Poor Prognosis In Colorectal Carcinoma Patients Research Articles

Lectin-Based Immunophenotyping and Whole Proteomic Profiling of CT-26 Colon Carcinoma Murine Model.

A murine colorectal carcinoma (CRC) model was established. CT26 colon carcinoma cells were injected into BALB/c mice's spleen to study the primary tumor and the mechanisms of cell spread of colon cancer to the liver. The CRC was verified by the immunohistochemistry of Pan Cytokeratin and Vimentin expression. Immunophenotyping of leukocytes isolated from CRC-bearing BALB/c mice or healthy controls, such as CD19+ B cells, CD11+ myeloid cells, and CD3+ T cells, was carried out using fluorochrome-labeled lectins. The binding of six lectins to white blood cells, such as galectin-1 (Gal1), siglec-1 (Sig1), Sambucus nigra lectin (SNA), Aleuria aurantia lectin (AAL), Phytolacca americana lectin (PWM), and galectin-3 (Gal3), was assayed. Flow cytometric analysis of the splenocytes revealed the increased binding of SNA, and AAL to CD3 + T cells and CD11b myeloid cells; and increased siglec-1 and AAL binding to CD19 B cells of the tumor-bearing mice. The whole proteomic analysis of the established CRC-bearing liver and spleen versus healthy tissues identified differentially expressed proteins, characteristic of the primary or secondary CRC tissues. KEGG Gene Ontology bioinformatic analysis delineated the established murine CRC characteristic protein interaction networks, biological pathways, and cellular processes involved in CRC. Galectin-1 and S100A4 were identified as upregulated proteins in the primary and secondary CT26 tumor tissues, and these were previously reported to contribute to the poor prognosis of CRC patients. Modelling the development of liver colonization of CRC by the injection of CT26 cells into the spleen may facilitate the understanding of carcinogenesis in human CRC and contribute to the development of novel therapeutic strategies.

Evaluation of HHLA2 and CD8 Immunohistochemical Expression in Colorectal Carcinoma and Their Prognostic Significance.

Colorectal carcinoma (CRC) is the third most common malignancy worldwide. Human endogenous retrovirus H long terminal repeat-associating protein 2 (HHLA2) is a novel immune checkpoint molecule. The association between HHLA2 expression and clinicopathological features and its prognostic significance in CRC patients are still controversial. The aim of this study is to evaluate the prognostic value of immunohistochemical (IHC) expression of HHLA2 and CD8 in CRC. This retrospective study included 134 cases diagnosed with primary CRC at the Gastrointestinal Surgery Center (GISC) department, Mansoura Faculty of Medicine, during the period from December 2014 to December 2018. Clinicopathological and survival data were collected. IHC for HHLA2 and CD8 was performed, and they were correlated with clinicopathological parameters and patient prognosis. Among 134 CRC cases, high HHLA2 expression was detected in 73 (54.5%). High HHLA2 expression was significantly related to the depth of invasion (P = 0.005*), lymph node metastasis (P = 0.01*), tumor stage )P = 0.002*), and distant recurrence )P = 0.012*). Multivariate analysis spotted HHLA2 high expression as an independent prognostic predictor for OS in CRC (P = 0.03*) and DFS (P = 0.008*). CD8 shows a significant correlation with tumor infiltrating lymphocytes (TILs) (P ≤ 0.001*), absence of metastasis ((P = 0.029*), absence of tumor deposits (P=0.014*). However, CD8 shows no significant association with survival or HHLA2. HHLA2 is an independent prognostic factor for the overall survival and disease free survival of CRC patients and can predict poor prognosis in CRC patients.

HOXD9 Stimulates the Process of Colorectal Carcinoma by Regulating KLK9

Colorectal carcinoma (CRC) is a common tumor in the digestive system. This study aims to elucidate the possible relationship between abnormally expressed HOXD9 and the malignant process of CRC. HOXD9 levels were analyzed in CRC and adjacent non-tumoral tissues to evaluate its prognostic value in CRC patients. Knockdown of HOXD9 was performed, and the proliferative and migratory capacities of LoVo and LS513 cells were assessed using CCK-8, transwell, and wound healing assays. Bioinformatic analysis and dual-luciferase reporter assay revealed the interaction between HOXD9 and KLK9. Rescue experiments were conducted to elucidate the co-regulation of HOXD9 and KLK9 on CRC cell behaviors. HOXD9 was upregulated in CRC tissues, and high level of HOXD9 predicted poor prognosis in CRC patients. HOXD9 was identically upregulated in CRC cell lines, especially LoVo and LS513 cells, which were used for generating HOXD9 knockdown models by transfection of sh-HOXD9. Knockdown of HOXD9 weakened proliferative and migratory capacities in CRC cells. KLK9 was the target binding HOXD9, which was downregulated in CRC tissues and cell lines. Knockdown of KLK9 reversed the inhibited proliferative and migratory capacities in CRC cells owing to HOXD9 knockdown. Highly expressed HOXD9 in CRC tissues is closely linked to the prognosis. HOXD9 stimulates CRC cells to proliferate and migrate by upregulating KLK9.

N6-methyladenosine methyltransferase KIAA1429 elevates colorectal cancer aerobic glycolysis via HK2-dependent manner

ABSTRACT Emerging evidence has emphasized the critical roles played by N6-methyladenosine RNA (m6A) modification in colorectal carcinoma (CRC) initiation and progression. However, the roles and mechanism of m6A and KIAA1429 in CRC progression require further clarification. Here, our research aimed to investigate the functions of KIAA1429 in CRC tumorigenesis. Results indicated that KIAA1429 up-regulation closely correlated to the poor prognosis of CRC patients. Bio-functional assays demonstrated that KIAA1429 promoted the aerobic glycolysis, including glucose uptake, lactate production, ATP generation and extracellular acidification rate (ECAR). Mechanistically, KIAA1429 positively up-regulated HK2 level via increasing its mRNA stability by binding the m6A site of HK2 mRNA via m6A-independent manner. Collectively, our work indicates that KIAA1429 has the potential to promote CRC carcinogenesis by targeting HK2 via m6A-independent manner, providing insight into the critical roles of m6A in CRC.

PPIP5K2 promotes colorectal carcinoma pathogenesis through facilitating DNA homologous recombination repair.

Colorectal carcinoma (CRC) is the second most deadly cancer worldwide. Therapies that take advantage of DNA repair defects have been explored in various tumors but not yet systematically in CRC. Here, we found that Diphosphoinositol Pentakisphosphate Kinase 2 (PPIP5K2), an inositol pyrophosphate kinase, was highly expressed in CRC and associated with a poor prognosis of CRC patients. In vitro and in vivo functional studies demonstrated that PPIP5K2 could promote the proliferation and migration ability of CRC cells independent of its inositol pyrophosphate kinase activity. Mechanically, S1006 dephosphorylation of PPIP5K2 could accelerate its dissociation with 14-3-3 in the cytoplasm, resulting in more nuclear distribution. Moreover, DNA damage treatments such as doxorubicin (DOX) or irradiation (IR) could induce nuclear translocation of PPIP5K2, which subsequently promoted homologous recombination (HR) repair by binding and recruiting RPA70 to the DNA damage site as a novel scaffold protein. Importantly, we verified that S1006 dephosphorylation of PPIP5K2 could significantly enhance the DNA repair ability of CRC cells through a series of DNA repair phenotype assays. In conclusion, PPIP5K2 is critical for enhancing the survival of CRC cells via facilitating DNA HR repair. Our findings revealed an unrecognized biological function and mechanism model of PPIP5K2 dependent on S1006 phosphorylation and provided a potential therapeutic target for CRC patients.

Loss of expression of Syndecan-1 is associated with Tumor Recurrence, Metastatic Potential, and Poor Survival in patients with Colorectal carcinoma.

Objective:The loss of expression of syndecansyndecan-1 is associated with poor prognosis in many types of human cancer. The objective of this study was to evaluate the relation between syndecan-1 immunoexpression and several clinicopathological parameters in a subset of colorectal carcinoma (CRC) patients.Methods:Pathology tissue blocks of 202 primary tumors, 41 adenomas, and 37 normal colonic mucosae were used in this study. The cases diagnosed in the period 1995–2015 was included in the study. Immunohistochemistry analysis was performed using anti-CD138/syndecan-1 (B-A38) mouse monoclonal antibody. A semiquantitative method was used to score the syndecan-1 expression based on an evaluation of the percentage and intensity of the membranous and cytoplasmic expression. The data collected from Pathology Department at King Abdulaziz University Hospital, Jeddah, Saudi Arabia. This is a retrospective cohort study that was conducted from July 2018 until August 2019.Results:Loss of syndecan-1 immunoexpression was observed in 72 (42.6%), 5 (12.2%), and 3 (8.1%) cases of CRC, adenomas, and normal mucosae, respectively. Low expression of syndecan-1 showed an association with nodal (p=0.003) and distant (p=0.001) metastasis, lymphovascular invasion (p=0.001), and tumor recurrence (p=0.006). Low syndecan-1 expression were associated with short overall survival (OS) (log rank 4.019, p=0.045) and disease-free survival (DFS) probabilities (log rank 4.748, p=0.029).Conclusion:Loss of syndecan-1 immunoexpression is associated with metastatic potential, tumor recurrence and shorter survival in CRC and is considered a potential biomarker of poor prognosis in CRC patients.

Clinical value and potential association of Rab1A and FoxM1 aberrant expression in colorectal cancer

Colorectal carcinoma (CRC) is one of the most common malignancies with a dismal 5-year survival rate. Our recent study indicated that Rab1A expression was closely related to GLI1 expression. A previous study shows that aberrant overexpression of GLI1 promotes colorectal cancer metastasis via FoxM1 overexpression. However, the potential correlation between Rab1A and FoxM1 in CRC remains elusive. Immunohistochemistry was performed to investigate the association of the expression of Rab1A and FoxM1 and to determine the prognosis in 135 CRC tissue and adjacent normal tissues. Using Oncomine datasets, we found that Rab1A and FoxM1 mRNA were obviously upregulated in CRC tissues compared to normal tissues. Additionally, the expression of Rab1A and FoxM1 was significantly higher in CRC tissues than that in normal tissues. Rab1A expression was positively correlated with FoxM1 expression in CRC, especially in TNM stage III. In addition, Rab1A and FoxM1 overexpression was found to be significantly correlated with poor prognosis in CRC patients. Besides, both high expression of Rab1A and FoxM1 led to a worse prognosis than anyone low group, and both low expression of Rab1A and FoxM1 had a better prognosis than the anyone low group. Therefore, Rab1A and FoxM1 play crucial roles and could be used as clinical biomarkers in CRC.

Silence of ATAD2 inhibits proliferation of colorectal carcinoma via the Rb-E2F1 signaling.

This study aims to clarify the potential function of ATAD2 (ATPase family, AAA domain containing 2) in regulating proliferative and apoptotic abilities of colorectal carcinoma (CRC). ATAD2 levels in CRC specimens and cell lines were detected by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR). Overall survival in CRC patients with high or low level of ATAD2 was assessed by Kaplan-Meier method. The correlation between ATAD2 level and clinical characteristics of CRC patients was analyzed by χ2 test. Univariable and multivariable Cox regression models were generated to illustrate potential risk factors for the overall survival of CRC. After knockdown of ATAD2 in SW620 cells, relative levels of Cyclin D1, ppRb, pRb, E2F1, Cyclin E and cleaved Caspase 3 were detected by Western blot. Regulatory effects of ATAD2 on viability, clonality, cell cycle distribution, and apoptosis in SW620 and HCT15 cells were examined by a series of functional experiments. Upregulated ATAD2 in CRC was correlated to tumor size, tumor node metastasis (TNM) staging, and histological classification of CRC. High level of ATAD2 predicted poor prognosis in CRC patients. Cox regression test suggested that ATAD2 level, tumor size, TNM staging and histological classification were independent factors influencing overall survival in CRC. Knockdown of ATAD2 reduced viability and clonality in SW620 and HCT15 cells. In addition, cell cycle was arrested in G1 phase and apoptosis was stimulated in CRC cells with ATAD2 knockdown. In SW620 cells transfected with ATAD2 shRNA, protein levels of Cyclin D1, ppRb, E2F1 and Cyclin E were downregulated, and cleaved Caspase 3 was upregulated. ATAD2 is upregulated in CRC tissues and correlated to poor prognosis of CRC patients. It exerts an anti-proliferation role in CRC by arresting cell cycle in G1/S phase and triggering apoptosis via the Rb-E2F1 signaling.

MicroRNA-95-3p inhibits cell proliferation and metastasis in colorectal carcinoma by HDGF

BackgroundMicroRNAs (miRNAs) play an important regulatory role in carcinogenesis and cancer progression. MiR-95-3p has been reported to be an oncogene in hepatocellular carcinoma. However, the role of miR-95-3p in colorectal carcinoma (CRC) remains unclear. MethodsmiR-95-3p was validated in an independent validation sample cohort of 215 CRC tissues. Functional assays, Cell proliferation (MTT) assay colony formation, wound healing, transwell and animal xenograft assays were used to determine the oppressor role of miR-95-3p in human CRC progression. Furthermore, Bioinformatics analysis, western blotting and dual-luciferase reporter assay were used to determine the mechanism by which miR-95-3p suppresses progression of CRC cells. ResultsIn this study, we found that miR-95-3p was downregulated in CRC tissues. The low level of miR-95-3p in CRC tumors was correlated with aggressive clinicopathological characteristics, and it predicted poor prognosis in CRC patients. The overexpression of miR-95-3p significantly inhibited CRC cell proliferation, colony formation and metastasis in vitro and in vivo. Bioinformatic analysis further identified hepatoma-derived growth factor (HDGF) as a novel target of miR-95-3p in CRC cells. These findings suggest that miR-95-3p regulates CRC cell survival, partially through the downregulation of HDGF. ConclusionsTherefore, the miR-95-3p/HDGF axis might serve as a novel therapeutic target in patients with CRC.

FBX8 degrades GSTP1 through ubiquitination to suppress colorectal cancer progression

F-box only protein 8 (FBX8), as a critical component of the SKP1-CUL1-F-box (SCF) E3 ubiquitin ligases, has been associated with several malignancies through interacting with a member of proteins. However, the substrates of FBX8 for destruction in the progression of colorectal carcinoma (CRC) need to be explored. Here, we show that loss of FBX8 accelerates chemical-induced colon tumorigenesis. FBX8 directly targets GSTP1 for ubiquitin-mediated proteasome degradation in CRC. GSTP1 promotes the proliferation, invasion, and metastasis of CRC cells. Furthermore, GSTP1 is upregulated in CRC tissue samples and predicts poor prognosis of CRC patients. The inactivation of FBX8 negatively correlated with increased levels and stability of GSTP1 in clinical CRC tissues and FBX8 knockout transgenic mice. These findings identify a novel ubiquitination pathway as FBX8-GSTP1 axis that regulates the progression of CRC, which might be a potential prognostic biomarker for CRC patients.

STAT1-mediated upregulation of lncRNA LINC00174 functions a ceRNA for miR-1910-3p to facilitate colorectal carcinoma progression through regulation of TAZ

Several of the thousands of human long noncoding RNAs (lncRNAs) have been functionally characterized, yet their potential involvement in colorectal carcinoma (CRC) remains poorly understood. In present study, we aim to investigate the role of lncRNA LINC00174 in CRC carcinogenesis. We observed that increased expression of LINC00174 in CRC tissues and cells in comparison to their corresponding controls. Moreover, the aberrant overexpression of LINC00174 indicated the poor prognosis of CRC patients. Silence of LINC00174 was able to repress CRC cell growth in vitro and in vivo. We first reported that transcription factor STAT1 mediated LINC00174 expression in CRC. In addition, rescue assay was performed to further confirm that LINC00174 contributed to CRC progression by regulating miR-1910-3p/TAZ signal pathway. Taken together, our study discovered the oncogenic role of LINC00174 in clinical specimens and cellular experiments, showing the potential LINC00174/miR-1910-3p/TAZ pathway. This results and findings provide a novel insight for CRC tumorigenesis.

Overexpression of NOX4 predicts poor prognosis and promotes tumor progression in human colorectal cancer.

NADPH oxidase 4 (NOX4), a major source of reactive oxygen species (ROS) production, has been increasingly reported to be involved in tumorigenesis and/or tumor progression, but limited data are available regarding the role of NOX4 in colorectal carcinoma (CRC). We retrieved six independent investigations from Oncomine database and found that NOX4 is highly expressed in CRC tissues compared with corresponding normal controls. Similar results were also found in clinical specimens at both mRNA and protein levels. Immunohistochemical analysis indicated that NOX4 overexpression was highly correlated with T classification, N classification, distant metastasis, and poor prognosis of CRC patients, which was also confirmed by GSE14333 and GSE17536 datasets from the Gene Expression Omnibus. Furthermore, we demonstrated that when NOX4 expression was knocked down by siRNAs, cell proliferation, cell-cycle and apoptosis, migration and invasion were significantly altered in CRC cell lines HCT116 and LOVO. Meanwhile, NOX4 promoted cancer cell proliferation and apoptosis, migration and invasion by regulating the expression of relevant genes. By these approaches we aim to elucidate NOX4 may be a reliable prognostic factor or therapeutic target in CRC.

Circadian gene hClock enhances proliferation and inhibits apoptosis of human colorectal carcinoma cells invitro and invivo.

Colorectal carcinoma (CRC) is one of the most prevalent types of malignancy-associated mortality worldwide. Previous studies have demonstrated that amplification and overexpression of the human circadian locomotor output cycles kaput gene (hClock) was closely associated with a high risk for CRC as well as poor prognosis in CRC patients. However, the underlying molecular mechanisms of CRC remain to be fully elucidated. In the present study, hClock was exogenously overexpressed in the CRC cell line SW480 via infection of a lentivirus vector expressing hClock; in addition, a lentivirus vector-based RNA interference approach, using short hairpin RNA, was performed in order to knockdown hClock in SW620 cells. The results showed that upregulation of hClock promoted proliferation and inhibited apoptosis in SW480 cells in vitro and in vivo, while downregulation of hClock inhibited SW620 cell proliferation and accelerated apoptosis in vitro. Upregulation of hClock enhanced the activity of the anti-apoptotic gene phosphorpylated (p-) AKT and inhibited the expression of the pro-apoptotic gene B cell lymphoma-2 (Bcl-2)-associated X protein and Bcl-2 homology 3 interacting domain death agonist. Furthermore, targeted inhibition of hClock activity reduced p-AKT expression. In conclusion, the results of the present study suggested that the circadian gene hClock promoted CRC progression and inhibit tumor cell apoptosis in vitro and in vivo, while silencing hClock was able to reverse this effect.

AUTHORS' REPLY

<h3>Background</h3> Glutamate-cysteine ligase modifier (GCLM) subunits, combined with catalytic (GCLC) subunits, has been known for the first rate-limiting enzyme for glutathione (GSH) synthesis. Increasing studies have shown that the non-metabolic functions of many metabolic enzymes also play a crucial role in tumor progress. However, the non-metabolic functions of GCLM remain unexplored. Hence, we wonder if there are unorthodox functions of GCLM under redox stress participating colorectal Carcinoma(CRC) tumorigenesis. <h3>Methods</h3> Western blot, real-time quantitative PCR and immunohistochemical analysis were used to detect the relative expression of GCLM in cell lines and patients‘ specimens. The MTS assay, migration and invasion assay, sphere formation assay were performed to detect the functions of GCLM <i>in vitro</i>, and the xenograft models and PDX models in nude mice <i>in vivo</i>. Cytosolic and nuclear extraction, immunofluorescent analysis were used to show the location of GCLM. Chromatin immunoprecipitation (ChIP) assay was used to screen and verify the GCLM target genes. <h3>Results</h3> We found that glucose deprivation condition upregulated the expression of GCLM rather than hypoxia or H2O2. Compared to adjacent tumor tissues, the expression of GCLM was upregulated in CRC tumor tissues. The high expression of GCLM predicted poor prognosis in CRC patients. Inhibition of GCLM results in decreased proliferation rate, migration and invasion ability and sphere formation in HCT116 and DLD1 <i>in vitro</i>. Similarly, GCLM inhibition suppressed CRC tumorigenesis and metastasis in xenograft models and PDX models<i> in vivo</i>. The cancer cell stem cell markers were also downregulated after GCLM inhibition. Interestingly, we found that glucose starvation could rescue the anti-cancer phenotypes of GCLM inhibition and led to nucleus-translocation and accumulation of GCLM. ChIP assay showed GCLM could interact with octamer-binding transcription factor 4 (<i>OCT4)</i> promoter<i>,</i> a cancer stem cell marker, to increase its expression, and the target gene of <i>OCT4</i> showed a positive correlation with GCLM expression in CRC patients. <h3>Conclusions</h3> Our data showed that GCLM displayed nucleus accumulation in the condition of glucose deprivation which increased the transcription of <i>OCT4.</i> This study revealed an unorthodox oncogenic function of GCLM in colorectal carcinoma and suggested GCLM as a potential therapeutic target in CRC.