Clinical Colorectal Cancer Research Articles

Kolorectal kanser taramasında panimmun inflamasyon değerinin ve sistemik immün-inflamasyon indeksinin önemi

Aim: It has been known for a long time that systemic inflammation is an important risk factor in cancer development. Colorectal cancer (CRC) is one of the most common causes of cancer-related morbidity and mortality in the world. In this study, we aimed to compare the inflammatory parameters tested in CRC patients at the time of diagnosis such as systemic immune-inflammation index (SII) and pan-immune inflammation value (PIV) with those of the healthy control group. 
 Materials and Methods: The data of 162 patients diagnosed with CRC in the internal medicine clinic between 2012-2016 were analysed retrospectively and a total of 139 patients who met the inclusion criteria were included in the study. Hemogram values, histopathology and tumour stage according to TNM classification of all patients at the time of diagnosis were recorded. The patients’ neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), lymphocyte-monocyte ratio (LMR), SII and PIV were calculated using hemogram parameters such as neutrophils, monocytes, platelets and lymphocytes.
 Results: A total of 139 CRC patients and 139 healthy control subjects with similar age and sex distribution were included in the study. The mean age of all subjects included in the study was 61.7±11.8 years, and 170 subjects (61.2%) were male. In the CRC group, SII, PIV, NLR, and PLR levels were significantly higher, and LMR level was significantly lower than the control group (p

Tumor cell-derived SPON2 promotes M2-polarized tumor-associated macrophage infiltration and cancer progression by activating PYK2 in CRC

BackgroundTumor-associated macrophages (TAMs) are key regulators of the complex interplay between cancer and the immune microenvironment. Tumor cell-derived spondin 2 (SPON2) is an extracellular matrix glycoprotein that has complicated roles in recruitment of macrophages and neutrophils during inflammation. Overexpression of SPON2 has been shown to promote tumor cell migration in colorectal cancer (CRC). However, the mechanism by which SPON2 regulates the accumulation of TAMs in the tumor microenvironment (TME) of CRC is unknown.MethodsImmunohistochemistry was used to examine SPON2 expression in clinical CRC tissues. In vitro migration assays, transendothelial migration assays (iTEM), and cell adhesion assays were used to investigate the effects of SPON2 on monocyte/macrophage migration. Subcutaneous tumor formation and orthotopic implantation assays were performed in C57 BL/6 mice to confirm the effects of SPON2 on TAM infiltration in tumors.ResultsSPON2 expression is positively correlated with M2-TAM infiltration in clinical CRC tumors and poor prognosis of CRC patients. In addition, SPON2 promotes cytoskeletal remodeling and transendothelial migration of monocytes by activating integrin β1/PYK2 axis. SPON2 may indirectly induce M2-polarization through upregulating cytokines including IL10, CCL2 and CSF1 expression in tumor cells. Blocking M2 polarization and Macrophage depletion inhibited the SPON2-induced tumors growth and invasion. Furthermore, blocking the SPON2/integrin β1/PYK2 axis impairs the transendothelial migration of monocytes and cancer-promoting functions of TAMs in vivo.ConclusionsOur findings demonstrate that SPON2-driven M2-TAM infiltration plays an important role during CRC tumor growth and metastasis. SPON2 may be a valuable biomarker guiding the use of macrophage-targeting strategies and a potential therapeutic target in advanced CRC.

Pharmacological targeting PTK6 inhibits the JAK2/STAT3 sustained stemness and reverses chemoresistance of colorectal cancer

BackgroundChemoresistance is the major cause of chemotherapy failure in patients with colorectal cancer (CRC). Protein tyrosine kinase 6 (PTK6) is aberrantly overexpressed in clinical CRC tissues undergoing chemotherapy. We studied if PTK6 contributed to the chemoresistance of CRC in human and mice.MethodsWe obtained tissue samples from patients with CRC and measured the expression of PTK6 by immunohistochemistry. Gain- and loss-of-function assays were performed to study the biological functions of PTK6. We constructed the FLAG-tagged wild type (WT), kinase-dead, and inhibition-defective recombinant mutants of PTK6 to study the effect phosphorylated activation of PTK6 played on CRC cell stemness and chemoresistance. We used small molecule inhibitor XMU-MP-2 to test the influence of PTK6 on sensitivity of CRC cells to 5-FU/L-OHP in both nude mouse and patient-derived xenograft (PDX) animal models.ResultsPTK6 is overexpressed in CRC tissues and plays a stimulatory role in the proliferation and chemoresistance of CRC cells both in vitro and in vivo. PTK6, especially the phosphorylated PTK6, can promote the stemness of CRC cells through interacting with JAK2 and phosphorylating it to activate the JAK2/STAT3 signaling. Pharmacological inhibition of PTK6 using XMU-MP-2 effectively reduces the stemness property of CRC cells and improves its chemosensitivity to 5-FU/L-OHP in both nude mice subcutaneously implanted tumor model and PDX model constructed with NOD-SCID mice.ConclusionsPTK6 interacts with JAK2 and phosphorylates it to activate JAK2/STAT3 signaling to promote the stemness and chemoresistance of CRC cells. Pharmacological inhibition of PTK6 by small molecule inhibitor dramatically enhances the sensitivity to chemotherapy in nude mice and PDX models.

In-Situ-Sprayed Dual-Functional Immunotherapeutic Gel for Colorectal Cancer Postsurgical Treatment.

Surgery remains the most preferred treatment options for colorectal cancer (CRC). Paradoxically, local recurrence and distant metastasis are usually accelerated postsurgery as a consequence of local and systemic immunosuppression caused by surgery. Therefore, modulating tumor postoperative immune microenvironment and activating systemic antitumor immunity are necessary supplementaries for CRC therapy. Here, an in-situ-sprayed immunotherapeutic gel loaded with anti-OX40 antibody (iSGels@aOX40) is reported for CRC postsurgical treatment. The iSGel is formed instantly after spraying with strong adhesion ability via crosslinking between tannic acid (TA) and poly(l-glutamic acid)-g-methoxy poly(ethylene glycol)/phenyl boronic acid (PLG-g-mPEG/PBA). TA not only serves as one component of the iSGel but also relieves the postsurgical immunosuppressive microenvironment by inhibiting the activity of cyclo-oxygenase-2 (COX-2). The aOX40 serves as an immune agonistic antibody and is released from the iSGel in a constant manner lasting for over 20 days. In a subcutaneous murine CRC model, the iSGels@aOX40 results in complete inhibition on tumor recurrence. In addition, the cured mice show resistance to tumor re-challenge, suggesting that immune memory effects are established after the iSGels@aOX40 treatment. In an orthotopic CRC peritoneal metastatic model, the iSGels@aOX40 also remarkably inhibits the growth of the abdominal metastatic tumors, suggesting great potential for clinical CRC therapy.

Glutamate receptor, ionotropic, N‑methyl D‑aspartate‑associated protein 1 promotes colorectal cancer cell proliferation and metastasis, and is negatively regulated by miR‑296‑3p.

N-methyl D-aspartate receptors (NMDARs) are closely associated with the development, growth and metastasis of cancer. Glutamate receptor, ionotropic, N-methyl D-aspartate-associated protein 1 (GRINA) is a member of the of the NMDAR family, and its aberrant expression is associated with gastric cancer. However, the role of GRINA in colorectal cancer (CRC) is not completely understood. In the present study, expression profiles of GRINA in several CRC databases were obtained and further verified using clinical CRC samples. The effects of GRINA overexpression on CRC progression both in vivo and in vitro were assessed. Briefly, cell proliferation was detected using MTT assay, and cell migration and invasion ability were evaluated by wound healing and Transwell assay. In addition, the molecular mechanism underlying the upregulated expression of GRINA in CRC was investigated. The regulatory association between GRINA and miR-296-3p was detected by luciferase assay, reverse transcription-quantitative PCR and western blotting. The results demonstrated that GRINA expression levels were significantly increased in tumor samples compared with those in healthy samples, and upregulated expression of GRINA was associated with a less favorable prognostic outcome in patients with CRC. GRINA overexpression significantly increased CRC cell proliferation, invasion and migration. Additionally, it was determined that GRINA was post-transcriptionally regulated by microRNA (miR)-296-3p. Together, the results of the present study suggested the potential importance of the miR-296-3p/GRINA axis and highlighted potential novel targets for the management of CRC.

MYB mediates downregulation of the colorectal cancer metastasis suppressor heterogeneous nuclear ribonucleoprotein L-like during epithelial-mesenchymal transition.

Heterogeneous nuclear ribonucleoprotein L‐like (HNRNPLL), a suppressor of colorectal cancer (CRC) metastasis, is transcriptionally downregulated when CRC cells undergo epithelial‐mesenchymal transition (EMT). Here we show that decrease of MYB mediates the downregulation of HNRNPLL during EMT. The promoter activity was attributed to a region from −273 to −10 base pairs upstream of the transcription start site identified by 5'‐RACE analysis, and the region contained potential binding sites for MYB and SP1. Luciferase reporter gene assays and knockdown or knockout experiments for genes encoding the MYB family proteins, MYB, MYBL1, and MYBL2, revealed that MYB was responsible for approximately half of the promoter activity. On the other hand, treatment with mithramycin A, an inhibitor for SP1 and SP3, suppressed the promoter activity and their additive contribution was confirmed by knockout experiments. The expression level of MYB was reduced on EMT while that of SP1 and SP3 was unchanged, suggesting that the downregulation of HNRNPLL during EMT was mediated by the decrease of MYB expression while SP1 and SP3 determine the basal transcription level of HNRNPLL. Histopathological analysis confirmed the accumulation of MYB‐downregulated cancer cells at the invasion front of clinical CRC tissues. These results provide an insight into the molecular mechanism underlying CRC progression.

PGE2-JNK signaling axis non-canonically promotes Gli activation by protecting Gli2 from ubiquitin-proteasomal degradation

Both bench and bedside investigations have challenged the supportive role of Hedgehog (Hh) activity in the progression of colorectal cancers, thus raising a critical need to further deeply determine the contribution of Hh to the growth of colorectal cancer. Combining multiple complementary means, including in vitro and in vivo inflammatory colorectal cancer models, and pathological analysis of clinical colorectal cancer patients samples. We report that colorectal cancer cells hijack prostaglandin E2 (PGE2) to non-canonically promote Hh transcriptional factor Gli activity and Gli-dependent proliferation of colorectal cancer cells in a Smo-independent manner. Mechanistically, PGE2 activates c-Jun N-terminal kinase (JNK), which in turn enables Gli2 to evade ubiquitin-proteasomal degradation by phosphorylating Gli2 at Thr1546. This study not only presents evidence for understanding the contribution of Hh to colorectal cancers, but also provides a novel molecular portrait underlying how PGE2-activated JNK fine-tunes the evasion of Gli2 from ubiquitin-proteasomal degradation. Therefore, it proposes a rationale for the future evaluation of chemopreventive and selective therapeutic strategies for colorectal cancers by targeting PGE2-JNK-Gli signaling route.

Blockage of Cholinergic Signaling via Muscarinic Acetylcholine Receptor 3 Inhibits Tumor Growth in Human Colorectal Adenocarcinoma.

Simple SummaryDarifenacin is a selective muscarinic acetylcholine receptor 3 (M3R) antagonist in clinical use for benign bladder disorders. Here, the effects of M3R blockage by darifenacin on human colon adenocarcinoma cells are analyzed in vitro and in vivo. M3R expression was found in the majority of clinical colorectal cancer samples, making it attractive for drug targeting. In mice, darifenacin inhibited orthotopic and metastatic tumor xenograft growth. In vitro analyses on colon carcinoma cell lines showed that M3R targeting counteracts acetylcholine-induced p38, ERK1/2 and Akt signaling, and disrupts MMP-1 expression and tumor cell invasion. Our data suggest that targeting cholinergic signaling by darifenacin may be beneficial in the treatment of colorectal cancer.Cholinergic signaling via the muscarinic M3 acetylcholine receptor (M3R) is involved in the development and progression of colorectal cancer (CRC). The present study aimed to analyze the blocking of M3R signaling in CRC using darifenacin, a selective M3R antagonist. Darifenacin effects were studied on HT-29 and SW480 CRC cells using MTT and BrdU assays, Western blotting and real time RT-PCR. In vivo, blocking of M3R was assessed in an orthotopic CRC xenograft BALB/cnu/nu mouse model. M3R expression in clinical tumor specimens was studied by immunohistochemistry on a tissue microarray of 585 CRC patients. In vitro, darifenacin decreased tumor cell survival and proliferation in a dose-dependent manner. Acetylcholine-induced p38, ERK1/2 and Akt signaling, and MMP-1 mRNA expression were decreased by darifenacin, as well as matrigel invasion of tumor cells. In mice, darifenacin reduced primary tumor volume and weight (p < 0.05), as well as liver metastases, compared to controls. High expression scores of M3R were found on 89.2% of clinical CRC samples and correlated with infiltrative tumor border and non-mucinous histology (p < 0.05). In conclusion, darifenacin inhibited components of tumor growth and progression in vitro and reduced tumor growth in vivo. Its target, M3R, was expressed on the majority of CRC. Thus, repurposing darifenacin may be an attractive addition to systemic tumor therapy in CRC patients expressing M3R.

Alternative Splicing Events in Tumor Immune Infiltration in Colorectal Cancer

Despite extensive research, the exact mechanisms involved in colorectal cancer (CRC) etiology and pathogenesis remain unclear. This study aimed to examine the correlation between tumor-associated alternative splicing (AS) events and tumor immune infiltration (TII) in CRC. We analyzed transcriptome profiling and clinical CRC data from The Cancer Genome Atlas (TCGA) database and lists of AS-related and immune-related signatures from the SpliceSeq and Innate databases, respectively to develop and validate a risk model of differential AS events and subsequently a TII risk model. We then conducted a two-factor survival analysis to study the association between TII and AS risk and evaluated the associations between immune signatures and six types of immune cells based on the TIMER database. Subsequently, we studied the distribution of six types of TII cells in high- and low-risk groups for seven AS events and in total. We obtained the profiles of AS events/genes for 484 patients, which included 473 CRC tumor samples and 41 corresponding normal samples, and detected 22581 AS events in 8122 genes. Exon Skip (ES) (8446) and Mutually Exclusive Exons (ME) (74) exhibited the most and fewest AS events, respectively. We then classified the 433 patients with CRC into low-risk (n = 217) and high-risk (n = 216) groups based on the median risk score in different AS events. Compared with patients with low-risk scores (mortality = 11.8%), patients with high-risk scores were associated with poor overall survival (mortality = 27.6%). The risk score, cancer stage, and pathological stage (T, M, and N) were closely correlated with prognosis in patients with CRC (P < 0.001). We identified 6479 differentially expressed genes from the transcriptome profiles of CRC and intersected 468 differential immune-related signatures. High-AS-risk and high-TII-risk predicted a poor prognosis in CRC. Different AS types were associated with different TII risk characteristics. Alternate Acceptor site (AA) and Alternate Promoter (AP) events directly affected the concentration of CD4T cells, and the level of CD8T cells was closely correlated with Alternate Terminator (AT) and Exon Skip (ES) events. Thus, the concentration of CD4T and CD8T cells in the CRC immune microenvironment was not specifically modulated by AS. However, B cell, dendritic cell, macrophage, and neutrophilic cell levels were strongly correlated with AS events. These results indicate adverse associations between AS event risk levels and immune cell infiltration density. Taken together, our findings show a clear association between tumor-associated alternative splicing and immune cell infiltration events and patient outcome and could form a basis for the identification of novel markers and therapeutic targets for CRC and other cancers in the future.

Comprehensive Analysis of Prognostic and Genetic Signatures for General Transcription Factor III (GTF3) in Clinical Colorectal Cancer Patients Using Bioinformatics Approaches.

Colorectal cancer (CRC) has the fourth-highest incidence of all cancer types, and its incidence has steadily increased in the last decade. The general transcription factor III (GTF3) family, comprising GTF3A, GTF3B, GTF3C1, and GTFC2, were stated to be linked with the expansion of different types of cancers; however, their messenger (m)RNA expressions and prognostic values in colorectal cancer need to be further investigated. To study the transcriptomic expression levels of GTF3 gene members in colorectal cancer in both cancerous tissues and cell lines, we first performed high-throughput screening using the Oncomine, GEPIA, and CCLE databases. We then applied the Prognoscan database to query correlations of their mRNA expressions with the disease-specific survival (DSS), overall survival (OS), and disease-free survival (DFS) status of the colorectal cancer patient. Furthermore, proteomics expressions of GTF3 family members in clinical colorectal cancer specimens were also examined using the Human Protein Atlas. Finally, genomic alterations of GTF3 family gene expressions in colorectal cancer and their signal transduction pathways were studied using cBioPortal, ClueGO, CluePedia, and MetaCore platform. Our findings revealed that GTF3 family members’ expressions were significantly correlated with the cell cycle, oxidative stress, WNT/β-catenin signaling, Rho GTPases, and G-protein-coupled receptors (GPCRs). Clinically, high GTF3A and GTF3B expressions were significantly correlated with poor prognoses in colorectal cancer patients. Collectively, our study declares that GTF3A was overexpressed in cancer tissues and cell lines, particularly colorectal cancer, and it could possibly step in as a potential prognostic biomarker.

Comparison of prognostic impact between positive intraoperative peritoneal and lavage cytologies in colorectal cancer.

The prognostic value of positive intraoperative peritoneal cytology and lavage cytology, including the differences in their prognostic impact, in colorectal cancer is controversial. We aimed to investigate the prognostic values of positive peritoneal cytology and lavage cytology findings for colorectal cancer and compare their prognostic impact. We retrospectively evaluated 592 clinical stage II-IV colorectal cancer patients who underwent peritoneal cytology (n = 225) or lavage cytology (n = 367) between November 1993 and December 2018. The prognostic factors for cancer-specific survival were identified, and the differences in cancer-specific survival were examined between the patients. The cytology-positive rate was 10.8% (64/592), 17.8% (40/225), and 6.5% (24/367) in the overall, peritoneal cytology, and lavage cytology groups, respectively. Both positive peritoneal cytology (hazard ratio: 2.196) and lavage cytology (hazard ratio: 2.319) were independent prognostic factors. The peritoneal cytology-positive group showed significantly poorer cancer-specific survival than the cytology-negative group (5-year: 3.5% vs. 59.5%; 10-year: 3.5% vs. 46.1%, p < 0.001). Similar results were obtained for lavage cytology (5-year: 14.1% vs. 73.9%; 10-year: 4.7% vs. 63.5%, p < 0.001). The cancer-specific survival was not significantly different between the peritoneal cytology-positive and lavage cytology-positive groups (p = 0.058). Both positive peritoneal and lavage cytology were associated with poorer cancer-specific survival across all colorectal cancer stages. Positive peritoneal and lavage cytology are associated with worse cancer-specific survival in colorectal cancer. The prognostic impact was comparable between positive lavage and peritoneal cytology. Thus, cytology should be a standard assessment modality for colorectal cancer.

Accurate diagnosis of colorectal cancer based on histopathology images using artificial intelligence

BackgroundAccurate and robust pathological image analysis for colorectal cancer (CRC) diagnosis is time-consuming and knowledge-intensive, but is essential for CRC patients’ treatment. The current heavy workload of pathologists in clinics/hospitals may easily lead to unconscious misdiagnosis of CRC based on daily image analyses.MethodsBased on a state-of-the-art transfer-learned deep convolutional neural network in artificial intelligence (AI), we proposed a novel patch aggregation strategy for clinic CRC diagnosis using weakly labeled pathological whole-slide image (WSI) patches. This approach was trained and validated using an unprecedented and enormously large number of 170,099 patches, > 14,680 WSIs, from > 9631 subjects that covered diverse and representative clinical cases from multi-independent-sources across China, the USA, and Germany.ResultsOur innovative AI tool consistently and nearly perfectly agreed with (average Kappa statistic 0.896) and even often better than most of the experienced expert pathologists when tested in diagnosing CRC WSIs from multicenters. The average area under the receiver operating characteristics curve (AUC) of AI was greater than that of the pathologists (0.988 vs 0.970) and achieved the best performance among the application of other AI methods to CRC diagnosis. Our AI-generated heatmap highlights the image regions of cancer tissue/cells.ConclusionsThis first-ever generalizable AI system can handle large amounts of WSIs consistently and robustly without potential bias due to fatigue commonly experienced by clinical pathologists. It will drastically alleviate the heavy clinical burden of daily pathology diagnosis and improve the treatment for CRC patients. This tool is generalizable to other cancer diagnosis based on image recognition.

C-Src facilitates tumorigenesis by phosphorylating and activating G6PD.

Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme in pentose phosphate pathway (PPP), excessive activation of which has been considered to be involved in tumorigenesis. Here, we show that tyrosine kinase c-Src interacts with and phosphorylates G6PD at Tyr 112. This phosphorylation enhances catalytic activity of G6PD by dramatically decreasing its Km value and increasing its Kcat value for substrate glucose-6-phosphate. Activated G6PD therefore augments the PPP flux for NADPH and ribose-5-phosphate production which is required for detoxification of intracellular reactive oxygen species (ROS) and biosynthesis of cancer cells, and eventually contributes to tumorigenesis. Consistently, c-Src activation is closely correlated with tyrosine phosphorylation and activity of G6PD in clinical colorectal cancer samples. We thus uncover another aspect of c-Src in promoting cell proliferation and tumorigenesis, deepening our understanding of c-Src as a proto-oncogene.

Low level of stromal lectin-like oxidized LDL receptor 1 and CD8+ cytotoxic T-lymphocytes indicate poor prognosis of colorectal cancer.

BackgroundLectin‐like oxidized LDL receptor‐1 (LOX‐1) has been identified as a new marker for functional myeloid‐derived suppressor cells (MDSCs) that exhibit an immunosuppressive phenotype in the tumor microenvironment (TME). However, the role of LOX‐1+ cells in the TME of colorectal cancer (CRC) remains unknown.AimThis study aimed to determine the expression and significance of LOX‐1 in the TME of clinical CRC specimens.Methods and resultsWe performed immunohistochemical and genetic analyses of LOX‐1, CD8, KRAS, and BRAF in 128 resected CRC specimens and determined the expression of IFN‐γ and IL‐10 using real‐time reverse transcription‐polymerase chain reaction. We analyzed the correlation between LOX‐1, TME factors, gene alteration, clinicopathological factors, and disease prognosis. The co‐expression pattern of LOX‐1, hematopoietic markers, and a fibroblast marker was evaluated using multiplex immunofluorescence staining. Low stromal LOX‐1 expression and low intratumoral CD8+ cytotoxic T‐lymphocyte (CTL) status correlated with poor prognosis. Moreover, stromal LOX‐1‐low/CD8+ CTL‐low status was the most important independent prognostic factor of poor overall survival. Most of the LOX‐1+ stromal cells were positive for CD163+, indicating they were CD163+ M2 macrophages.ConclusionsThe MDSC marker, LOX‐1, was mainly expressed by M2 macrophages in CRC tissues. LOX‐1+ macrophages and CD8+ CTLs may serve as useful biomarkers for predicting the prognosis of CRC.

A role of TTI1 in the colorectal cancer by promoting proliferation.

BackgroundColorectal cancer (CRC) is one of the most malignant cancer worldwide, which leads to a high incidence and mortality. The molecular mechanism in CRC is still limited. The aim of this study was to identify hub genes and its related function in CRC.MethodsThe expression dataset (GSE44076) was downloaded from Gene Expression Omnibus (GEO) and differentially expressed genes (DEGs) analysis was done using R ‘limma’ packages. Weighted gene co-expression network analysis (WGCNA) was done and tumor-specific modules were picked up for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. The hub gene was selected with higher inter-connectivity. Expression levels of TTI1 were verified by in clinical CRC tissues. The cell counting kit-8 (CCK-8) assay was to measure the proliferative ability of TTI1.ResultsEight hundred and eight up-regulated and 929 down-regulated DEGs were screened out. Up-regulated genes enriched in cell proliferation and down-regulated genes enriched in oxidation-reduction process. After WGCNA, the yellow module was found to be the most significant tumor-specific module. Function analysis showed genes in the yellow module enriched in oxidation-reduction, cell proliferation and extracellular matrix (ECM)-receptor interaction. TTI1 was demonstrated as the hub gene. Real-time quantitative reverse transcription((qRT-PCR) results showed TTI1 significantly expressed higher in CRC tissues than adjacent normal tissues. TTI1 dramatically correlated with proliferation in CRC.ConclusionsThese findings regarded TTI1 as a vital promoting factor in CRC development and provided a potential biomarker for CRC treatment.

Activation of WNT7b autocrine eases metastasis of colorectal cancer via epithelial to mesenchymal transition and predicts poor prognosis

BackgroundAberrant activation of the Wnt/β-catenin signaling pathway is one of the most frequent abnormalities in human cancer, including colorectal cancer (CRC). Previous studies revealed pivotal functions of WNT family members in colorectal cancer, as well as their prognostic values. Nevertheless, the prognostic role and mechanisms underlying WNT7b in colorectal cancer development remains unclear.MethodsIn this study, WNT7b expression was measured by immunohistochemical staining of 100 cases of surgically resected human colorectal cancerous tissues as well as matched adjacent normal tissues constructed as tissue microarrays. In vitro studies, we attempted to substantiate the WNT7b expressional pattern previously found in immunohistochemistry staining. We used the colorectal cancer cell-line HCT116 and normal colorectal cell-line FHC for immunofluorescence staining and nuclear/cytoplasmic separated western blotting. We measured epithelial–mesenchymal transition (EMT) markers and migration capacity of HCT116 in the context of WNT7b knocked-down using short interfering RNA. Finally, clinical and prognostic values of WNT7b activation levels were examined.ResultsWNT7b was expressed in the nucleus in adjacent normal tissues. In CRC tissues, nuclear expression of WNT7b was similar; however, membrane and cytoplasmic expression was strikingly enhanced. Consistently, in vitro analysis confirmed the same expression pattern of WNT7b. Compared with FHC cells, HCT116 cells displayed higher levels of WNT7b membrane and cytoplasmic enrichment, as well as higher migration capacity with a sensitized EMT process. Either partial knockdown of WNT7b or blockade of the Wnt/β-catenin signaling pathway reversed EMT process and inhibited the migration of HCT116 cells. Finally, elevated secretion levels of WNT7b were significantly associated with lymphatic and remote metastasis and predicted worse prognosis in the CRC cohort.ConclusionIn summary, we demonstrated that the activation of WNT7b autocrine probably contributes to CRC metastasis by triggering EMT process through the Wnt/β-catenin signaling pathway. High levels of WNT7b autocrine secretion predicts poor outcome in patients with CRC. This molecule is a promising candidate for clinical CRC treatments.

Building Cancer Prevention and Control Research Capacity in Rural Appalachian Kentucky Primary Care Clinics During COVID-19: Development and Adaptation of a Multilevel Colorectal Cancer Screening Project.

This study describes the development of a colorectal cancer (CRC) screening multilevel intervention with four primary care clinics in rural Appalachian Kentucky. We also discuss barriers experienced by the clinics during COVID-19 and how clinic limitations and needs informed project modifications. Four primary care clinics were recruited, key informant interviews with clinic providers were conducted, electronic health record (EHR) capacity to collect data related to CRC screening and follow-up was assessed, and a series of meetings were held with clinic champions to discuss implementation of strategies to impact clinic CRC screening rates. Analysis of interviews revealed multilevel barriers to CRC screening. Patient-level barriers included fatalism, competing priorities, and financial and literacy concerns. The main provider- and clinic-level barriers were provider preference for colonoscopy over stool-based testing and EHR tracking concerns. Clinics selected strategies to address barriers, but the onset of COVID-19 necessitated modifications to these strategies. Due to COVID-19, changes in clinic staffing and workflow occurred, including provider furloughs, a state-mandated pause in elective procedures, and an increase in telehealth. Clinics adapted screening strategies to match changing needs, including shifting from paper to digital educational tools and using telehealth to increase annual wellness visits for screening promotion. While significant delays persist for scheduling colonoscopies, clinics were encouraged to promote stool-based tests as a primary screening modality for average-risk patients.

Cyclin A2 maintains colon homeostasis and is a prognostic factor in colorectal cancer.

To clarify the function of cyclin A2 in colon homeostasis and colorectal cancer (CRC), we generated mice deficient for cyclin A2 in colonic epithelial cells (CECs). Colons of these mice displayed architectural changes in the mucosa and signs of inflammation, as well as increased proliferation of CECs associated with the appearance of low- and high-grade dysplasias. The main initial events triggering those alterations in cyclin A2-deficient CECs appeared to be abnormal mitoses and DNA damage. Cyclin A2 deletion in CECs promoted the development of dysplasia and adenocarcinomas in a murine colitis-associated cancer model. We next explored the status of cyclin A2 expression in clinical CRC samples at the mRNA and protein levels and found higher expression in tumors of patients with stage 1 or 2 CRC compared with those of patients with stage 3 or 4 CRC. A meta-analysis of 11 transcriptome data sets comprising 2239 primary CRC tumors revealed different expression levels of CCNA2 (the mRNA coding for cyclin A2) among the CRC tumor subtypes, with the highest expression detected in consensus molecular subtype 1 (CMS1) and the lowest in CMS4 tumors. Moreover, we found high expression of CCNA2 to be a new, independent prognosis factor for CRC tumors.

Crenolanib Regulates ERK and AKT/mTOR Signaling Pathways in RAS/BRAF-Mutated Colorectal Cancer Cells and Organoids.

Recently developed molecularly targeted therapies such as EGFR inhibitors have notably improved the prognosis of patients with cancer. However, patients with KRAS and BRAF mutations do not currently benefit from these therapies. Here, we aimed to examine potential effects of crenolanib as a new molecularly targeted therapy in colorectal cancer. We used multiple colorectal cancer cell lines to investigate the growth-inhibitory effect of crenolanib and its effect in combination with other cytotoxic agents. Primary cultures of patient-derived organoids (PDO), a model that reflects the heterogeneity of clinical colorectal cancer, were used to further validate the effects of crenolanib. Unlike cetuximab, crenolanib remarkably suppressed ERK and AKT/mTOR pathways in HT29 cells with BRAF mutation and in HCT116 cells with KRAS mutation with corresponding growth-suppressing effects. Additive or synergistic effects were observed in treatments with combination of crenolanib and other cytotoxic drugs. Moreover, crenolanib suppressed the expression of stem cell markers, such as OCT4, NANOG, and SOX2. These observations were substantiated in seven PDOs with KRAS mutation and two PDOs without KRAS/BRAF mutations, with crenolanib suppressing the growth of all PDOs regardless of their KRAS mutation status. Furthermore, crenolanib abrogated PDGF- and TGFβ-induced increase of OCT4-positive cells in PDOs. Together, these findings suggest that crenolanib may have clinical utility for patients with colorectal cancer, especially patients with KRAS/BRAF mutations. IMPLICATIONS: These findings indicate that crenolanib can be a useful target agent for patients with colorectal cancer, especially patients with KRAS/BRAF mutations.

AMIGO2 as a novel indicator of liver metastasis in patients with colorectal cancer.

Our previous study showed that adhesion molecule with immunoglobulin like domain 2 (AMIGO2) is a pivotal driver gene of liver metastasis via regulating tumor cell adhesion to liver endothelial cells in mouse models. The aim of the present study was to clarify the role of AMIGO2 in liver metastasis in patients the colorectal cancer (CRC). Two human CRC cell lines, Caco-2 (AMIGO2-low) and HCT116 (AMIGO2-high), were used in this study. AMIGO2-overexpressing Caco-2 and AMIGO2-knockdown HCT116 cells were generated by transfection with an AMIGO2 expression vector or AMIGO2 small interfering RNA, respectively. Cell proliferation, invasion and adhesion to human liver endothelial cells were examined in in vitro studies. Immunohistochemical analysis was also performed to evaluate the association between AMIGO2 expression and liver metastasis in patients with CRC. In vitro studies revealed that cell proliferation, invasion and adhesion to liver endothelial cells were accelerated by upregulation of AMIGO2 expression, but suppressed by downregulation of AMIGO2 expression in human CRC cells. Immunohistochemical analysis using clinical CRC specimens revealed that AMIGO2 expression was associated with the frequency of liver metastasis (P<0.01), but not that of pulmonary metastasis (P=0.611) and peritoneal dissemination (P=0.909). In addition, AMIGO2 expression levels in tumor cells were significantly higher in liver metastatic foci than primary lesions (P=0.012). In conclusion, the present results indicated that AMIGO2 expression may contribute to the formation of liver metastasis in CRC.