRNA PCAT6 in CRC cell lines was demonstrated by PCR experiments. PCAT6 represents a promising prognostic biomarker of poor CRC patient survival outcomes, with DNA methylation and RNA-mediated gene silencing being potentially promising mechanistic pathways whereby this lncRNA may shape patient outcomes.

Integrative Multi-Omics Analysis Identifies Transmembrane p24 Trafficking Protein 1 (TMED1) as a Potential Prognostic Marker in Colorectal Cancer.

Several TMED protein family members are overexpressed in malignant tumors and associated with tumor progression. TMED1 belongs to the TMED protein family and is involved in protein vesicular trafficking. However, the expression level and biological role of TMED1 in colorectal cancer (CRC) have yet to be fully elucidated. In this study, the integration of patient survival and multi-omics data (immunohistochemical staining, transcriptomics, and proteomics) revealed that the highly expressed TMED1 was related to the poor prognosis in CRC. Crystal violet staining indicated the cell growth was reduced after knocking down TMED1. Moreover, the flow cytometry results showed that TMED1 knockdown could increase cell apoptosis. The expression of TMED1 was positively correlated with other TMED family members (TMED2, TMED4, TMED9, and TMED10) in CRC, and the protein-protein interaction network suggested its potential impact on immune regulation. Furthermore, TMED1 expression was positively associated with the infiltration levels of regulatory T cells (Tregs), cancer-associated fibroblasts (CAFs), and endothelial cells and negatively correlated with the infiltration levels of CD4+ T cells, CD8+ T cells, and B cells. At last, the CTRP and GDSC datasets on the GSCA platform were used to analyze the relationship between TMED1 expression and drug sensitivity (IC50). The result found that the elevation of TMED1 was positively correlated with IC50 and implied it could increase the drug resistance of cancer cells. This research revealed that TMED1 is a novel prognostic biomarker in CRC and provided a valuable strategy for analyzing potential therapeutic targets of malignant tumors.

Fabrication of hyaluronic acid-inulin coated Enterococcus faecium for colon-targeted delivery to fight Fusobacterium nucleatum

The abundance of Fusobacterium nucleatum (F. nucleatum) is highly associated with the development and poor prognosis of colorectal cancer (CRC), which is regarded as a promising target for CRC. However, until now, the novel strategy to clear F. nucleatum in the colon and CRC has not been well proposed. Herein, a probiotic strain Enterococcus faecium (E. faecium, EF47) is verified to secrete various organic acids and bacteriocins to exert superior antimicrobial activity towards F. nucleatum. However, the oral delivery of EF47 is affected by the complex digestive tract environment, so we design the hyaluronic acid-inulin (HA-IN) coated EF47 for colon-targeted delivery to fight F. nucleatum. IN can protect EF47 from the harsh gastrointestinal tract environment and is degraded specifically in the colon, acting as prebiotics to further promote the proliferation of EF47. The exposed HA can also enhance the targeting effect to the tumor area via the interaction with the CD44 receptor on the tumor cells, which is confirmed to increase the adhesive ability in tumor tissues and inhibit the growth of F. nucleatum. Therefore, this colon-targeted delivery system provides a novel platform to realize high-activity and adhesive delivery of probiotics to assist the therapeutic efficiency of CRC.

Endoscopic obstruction predominantly occurs in right-side colon cancer and endoscopic obstruction with tumor size ≤ 5cm seems poor prognosis in colorectal cancer.

Endoscopic obstruction (eOB) is associated with a poor prognosis in colorectal cancer (CRC). Our study aimed to investigate the association between tumor location and eOB, as well as the prognostic differences among non-endoscopic obstruction (N-eOB), eOB with tumor size ≤ 5cm, and eOB with tumor size > 5cm in non-elderly patients. We retrospectively reviewed the clinicopathological variables of 230 patients with CRC who underwent curative surgery. The multivariable logistic regression model was used to identify risk factors for eOB. The association between eOB with tumor size ≤ 5cm and disease-free survival (DFS) was evaluated using multivariate cox regression analysis. A total of 87 patients had eOB while 143 had N-eOB. In multivariate analysis, preoperative carcinoembryonic antigen (p = 0.014), tumor size (p = 0.010), tumor location (left-side colon; p = 0.033; rectum; p < 0.001), and pT stage (T3, p = 0.009; T4, p < 0.001) were significant factors of eOB. The DFS rate for eOB with tumor size ≤ 5cm was significantly lower (p < 0.001) in survival analysis. The eOB with tumor size ≤ 5cm (p = 0.012) was an unfavorable independent factor for DFS. The patients with eOB were significantly associated with right-side colon cancer as opposed to left-side colon cancer and rectal cancer. The eOB with tumor size ≤ 5 cm was an independent poor prognostic factor. Further studies are needed to target these high-risk groups.

METTL16 Promotes Stability of SYNPO2L mRNA and leading to Cancer Cell Lung Metastasis by Secretion of COL10A1 and attract the Cancer-Associated Fibroblasts.

The occurrence of metastasis is a major factor contributing to poor prognosis in colorectal cancer. Different stages of the disease play a crucial role in distant metastasis. Furthermore, m6A has been demonstrated to play a significant role in regulating tumor metastasis. Therefore, we conducted an analysis of transcriptome data from high-stage and low-stage colorectal cancer patients in The Cancer Genome Atlas (TCGA) to identify genes associated with m6A-related regulation. We identified SYNPO2L as a core gene regulated by m6A, and it is correlated with adverse prognosis and metastasis in patients. Additionally, we demonstrated that the m6A writer gene Mettl16 can regulate the stability of SYNPO2L through interaction with YTHDC1. Subsequently, using Weighted Gene Co-expression Network Analysis (WGCNA), we discovered that SYNPO2L can regulate COL10A1, mediating the actions of Cancer-Associated Fibroblasts. SYNPO2L promotes the secretion of COL10A1 and the infiltration of tumor-associated fibroblasts, thereby facilitating Epithelial-Mesenchymal Transition (EMT) in tumor cells and making them more prone to distant metastasis.

Citraconate promotes the malignant progression of colorectal cancer by inhibiting ferroptosis.

Metastasis is a principal factor in the poor prognosis of colorectal cancer. Recent studies have found microbial metabolites regulate colorectal cancer metastasis. By analyzing metabolomics data, we identified an essential fecal metabolite citraconate that potentially promotes colorectal cancer metastasis. Next, we tried to reveal its effect on colorectal cancer and the underlying mechanism. Firstly, the response of colorectal cancer cells (HCT116 and MC38 cells) to citraconate was assessed by Cell Counting Kit-8 assay, clonogenic assay, transwell migration and invasion assay. Moreover, we utilized an intra-splenic injection model to evaluate the effect of citraconate on colorectal cancer liver metastasis in vivo. Then molecular approaches were employed, including RNA sequencing, mass spectrometry-based metabolomics, western blot, quantitative real-time PCR, cell ferrous iron colorimetric assay and intracellular malondialdehyde measurement. In vitro, citraconate promotes the growth of colorectal cancer cells. In vivo, citraconate aggravated liver metastasis of colorectal cancer. Mechanistically, downstream genes of NRF2, NQO1, GCLC, and GCLM high expression induced by citraconate resulted in resistance to ferroptosis of colorectal cancer cells. In summary, citraconate promotes the malignant progression of colorectal cancer through NRF2-mediated ferroptosis resistance in colorectal cancer cells. Furthermore, our study indicates that fecal metabolite may be crucial in colorectal cancer development.

LINC00339 accelerates invasion and migration of colorectal cancer via mediating miRNA-30a-5p.

To analyze the biological function of LINC00339 in the progression of colorectal cancer (CRC). We aim to provide directions in the early-stage treatment of CRC. LINC00339 level in 60 paired CRC tissues and paracancerous tissues was examined by quantitative real-time polymerase chain reaction (qRT-PCR). The correlation between the LINC00339 level and clinical parameters was analyzed. Moreover, the LINC00339 level in CRC cell lines was determined as well. LINC00339 expression was changed in HCT-8 and HCT-116 cell lines by transfection of LINC00339 overexpression plasmid or anti-LINC00339. The regulatory effects of LINC00339 on the migratory and invasive abilities of CRC cells were evaluated through a series of functional experiments. Dual-luciferase reporter gene assay and rescue experiments were conducted to verify the interaction of LINC00339 and miRNA-30a-5p in mediating the progression of CRC. LINC00339 was upregulated in CRC tissues relative to paracancerous tissues. CRC patients with higher levels of LINC00339 had higher rates of lymph node metastasis and distant metastasis, and worse prognosis than those with lower levels. Knockdown of LINC00339 attenuated migratory and invasive abilities of HCT-116 cells. Overexpression of LINC00339 in HCT-8 obtained the opposite trends. In addition, we verified a negative correlation between LINC00339 and miRNA-30a-5p in CRC tissues. LINC00339 served as a ceRNA to absorb miRNA-30a-5p. Rescue experiments confirmed that miRNA-30a-5p knockdown revered the regulatory effects of LINC00339 on the migratory and invasive abilities of CRC cells. LINC00339 was closely correlated to metastasis and poor prognosis of CRC. It accelerates CRC cells to migrate and invade via mediating miRNA-30a-5p.

Low expression of ZHX3 is associated with progression and poor prognosis in colorectal cancer

Accumulating studies suggest that ZHX3, the member of ZHX family, is involved in a variety of biological functions such as development and differentiation. Recently, ZHX3 may also be involved in the progression of several cancer types including renal cancer, gastric cancer, liver cancer and breast cancer. However, the potential role of ZHX3 in colorectal cancer (CRC) is still unknown. In this study, we analyzed the protein levels of ZHX3 by immunohistochemistry and evaluated its relationship with the clinicopathological features and prognosis in 286 CRC patients. In vitro cell proliferation assay, plate colony formation assay and xenograft model in nude mice were applied to evaluate CRC cell proliferative ability. Our results showed that the expression of ZHX3 was significantly downregulated in CRC tissues compared with paired adjacent nontumor tissues. Furthermore, the ZHX3 expression was found to have a strong correlation with tumor size, tumor invasion depth and TNM stage. Kaplan-Meier analysis demonstrated that low ZHX3 expression was related to a poorer overall survival and disease-free survival in CRC patients. In addition, cox's proportional hazards analysis indicated that low ZHX3 expression was an independent prognostic indicator of poor prognosis. Functionally, reduced expression of ZHX3 promotes the proliferation of CRC cells both in vitro and in vivo. Conversely, overexpression of ZHX3 inhibited the growth of CRC cells, indicated that ZHX3 was significantly correlated with CRC progression. Our results indicate for the first time that ZHX3 may be a potential marker of cancer prognosis and CRC recurrence.

Identification of necroptosis-related lncRNAs for prognosis prediction and screening of potential drugs in patients with colorectal cancer

BACKGROUND Tumor recurrence and metastasis lead to a poor prognosis in colorectal cancer (CRC). Necroptosis is closely related to the tumor microenvironment (TME) and affects tumor recurrence and metastasis. We aimed to stratify CRC patients according to necroptosis-related long noncoding RNAs (lncRNAs), which can be used to not only evaluate prognosis and improve precision medicine in clinical practice but also screen potential immunotherapy drugs. AIM To stratify CRC patients according to necroptosis-related lncRNAs (NRLs), which can be used to not only evaluate prognosis and improve precision medicine in clinical practice but also screen potential immunotherapy drugs. METHODS LncRNA expression profiles were collected from The Cancer Genome Atlas. NRLs were identified by coexpression analysis. Cox regression analysis identified a NRL signature. Then, the value of this signature was comprehensively and multidimensionally evaluated, and its reliability for CRC prognosis prediction was assessed with clinical CRC data and compared with that of six other lncRNA signatures. Gene set enrichment analysis, TME analysis and half-maximal inhibitory concentration (IC50) prediction were also performed according to the risk score (RS) of the signature. RESULTS An 8-lncRNA signature significantly associated with overall survival (OS) was constructed, and its reliability was validated with clinical CRC data. Most of the areas under the receiver operating characteristic curves (AUCs) values for 1-, 3- and 5-year OS for this signature were higher than those for the other six lncRNA signatures. OS, disease-specific survival and the progression-free interval were all significantly poorer in the high-risk group. The RS of the signature showed good concordance with the predicted prognosis, with AUCs for 1-, 3- and 5-year OS of 0.79, 0.81 and 0.77, respectively. Additionally, the calibration plots for this signature combined with clinical factors showed that this combination could effectively improve the ability to predict OS. The RS was correlated with tumor stage, lymph node metastasis and distant metastasis. Most of the enriched Kyoto Encyclopedia of Genes and Genomes and Gene Ontology terms were tumor metastasis-related pathways in the high-risk group; these patients showed greater infiltration of immunosuppressive cells, such as cancer-associated fibroblasts, hematopoietic stem cells and M2 macrophages, but less infiltration of infiltrating antitumor effector immune cells, such as cluster of differentiation 8+ T cells and regulatory T cells (Tregs). We explored additional potential immune checkpoint genes and potential immunotherapeutic and chemotherapeutic drugs with relatively low IC50 values. CONCLUSION We identified an NRL signature with strong fidelity that could stably predict prognosis and might be an indicator of the TME of CRC. Furthermore, additional potential immunotherapeutic and chemotherapeutic drugs were explored.

High expression of CDKN2A is associated with poor prognosis in colorectal cancer and may guide PD-1-mediated immunotherapy

BackgroundColorectal cancer (CRC) is one of the most common malignancies worldwide. Immunotherapy targeting the programmed death protein 1(PD-1) and its ligand (PD-L1), is a promising treatment option for many cancers, but has exhibited poor therapeutic efficacy in CRC. This study aimed to identify and validate the prognostic value of immune-related genes and PD-1-associated genes for immunotherapy treatment of CRC.MethodsAn extensive analysis of prognostic immune-related DEGs and PD-1-related genes has highlighted CDKN2A as a vital overlapping gene. To further explore its expression in CRC and its prognostic value, we conducted qRT-PCR, Western blot experiments, and consulted various databases. Subsequently, we conducted gene expression analysis, survival and prognostic analysis, enrichment analysis, immune infiltration assessment, and TIDE analysis to assess the significance of CDKN2A.ResultsIn CRC, CDKN2A was highly expressed compared to normal tissue. It was found that CDKN2A expression was related to clinicopathological features such as inflammation and tumor stage. Furthermore, a significant correlation was identified between CDKN2A and immune infiltration, specifically involving CD4 T cells, CD8 T cells, and macrophages. The analysis of the GSEA of CRC samples with high CDKN2A expression identified enrichment of genes involved in MYC target-v2 and metabolism pathways. Furthermore, UBE2I, CDK4, CDK6, TP53, and CCND1 were found to be significantly coexpressed with CDKN2A, suggesting a potential role that these gene play in CRC and immunotherapy.ConclusionsOur study revealed that high CDKN2A expression in CRC is a potentially valuable prognostic biomarker, which may guide PD-1-mediated immunotherapy.

CRISPR–Cas9-based functional interrogation of unconventional translatome reveals human cancer dependency on cryptic non-canonical open reading frames

Emerging evidence suggests that cryptic translation beyond the annotated translatome produces proteins with developmental or physiological functions. However, functions of cryptic non-canonical open reading frames (ORFs) in cancer remain largely unknown. To fill this gap and systematically identify colorectal cancer (CRC) dependency on non-canonical ORFs, we apply an integrative multiomic strategy, combining ribosome profiling and a CRISPR–Cas9 knockout screen with large-scale analysis of molecular and clinical data. Many such ORFs are upregulated in CRC compared to normal tissues and are associated with clinically relevant molecular subtypes. We confirm the in vivo tumor-promoting function of the microprotein SMIMP, encoded by a primate-specific, long noncoding RNA, the expression of which is associated with poor prognosis in CRC, is low in normal tissues and is specifically elevated in CRC and several other cancer types. Mechanistically, SMIMP interacts with the ATPase-forming domains of SMC1A, the core subunit of the cohesin complex, and facilitates SMC1A binding to cis-regulatory elements to promote epigenetic repression of the tumor-suppressive cell cycle regulators encoded by CDKN1A and CDKN2B. Thus, our study reveals a cryptic microprotein as an important component of cohesin-mediated gene regulation and suggests that the ‘dark’ proteome, encoded by cryptic non-canonical ORFs, may contain potential therapeutic or diagnostic targets.

METTL3/IGF2BP2 axis affects the progression of colorectal cancer by regulating m6A modification of STAG3

Colorectal cancer (CRC) is among the commonest malignant tumors of humans. Existing evidence has linked the poor prognosis of CRC with high expression of stromal antigen 3 (STAG3), but, the exact biological effect of STAG3 in CRC is still unclear. The aim of this research is to reveal the biological function and molecular mechanism of STAG3 in CRC. To investigate the differential expression of STAG3 in CRC tissues and cell lines compared to normal colon tissues and cell lines, Western blot (WB) and quantitative real-time PCR (qRT-PCR) techniques were utilized. STAG3 N6-methyladenosine (m6A) modification level were identified using m6A RNA immunoprecipitation (MeRIP). Additionally, the functional roles of methyltransferase-like protein 3 (METTL3) and insulin-like growth factor 2 mRNA binding protein 2 (IGF2BP2) in CRC were explored by manipulating their levels via knockdown or overexpression. Cell proliferation was evaluated through Cell Counting Kit 8 (CCK-8) and clone formation experiments, while cell migration was assessed through wound healing experiments. Furthermore, cell apoptosis was detected using flow cytometry, and the protein expressions associated with proliferation and apoptosis were detected using WB. To identify the specific binding of target genes, RIP and pull-down assays were employed. Finally, the biological function of STAG3 in vivo was investigated through a xenotransplantation mouse tumor model. In CRC tissues and cell lines, STAG3 was up-regulated and accompanied by m6A methylation. Additionally, the expression of METTL3 was found to be upregulated in CRC tissues. Knocking down METTL3 resulted in a decrease in both the m6A level and protein expression of STAG3, inhibited cell proliferation and migration while promoting apoptosis, which were restored through STAG3 overexpression. Furthermore, online prediction indicated the interaction between STAG3 mRNA and IGF2BP2 protein, which was further verified by RIP experiments. IGF2BP2 downregulation led to decreased STAG3 protein expression, cell proliferation, and migration, but increased apoptosis. However, these impacts were reversed by STAG3 overexpression. Finally, subcutaneous tumor experiments conducted in nude mice also confirmed that METTL3 regulated CRC progression through STAG3 in vivo. The METTL3/IGF2BP2/STAG3 axis affects CRC progression in an m6A modification-dependent manner. This may guide targeted therapy in CRC patients.

ELK4 Promotes Colorectal Cancer Progression by Activating the Neoangiogenic Factor LRG1 in a Noncanonical SP1/3-Dependent Manner.

Although the MAPK/MEK/ERK pathway is prevalently activated in colorectal cancer (CRC), MEK/ERK inhibitors show limited efficiency in clinic. As a downstream target of MAPK, ELK4 is thought to work primarily by forming a complex with SRF. Whether ELK4 can serve as a potential therapeutic target is unclear and the transcriptional regulatory mechanism has not been systemically analyzed. Here,it isshown that ELK4 promotes CRC tumorigenesis. Integrated genomics- and proteomics-based approaches identified SP1 and SP3, instead of SRF, as cooperative functional partners of ELK4 at genome-wide level in CRC. Serum-induced phosphorylation of ELK4 by MAPKs facilitated its interaction with SP1/SP3. The pathological neoangiogenic factor LRG1isidentified as a direct target of the ELK4-SP1/SP3 complex. Furthermore, targeting the ELK4-SP1/SP3 complex by combination treatment with MEK/ERK inhibitor and the relatively specific SP1 inhibitor mithramycin A (MMA) elicited a synergistic antitumor effect on CRC. Clinically, ELK4isa marker of poor prognosis in CRC. A 9-gene prognostic model based on the ELK4-SP1/3 complex-regulated gene set showed robust prognostic accuracy.Theresults demonstrate that ELK4 cooperates with SP1 and SP3 to transcriptionally regulate LRG1 to promote CRC tumorigenesis in an SRF-independent manner, identifying the ELK4-SP1/SP3 complex as a potential target for rational combination therapy.

Efficient targeting of HIF-1α mediated by YC-1 and PX-12 encapsulated niosomes: potential application in colon cancer therapy

A number of molecular biofactors have been documented in pathogenesis and poor prognosis of colorectal cancer (CRC). Among them, the Hypoxia-Inducible Factor (HIF-1a) is frequently reported to become over-expressed, and its targeting could restrict and control a variety of essential hallmarks of CRC. Niosomes are innovative drug delivery vehicles with the encapsulating capacity for co-loading both hydrophilic and hydrophobic drugs at the same time. Also, they can enhance the local accumulation while minimizing the dose and side effects of drugs. YC-1 and PX-12 are two inhibitors of HIF-1a. The purpose of this work was to synthesize dual-loaded YC-1 and PX-12 niosomes to efficiently target HIF-1α in CRC, HT-29 cells. The niosomes were prepared by the thin-film hydration method, then the niosomal formulation of YC-1 and PX-12 (NIO/PX-YC) was developed and optimized by the central composition method (CCD) using the Box-Behnken design in terms of size, polydispersity index (PDI), entrapment efficiency (EE). Also, they are characterized by DLS, FESEM, and TEM microscopy, as well as FTIR spectroscopy. Additionally, entrapment efficiency, in vitro drug release kinetics, and stability were assessed. Cytotoxicity, apoptosis, and cell cycle studies were performed after the treatment of HT-29 cells with NIO/PX-YC. The expression of HIF-1αat both mRNA and protein levels were studied after NIO/PX-YC treatment. The prepared NIO/PX-YC showed a mean particle size of 185 nm with a zeta potential of about-7.10 mv and a spherical morphology. Also, PX-12 and YC-1 represented the entrapment efficiency of about %78 and %91, respectively, with a sustainable and controllable release. The greater effect of NIO/PX-YC than the free state of PX-YC on the cell survival rate, cell apoptosis, and HIF-1α gene/protein expression were detected (p < 0.05). In conclusion, dual loading of niosomes with YC-1 and PX-12 enhanced the effect of drugs on HIF-1α inhibition, thus boosting their anticancer effects.Graphical

High expression of NOLC1 as an independent prognostic factor for survival in patients with colorectal cancer

BackgroundAs a phosphorylated protein, NOLC1 is mainly located in the nucleus and is highly expressed in a variety of tumors, participating in the regulation of cell proliferation and aging. This study further investigated the role of NOLC1 in colorectal cancer tumors, aiming to provide sufficient scientific evidence for the clinical treatment of colorectal cancer.MethodsWe used TCGA, GEO, TNMplot, GEPIA, and other databases to explore the expression level of NOLC1 in colorectal cancer patients, as well as the correlation between the clinical characteristics of colorectal cancer patients and their expression, and conducted the prognostic analysis. Immunohistofluorescence (IHF) staining verified the analytical results. Subsequently, KEGG and GO enrichment analysis was used to identify the potential molecular mechanism of NOLC1 promoting the occurrence and development of colorectal cancer. The influence of NOLC1 expression on the immune microenvironment of colorectal cancer patients was further investigated using the TIMER database. GDSC database analysis was used to screen out possible anti-colorectal cancer drugs against NOLC1. Finally, we demonstrated the effect of NOLC1 on the activity and migration of colorectal cancer cells by Edu Cell proliferation assay and Wound Healing assay in vitro.ResultsOur results suggest that NOLC1 is overexpressed in colorectal cancer, and that overexpression of NOLC1 is associated with relevant clinical features. NOLC1, as an independent risk factor affecting the prognosis of colorectal cancer patients, can lead to a poor prognosis of colorectal cancer. In addition, NOLC1 may be associated with MCM10, HELLS, NOC3L, and other genes through participating in Wnt signaling pathways and jointly regulate the occurrence and development of colorectal cancer under the influence of the tumor microenvironment and many other influencing factors. Related to NOLC1: Selumetinib, Imatinib, and targeted drugs such as Lapatinib have potential value in the clinical application of colorectal cancer. NOLC1 enhances the proliferation and migration of colorectal cancer cells.ConclusionsHigh expression of NOLC1 as an independent prognostic factor for survival in patients with colorectal cancer. NOLC1 enhances the proliferation and migration of colorectal cancer cells. Further studies and clinical trials are needed to confirm the role of NOLC1 in the development and progression of colorectal cancer.

Identification of AOC3 and LRRC17 as Colonic Fibroblast Activation Markers and Their Potential Roles in Colorectal Cancer Progression.

Accumulation of cancer-associated fibroblasts (CAFs) in the tumor stroma is linked to poor prognosis in colorectal cancer (CRC). CAF-cancer cell interplay, facilitated by secretomes including transforming growth factor-beta 1 (TGF-β1), supports fibroblast activation, drives colorectal carcinogenesis, and contributes to CRC aggressive phenotypes. Although widely used, traditional CAF biomarkers are found to have heterogeneous and non-specific expression. Amine oxidase copper containing 3 (AOC3) and leucine-rich repeat-containing 17 (LRRC17) have been reported to be emerging markers of myofibroblasts. Our objective was to investigate the potential of AOC3 and LRRC17 as biomarkers for fibroblast activation thus predicting their roles in CRC progression. Immunofluorescence (IF) staining of AOC3 and LRRC17 was performed on myofibroblast line (CCD-112CoN), primary fibroblasts from colorectal tumor (CAFs), and adjacent normal tissue (normal fibroblasts-NFs). SW620 (epithelial CRC cell line) was used as a control. Conventional CAF biomarker (alpha-smooth muscle actin - α-SMA) was included in the IF analysis. Fluorescence intensity was compared between groups using ImageJ software. Proliferation and contractility of treated cells were assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and collagen gel contraction assays, respectively. Fibroblast contraction under TGF-β1 treatment was compared to those treated with complete medium (addition of 10% serum) and serum free (SF) medium. Positive AOC3, LRRC17, and α-SMA expression were observed in colonic fibroblasts, more prominent in CAFs, whereas negative staining was found in SW620. Significant downregulation of AOC3, and upregulations in LRRC17 and α-SMA expression was found in TGF-β1-treated fibroblasts compared to SF medium treatment (p-value<0.05). All fibroblasts exhibited higher proliferation in complete medium and under treatment with conditioned medium from SW620 than SF medium. Significant contraction of NFs was recorded in complete medium and TGF-β1 (p-value<0.01). Our results demonstrate AOC3 and LRRC17 as the potential markers of CAF activation which promote CRC progression.

CircAGFG1 absence decreases PKM2 expression to enhance oxaliplatin sensitivity in colorectal cancer in a miR-7-5p-dependent manner

Circular RNA (circRNA) ArfGAP with FG repeats 1 (circAGFG1) contributes to colorectal cancer (CRC) development. However, whether circAGFG1 regulates the resistance of CRC to oxaliplatin (L-OHP) remains unknown. CircAGFG1, microRNA-7-5p (miR-7-5p) and pyruvate kinase M2 (PKM2) RNA expression were quantified by quantitative real-time polymerase chain reaction. Protein expression was detected by western blot assay and immunohistochemistry assay. Glycolysis was analyzed through glucose uptake, lactate production and adenosine triphosphate (ATP) concentration assays. 50% inhibitory concentration of L-OHP was determined by cell counting kit-8 assay. Cell proliferation and apoptotic rate were analyzed by cell colony formation and flow cytometry analysis, respectively. Dual-luciferase reporter assay was used to identify the relationship among circAGFG1, miR-7- 5p and PKM2. The effect of circAGFG1 on L-OHP sensitivity in vivo was further evaluated by a xenograft model assay. CircAGFG1 and PKM2 expression were significantly increased, while miR-7-5p was decreased in L-OHP-resistant CRC tissues and cells. High circAGFG1 expression predicted a poor prognosis of CRC. CircAGFG1 knockdown or PKM2 depletion decreased glycolysis and cell proliferation and increased L-OHP sensitivity and cell apoptosis. PKM2 introduction rescued circAGFG1 silencing-induced effects in CRC cells. In terms of mechanism, circAGFG1 bound to miR-7-5p, which was identified to target PKM2. Also, circAGFG1 regulated PKM2 expression by interacting with miR-7-5p. Further, circAGFG1 knockdown improved the sensitivity of tumors to L-OHP in vivo. CircAGFG1 depletion inhibited L-OHP resistance by regulating the miR-7-5p/PKM2 pathway.

Integrated analysis of single cell and bulk RNA sequencing identifies CTHRC1+ INHBA+ CAF as drivers of colorectal cancer progression.

Cancer-associated fibroblasts (CAFs) are a key component of the tumor microenvironment and a critical factor in the progression of colorectal cancer (CRC). The aim of this study was to screen for CAFs specific genes that could serve as promising therapeutic targets for CRC patients. Our findings showed a significant increase in the proportion of fibroblasts in CRC tissues, and a high proportion of fibroblasts was associated with immune escape and poor prognosis in CRC. Collagen triple helix repeat containing 1 (CTHRC1) and inhibin subunit beta A (INHBA) were identified as key genes in the progression of CRC, primarily expressed in CAFs and significantly upregulated in CRC tissues. We defined CTHRC1 and INHBA as cancer-associated fibroblast-related genes (CAFRGs), which were associated with poor prognosis in CRC and macrophage polarization. CAFRGs promoted immune escape and metastasis in CRC and were good predictors of immune therapy response. Drug sensitivity analysis showed that the high expression group of CAFRGs was sensitive to 15 chemotherapy drugs, while the low expression group was sensitive to only 3. Clustering of fibroblasts in the tumor revealed that CTHRC1+ INHBA+ CAF was a poor prognostic factor in CRC and was associated with extracellular matrix remodeling and immune regulation. In conclusion, our study provides new theoretical basis for effective treatment strategies and therapeutic targets for CRC.

The Pathogenicity of Fusobacterium nucleatum Modulated by Dietary Fibers-A Possible Missing Link between the Dietary Composition and the Risk of Colorectal Cancer.

The dietary composition has been approved to be strongly associated with the risk of colorectal cancer (CRC), one of the most serious malignancies worldwide, through regulating the gut microbiota structure, thereby influencing the homeostasis of colonic epithelial cells by producing carcinogens, i.e., ammonia or antitumor metabolites, like butyrate. Though butyrate-producing Fusobacterium nucleatum has been considered a potential tumor driver associated with chemotherapy resistance and poor prognosis in CRC, it was more frequently identified in the gut microbiota of healthy individuals rather than CRC tumor tissues. First, within the concentration range tested, the fermentation broth of F. nucleatum exhibited no significant effects on Caco-2 and NCM460 cells viability except for a notable up-regulation of the expression of TLR4 (30.70%, p < 0.0001) and Myc (47.67%, p = 0.021) and genes encoding proinflammatory cytokines including IL1B (197.57%, p < 0.0001), IL6 (1704.51%, p < 0.0001), and IL8 (897.05%, p < 0.0001) in Caco-2 cells exclusively. Although no marked effects of polydextrose or fibersol-2 on the growth of F. nucleatum, Caco-2 and NCM460 cells were observed, once culture media supplemented with polydextrose or fibersol-2, the corresponding fermentation broths of F. nucleatum significantly inhibited the growth of Caco-2 cells up to 48.90% (p = 0.0003, 72 h, 10%) and 52.96% (p = 0.0002, 72 h, 10%), respectively in a dose-dependent manner. These two kinds of fibers considerably promoted butyrate production of F. nucleatum up to 205.67% (p < 0.0001, 6% polydextrose at 24 h) and 153.46% (p = 0.0002, 6% fibersol-2 at 12 h), which explained why and how the fermentation broths of F. nucleatum cultured with fibers suppressing the growth of Caco-2 cells. Above findings indicated that dietary fiber determined F. nucleatum to be a carcinogenic or antitumor bacterium, and F. nucleatum played an important role in the association between the dietary composition, primarily the content of dietary fibers, and the risk of CRC.