Molecular Deciphering of Colorectal Cancer: Exploring Molecular Classifications and Analyzing the Interplay among Molecular Biomarkers MMR/MSI, KRAS, NRAS, BRAF and CDX2 - A Comprehensive Literature Review.

Background: Colorectal cancer (CRC) exhibits molecular and morphological diversity, involving genetic, epigenetic alterations, and disruptions in signaling pathways. This necessitates a comprehensive review synthesizing recent advancements in molecular mechanisms, established biomarkers, as well as emerging ones like CDX2 for enhanced CRC assessment. Material and Methods: This review analyzes the last decade's literature and current guidelines to study CRC's molecular intricacies. It extends the analysis beyond traditional biomarkers to include emerging ones like CDX2, examining their interaction with carcinogenic mechanisms and molecular pathways, alongside reviewing current testing methodologies. Results: A multi-biomarker strategy, incorporating both traditional and emerging biomarkers like CDX2, is crucial for optimizing CRC management. This strategy elucidates the complex interaction between biomarkers and the tumor's molecular pathways, significantly influencing prognostic evaluations, therapeutic decision-making, and paving the way for personalized medicine in CRC. Conclusions: This review proposes CDX2 as an emerging prognostic biomarker and emphasizes the necessity of thorough molecular profiling for individualized treatment strategies. By enhancing CRC treatment approaches and prognostic evaluation, this effort marks a step forward in precision oncology, leveraging an enriched understanding of tumor behavior.

Individual transcription factors modulate both the micromovement of chromatin and its long-range structure.

The control of eukaryotic gene expression is intimately connected to highly dynamic chromatin structures. Gene regulation relies on activator and repressor transcription factors (TFs) that induce local chromatin opening and closing. However, it is unclear how nucleus-wide chromatin organization responds dynamically to the activity of specific TFs. Here, we examined how two TFs with opposite effects on local chromatin accessibility modulate chromatin dynamics nucleus-wide. We combine high-resolution diffusion mapping and dense flow reconstruction and correlation in living cells to obtain an imaging-based, nanometer-scale analysis of local diffusion processes and long-range coordinated movements of both chromatin and TFs. We show that the expression of either an individual transcriptional activator (CDX2) or repressor (SIX6) with large numbers of binding sites increases chromatin mobility nucleus-wide, yet they induce opposite coherent chromatin motions at the micron scale. Hi-C analysis of higher-order chromatin structures shows that induction of the pioneer factor CDX2 leads both to changes in local chromatin interactions and the distribution of A and B compartments, thus relating the micromovement of chromatin with changes in compartmental structures. Given that inhibition of transcription initiation and elongation by RNA Pol II has a partial impact on the global chromatin dynamics induced by CDX2, we suggest that CDX2 overexpression alters chromatin structure dynamics both dependently and independently of transcription. Our biophysical analysis shows that sequence-specific TFs can influence chromatin structure on multiple architectural levels, arguing that local chromatin changes brought by TFs alter long-range chromatin mobility and its organization.

CDX2 dose-dependently influences the gene regulatory network underlying human extraembryonic mesoderm development.

Loss of Cdx2 in vivo leads to stunted development of the allantois, an extraembryonic mesoderm-derived structure critical for nutrient delivery and waste removal in the early embryo. Here, we investigate how CDX2 dose-dependently influences the gene regulatory network underlying extraembryonic mesoderm development. By engineering human induced pluripotent stem cells (hiPSCs) consisting of wild-type (WT), heterozygous (CDX2-Het), and homozygous null CDX2 (CDX2-KO) genotypes, differentiating these cells in a 2D gastruloid model, and subjecting these cells to single-nucleus RNA and ATAC sequencing, we identify several pathways that are dose-dependently regulated by CDX2 including VEGF and non-canonical WNT. snATAC-seq reveals that CDX2-Het cells retain a WT-like chromatin accessibility profile, suggesting accessibility alone is not sufficient to drive this variability in gene expression. Because the loss of CDX2 or TBXT phenocopy one another in vivo, we compared differentially expressed genes in our CDX2-KO to those from TBXT-KO hiPSCs differentiated in an analogous experiment. This comparison identifies several communally misregulated genes that are critical for cytoskeletal integrity and tissue permeability. Together, these results clarify how CDX2 dose-dependently regulates gene expression in the extraembryonic mesoderm and reveal pathways that may underlie the defects in vascular development and allantoic elongation seen in vivo.

Combined Use of Helicobacter pylori Genotyping and CDX2 Expression as a Predictor of Malignant Potential in Gastric Intestinal Metaplasia.

OBJECTIVE: Gastrointestinal metaplasia (GIM) has a close relationship with gastric cancer (GC), but it is unclear how to judge which GIM could develop into GC. This study aimed to assess the role of CDX2 and its association with Helicobacter pylori (H.pylori) genotypes in GIM. METHODS: CagA and vacA genes were identified via PCR in 466 H. pylori-positive gastric tissues, including gastritis (n=104), GIM diagnosed endoscopically (GIM-1; n=82), gastric cancer (GC; n=173), and paired adjacent GIM tumors resected surgically (GIM-2; n=107). GIM was subclassified per the HID- AB pH2.5-PAS as follows: type I (n=23), type II (n=43), and type III (n=16) in GIM-1; type I (n=8), type II (n=40), and type III (n=59) in GIM-2. CDX2 expression was evaluated immunohistochemically. RESULTS: In GIM-1, the infection rate of vacAm2 (55.8%) and vacAs1m2 (53.5%) was higher in subtype II than in others (P<0.05), while that of vacAm1 (49.2%) and vacAs1m1 (33.9%) was higher in subtype III than in others. The cagA+ rate was higher in subtypes I (75.0%) and III (64.4%) than in subtype II (40.0%; P<0.05) respectively. CDX2 was upregulated in subtype I than in subtypes II and III in GIM-1 and GIM-2. In GIM-2 and GC, CDX2 was downregulated in vacAm1, vacAs1m1, and cagA+ (P<0.05). The predominant genotype was vacAs1m2 in subtype II of GIM-1, CDX2 expression remaining unaltered; however, the predominant genotype was cagA+ vacAs1m1 in subtypes II and III of GIM-2, negatively correlated with CDX2 expression. CONCLUSION: These GIM subtypes (cagA+ vacAs1m1 H. pylori-positive GIM with negative CDX2 expression) resemble GC and should be evaluated similar to cancerous GIM.

Tissue-location-specific transcription programs drive tumor dependencies in colon cancer.

Cancers of the same tissue-type but in anatomically distinct locations exhibit different molecular dependencies for tumorigenesis. Proximal and distal colon cancers exemplify such characteristics, with BRAFV600E predominantly occurring in proximal colon cancers along with increased DNA methylation phenotype. Using mouse colon organoids, here we show that proximal and distal colon stem cells have distinct transcriptional programs that regulate stemness and differentiation. We identify that the homeobox transcription factor, CDX2, which is silenced by DNA methylation in proximal colon cancers, is a key mediator of the differential transcriptional programs. Cdx2-mediated proximal colon-specific transcriptional program concurrently is tumor suppressive, and Cdx2 loss sufficiently creates permissive state for BRAFV600E-driven transformation. Human proximal colon cancers with CDX2 downregulation showed similar transcriptional program as in mouse proximal organoids with Cdx2 loss. Developmental transcription factors, such as CDX2, are thus critical in maintaining tissue-location specific transcriptional programs that create tissue-type origin specific dependencies for tumor development.

CDX2 alleviates hypoxia-induced apoptosis and oxidative stress in spermatogenic cells through suppression of reactive oxygen species-mediated Wnt/ß-catenin pathway.

Hypoxia-induced apoptosis and oxidative stress in spermatogenic cells are considered to be important factors leading to male infertility. It was reported that CDX2 expression was downregulated in hypoxia-stimulated spermatogenic cells. However, the effects of CDX2 on hypoxia-induced apoptosis and oxidative stress in spermatogenic cells are still unknown. This study aimed to explore the roles of CDX2 in hypoxia-induced injury of spermatogenic cells, as well as its mechanism of action. Spermatogenic cells were cultured under 1% oxygen for 48 h to established hypoxia damage model. Reactive oxygen species (ROS) generation was determined using 2',7'-dichlorofluorescein diacetate assay. Apoptosis was assessed using flow cytometry. Enzyme-linked immunosorbent assay was used to evaluate oxidative stress markers, including malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidases (GSH-Px). Protein levels were detected using western blotting. Hypoxia exposure induced increase in ROS generation, apoptosis rate, and oxidative stress in spermatogenic cells. ROS scavenger inhibited hypoxia-induced apoptosis, oxidative stress, and Wnt/ß-catenin pathway activation. Hypoxia exposure induced CDX2 downregulation. CDX2 overexpression suppressed hypoxia-induced ROS generation, apoptosis rate, oxidative stress, and Wnt/ß-catenin pathway activation. Moreover, CDX2 knockdown restores the inhibitory effects of si-ß-catenin or NAC on hypoxia-induced activation of the Wnt/ß-catenin pathway, apoptosis, and oxidative stress. In conclusion, our study suggests that CDX2 overexpression alleviates hypoxia-induced apoptosis and oxidative stress by suppression of ROS-mediated Wnt/ß-catenin pathway in spermatogenic cells.

TEAD4 regulates KRT8 and YAP in preimplantation embryos in mice but not in cattle.

In brief: Lineage specification plays a vital role in preimplantation development. TEAD4 is an essential transcription factor for trophectoderm lineage specification in mice but not in cattle. Abstract: Tead4, a critical transcription factor expressed during preimplantation development, is essential for the expression of trophectoderm-specific genes in mice. However, the functional mechanism of TEAD4 in mouse preimplantation development and its conservation across mammals remain unclear. Here, we report that Tead4 is a crucial transcription factor necessary for blastocyst formation in mice. Disruption of Tead4 through base editing results in developmental arrest at the morula stage. Additionally, RNA-seq analysis reveals dysregulation of 670 genes in Tead4 knockout embryos. As anticipated, Tead4 knockout led to a decrease in trophectoderm genes Cdx2 and Gata3. Intriguingly, we observed a reduction in Krt8, suggesting that Tead4 influences the integrity of the trophectoderm epithelium in mice. More importantly, we noted a dramatic decrease in nuclear Yap in outside cells for Tead4-deficient morula, indicating that Tead4 directly regulates Hippo signaling. In contrast, bovine embryos with TEAD4 depletion could still develop to blastocysts with normal expression of CDX2, GATA3, and SOX2, albeit with a decrease in total cell number and ICM cell number. In conclusion, we propose that Tead4 regulates mouse blastocyst formation via Krt8 and Yap, both of which are critical regulators of mouse preimplantation development.

Clinical impact of ampulla of Vater cancer subtype classification based on immunohistochemical staining.

BACKGROUND: The histological subtype is an important prognostic factor for ampulla of Vater (AoV) cancer. This study proposes a classification system for the histological subtyping of AoV cancer based on immunohistochemical (IHC) staining and its prognostic significance. METHODS: Seventy-five AoV cancers were analyzed for cytokeratin 7 (CK7), CK20, and causal-type homeobox transcription factor 2 (CDX2) expression by IHC staining. We differentiated the subtypes (INT, intestinal; PB, pancreatobiliary; MIX, mixed; NOS, not otherwise specified) into classification I: CK7/CK20, classification II: CK7/CK20 or CDX2, classification III: CK7/CDX2 and examined their associations with clinicopathological factors. RESULTS: Classifications I, II, and III subtypes were INT (7, 10, and 10 cases), PB (43, 37, and 38 cases), MIX (13, 19, and 18 cases), and NOS (12, 9, and 9 cases). Significant differences in disease-free survival among the subtypes were observed in classifications II and III using CDX2; the PB and NOS subtype exhibited shorter survival time compared with INT subtype. In classification III, an association was revealed between advanced T/N stage, poor differentiation, lymphovascular invasion (LVI), the PB and NOS subtypes, and recurrence risk. In classification III, the subtypes differed significantly in T/N stage and LVI. Patients with the PB subtype had advanced T and N stages and a higher incidence of LVI. CONCLUSIONS: Classification using CDX2 revealed subtypes with distinct prognostic significance. Combining CK7 and CDX2 or adding CDX2 to CK7/CK20 is useful for distinguishing subtypes, predicting disease outcomes, and impacting the clinical management of patients with AoV cancer.

Loss of SATB2 and CDX2 expression is associated with DNA mismatch repair protein deficiency and BRAF mutation in colorectal cancer.

The relationship between the expression of the SATB2 and CDX2 proteins and common molecular changes and clinical prognosis in colorectal cancer (CRC) still needs further clarification. We collected 1180 cases of CRC and explored the association between the expression of SATB2 and CDX2 and clinicopathological characteristics, molecular alterations, and overall survival of CRC using whole-slide immunohistochemistry. Our results showed that negative expression of SATB2 and CDX2 was more common in MMR-protein-deficient CRC than in MMR-protein-proficient CRC (15.8% vs. 6.0%, P = 0.001; 14.5% vs. 4.0%, P = 0.000, respectively). Negative expression of SATB2 and CDX2 was more common in BRAF-mutant CRC than in BRAF wild-type CRC (17.2% vs. 6.1%, P = 0.003; 13.8% vs. 4. 2%; P = 0.004, respectively). There was no relationship between SATB2 and/or CDX2 negative expression and KRAS, NRAS, and PIK3CA mutations. The lack of expression of SATB2 and CDX2 was associated with poor histopathological features of CRC. In multivariate analysis, negative expression of SATB2 (P = 0.030), negative expression of CDX2 (P = 0.043) and late clinical stage (P = 0.000) were associated with decreased overall survival of CRC. In conclusion, the lack of SATB2 and CDX2 expression in CRC was associated with MMR protein deficiency and BRAF mutation, but not with KRAS, NRAS and PIK3CA mutation. SATB2 and CDX2 are prognostic biomarkers in patients with CRC.

The CDX2 G allele and the FoKI F allele of the VDR gene are more prevalent and related to changes in vitamin D levels in patients with psoriasis vulgaris: A pilot study.

BACKGROUND AND AIMS: Psoriasis is a chronic, non-contagious autoimmune condition marked by dry, itchy,erythematous and scaly plaques. From modest, localized plaques to total body coverage, the severity of psoriasis varies. Plaque, guttate, inverted, pustular, and erythrodermic psoriasis are the five primary kinds. About 90% of cases are of plaque psoriasis, commonly known as psoriasis vulgaris. Study aims to determine the impact of an rs2228570 (FokI) variant and an rs11568820 (CDX2) variant on serum vitamin D levels (SVD) in patients with psoriasis, and the correlation between the two variants and disease severity. METHODS: A case-control study consisting of 95 psoriasis vulgaris patients and 84 healthy controls. The clinical investigation, molecular genetics analysis, and biochemical analysis were done for both groups. RESULTS: SVD levels were significantly decreased in psoriasis patients group. FokI genotypes analysis, we found no significant variance between groups. CDX2 G/G genotype is more prevalent in patients than controls. Moderate psoriasis vulgaris patients with CDX2 G/G genotypes have higher SVD levels than CDX2 G/A, and CDX2 A/A p = 0.003. CONCLUSION: The study found a difference in vitamin D levels between patients and healthy subjects, as well as a difference in vitamin D levels with different FoKI and CDX2 genotypes.

Novel inherited CDX2 variant segregating in a family with diverse congenital malformations of the genitourinary system.

Anorectal malformations (ARMs) constitute a group of congenital defects of the gastrointestinal and urogenital systems. They affect males and females, with an estimated worldwide prevalence of 1 in 5000 live births. These malformations are clinically heterogeneous and can be part of a syndromic presentation (syndromic ARM) or as a nonsyndromic entity (nonsyndromic ARM). Despite the well-recognized heritability of nonsyndromic ARM, the genetic etiology in most patients is unknown. In this study, we describe three siblings with diverse congenital anomalies of the genitourinary system, anemia, delayed milestones, and skeletal anomalies. Genome sequencing identified a novel, paternally inherited heterozygous Caudal type Homeobox 2 (CDX2) variant (c.722A > G (p.Glu241Gly)), that was present in all three affected siblings. The variant identified in this family is absent from population databases and predicted to be damaging by most in silico pathogenicity tools. So far, only two other reports implicate variants in CDX2 with ARMs. Remarkably, the individuals described in these studies had similar clinical phenotypes and genetic alterations in CDX2 CDX2 encodes a transcription factor and is considered the master regulator of gastrointestinal development. This variant maps to the homeobox domain of the encoded protein, which is critical for interaction with DNA targets. Our finding provides a potential molecular diagnosis for this family's condition and supports the role of CDX2 in anorectal anomalies. It also highlights the clinical heterogeneity and variable penetrance of ARM predisposition variants, another well-documented phenomenon. Finally, it underscores the diagnostic utility of genomic profiling of ARMs to identify the genetic etiology of these defects.

Histologic subtype-based evaluation of recurrence and survival outcomes in patients with adenocarcinoma of the ampulla of Vater.

Patients with ampulla of Vater adenocarcinoma exhibit diverse outcomes, likely since these malignancies can originate from any of the three converging epithelia at this site. Such variability presents difficulties in clinical decision-making processes and in devising therapeutic approaches. In this study, the potential clinical value of histomolecular phenotypes was determined by integrating histopathological analysis with protein expression (MUC1, CDX2, CK20, and MUC2), in a cohort of 87 patients diagnosed with stage IB to III ampulla of Vater adenocarcinoma who underwent curative surgical resection. Of the 87 patients, 54 were classified as pancreato-biliary (PB) subtype and 33 as intestinal subtype. The median follow-up time for all patients was 32.8 months (95% CI, 25.3-49.2). Patients with a histomolecular PB phenotype (CDX2 negative, MUC1 positive, MUC2 negative, and irrespective of the CK20 results) were associated with poor prognostic outcomes in both disease-free survival (DFS) (HR = 1.81; 95% CI, 1.04-3.17; p = 0.054) and overall survival (OS) (HR = 2.01; 95% CI, 1.11-3.66; p = 0.039) compared to those with histomolecular intestinal carcinomas. Patients with the PB subtype were more likely to have local recurrence alone (11 of 37, 29.7%) compared to those with the intestinal subtype (1 of 15, 6.7%). In the context of systemic disease, a notably greater proportion of patients exhibiting elevated carbohydrate antigen 19-9 levels were observed in the PB subtype compared to the intestinal subtype (p = 0.024). In the cohort of 38 patients who received first-line palliative chemotherapy, a diminished median overall survival (OS) was observed in the PB group compared to the intestinal group (10.3 vs. 28.3 months, HR = 2.47; 95% CI, 1.23-4.95; p = 0.025). By integrating histopathologic and molecular criteria, we can identify distinct and clinically relevant histomolecular phenotypes in adenocarcinomas of the ampulla of Vater, which could have considerable impact on existing therapeutic approaches.

A Case of Biphenotypic Adnexal Carcinoma With Bowenoid and Basaloid Features: Focus on the Expression of SOX9 and Wnt Signaling Pathway Molecules, Including CDX2.

ABSTRACT: An 87-year-old woman presented with a pedunculated nodule of 1.2 × 1.2 × 0.6 cm on her left cheek. Microscopic examination of the lesion revealed bowenoid and rosette-like basaloid components, resembling Bowen disease and neuroendocrine carcinoma, respectively. Immunohistochemically, both components were positive for Wnt signaling pathway molecules-nuclear/cytoplasmic beta-catenin, lymphoid enhancer binding factor 1 (LEF1), and caudal type homeobox 2 (CDX2)-and the adnexal marker SRY-box transcription factor 9 (SOX9). Unlike neuroendocrine tumors and basal cell carcinomas, the basaloid component in the present case was negative for chromogranin A, INSM1, synaptophysin, and p40. Previously reported cases of similar CDX2-positive lesions were diagnosed as squamous cell carcinoma with enteric adenocarcinomatous differentiation and basaloid cutaneous carcinoma with a primitive cytomorphology. However, the lesion in the present case was simultaneously positive for SOX9, indicating adnexal differentiation. In particular, the expression of multiple Wnt signaling pathway molecules indicates follicular differentiation despite the absence of morphological follicular features, such as shadow cells. Moreover, shared immunopositivity for SOX9, CDX2, nuclear/cytoplasmic beta-catenin, and LEF1 by both bowenoid and basaloid components indicated that the bowenoid component did not represent Bowen disease but a part of the adnexal tumor, and that the basaloid component was not a tumor-to-tumor metastasis. After complete excision, no recurrence has been observed for 5 months. The findings of the present case expand the histological spectrum of cutaneous adnexal tumors with follicular immunophenotypic differentiation.

The prognostic potential of CDX2 in colorectal cancer: Harmonizing biology and clinical practice.

Adjuvant chemotherapy following surgical intervention remains the primary treatment option for patients with localized colorectal cancer (CRC). However, a significant proportion of patients will have an unfavorable outcome after current forms of chemotherapy. While reflecting the increasing complexity of CRC, the clinical application of molecular biomarkers provides information that can be utilized to guide therapeutic strategies. Among these, caudal-related homeobox transcription factor 2 (CDX2) emerges as a biomarker of both prognosis and relapse after therapy. CDX2 is a key transcription factor that controls intestinal fate. Although rarely mutated in CRC, loss of CDX2 expression has been reported mostly in right-sided, microsatellite-unstable tumors and is associated with aggressive carcinomas. The pathological assessment of CDX2 by immunohistochemistry can thus identify patients with high-risk CRC, but the evaluation of CDX2 expression remains challenging in a substantial proportion of patients. In this review, we discuss the roles of CDX2 in homeostasis and CRC and the alterations that lead to protein expression loss. Furthermore, we review the clinical significance of CDX2 assessment, with a particular focus on its current use as a biomarker for pathological evaluation and clinical decision-making. Finally, we attempt to clarify the molecular implications of CDX2 deficiency, ultimately providing insights for a more precise evaluation of CDX2 protein expression.

Ectopic expression of HNF4α in Het1A cells induces an invasive phenotype.

Barrett's oesophagus (BO) is a pathological condition in which the squamous epithelium of the distal oesophagus is replaced by an intestinal-like columnar epithelium originating from the gastric cardia. Several somatic mutations contribute to the intestinal-like metaplasia. Once these have occurred in a single cell, it will be unable to expand further unless the altered cell can colonise the surrounding squamous epithelium of the oesophagus. The mechanisms by which this happens are still unknown. Here we have established an in vitro system for examining the competitive behaviour of two epithelia. We find that when an oesophageal epithelium model (Het1A cells) is confronted by an intestinal epithelium model (Caco-2 cells), the intestinal cells expand into the oesophageal domain. In this case the boundary involves overgrowth by the Caco-2 cells and the formation of isolated colonies. Two key transcription factors, normally involved in intestinal development, HNF4α and CDX2, are both expressed in BO. We examined the competitive ability of Het1A cells stably expressing HNF4α or CDX2 and placed in confrontation with unmodified Het1A cells. The key result is that stable expression of HNF4α, but not CDX2, increased the ability of the cells to migrate and push into the unmodified Het1A domain. In this situation the boundary between the cell types is a sharp one, as is normally seen in BO. The experiments were conducted using a variety of extracellular substrates, which all tended to increase the cell migration compared to uncoated plastic. These data provide evidence that HNF4α expression could have a potential role in the competitive spread of BO into the oesophagus as HNF4α increases the ability of cells to invade into the adjacent stratified squamous epithelium, thus enabling a single mutant cell eventually to generate a macroscopic patch of metaplasia.

Association of CDX2 and mucin expression with chemotherapeutic benefits in patients with stage II/III gastric cancer.

BACKGROUND: Better predictors of patients with stage II/III gastric cancer (GC) most likely to benefit from adjuvant chemotherapy are urgently needed. This study aimed to assess the ability of CDX2 and mucin markers to predict prognosis and fluorouracil-based adjuvant chemotherapy benefits. METHODS: CDX2 and mucin protein expressions were examined by immunohistochemistry and compared with survival and adjuvant chemotherapy benefits in a prospective evaluation cohort of 782 stage II/III GC patients. Then, the main findings were validated in an independent validation cohort (n = 386) and an external mRNA sequencing dataset (ACRG cohort, n = 193). RESULTS: In the evaluation cohort, CDX2, CD10, MUC2, MUC5AC, and MUC6 expressions were observed in 59.7%, 26.7%, 27.6%, 55.1%, and 57.7% of patients, respectively. However, only the expression of CDX2 was found to be associated with adjuvant chemotherapy benefits. Most importantly, CDX2-negative patients had a poorer prognosis when treated with surgery only, while the prognosis of CDX2-negative and CDX2-positive patients was similar when receiving postoperative adjuvant chemotherapy. Further analysis revealed that patients with CDX2 negative tumors benefited from chemotherapy (5-year overall survival rates: 60.0% with chemotherapy vs. 23.2% with surgery-only, p < 0.001), whereas patients with CDX2 positive tumors did not (pinteraction = 0.004). Consistent results were obtained in the validation and ACRG cohorts. CONCLUSIONS: Negative expression of CDX2 is an independent risk factor for survival in stage II/III GC, but subsequent adjuvant chemotherapy is able to compensate for this unfavorable effect. Therefore, active chemotherapy is more urgent for patients with negative CDX2 expression than for patients with positive CDX2 expression.

CDX2 Is a Prognostic Biomarker for Unresectable Metastatic Colorectal Cancer.

BACKGROUND/AIM: Colorectal cancer (CRC) with reduced expression of the homeobox transcription factor CDX2, a master gene essential for the development and maintenance of the intestinal tract, is known as a poor prognosis subtype of CRC. The recurrence rate is high in patients with CDX2low CRC. However, the prognostic significance of CDX2 in advanced CRC is unclear. This study aimed to elucidate the prognostic significance of CDX2 in unresectable metastatic CRC (mCRC). PATIENTS AND METHODS: Twenty-nine patients with unresectable mCRC who underwent primary site resection at the Kobe University Hospital during a 6-year period from January 2008 to January 2015 were included. The tissues from those patients were immunohistochemically stained with anti-CDX2 antibody (clone: CDX2-88). The patients were divided into CDX2high CRC group and CDX2low CRC group and their prognoses were analyzed. RESULTS: There were no clear differences in background between the two groups. A low CDX2 expression was associated with reduced overall survival (37.67 months vs. 25.32 months, p=0.03) and tended to associate with reduced progression-free survival (17.4 months vs. 12.9 months, p=0.37). Two patients received chemotherapy after resection of the primary lesion and obtained pathological complete response. CONCLUSION: CDX2 expression might be a possible prognostic biomarker for unresectable mCRC.

DNA methyltransferase (Dnmt) silencing causes increased Cdx2 and Nanog levels in surviving embryos.

Epigenetic mechanisms are one of the essential regulators of gene expression which do not involve altering the primary nucleotide sequence. DNA methylation is considered among the most prominent epigenetic mechanisms in controlling the functions of genes related to cell differentiation, cell cycle, cell survival, autophagy, and embryo development. DNA methyl transferases (Dnmts) control DNA methylation, the levels of which are differentially altered during embryonic development, and may determine cell differentiation fate as in the case of pluripotent inner cell mass (ICM) or trophectoderm (TE). In this study, we aimed to analyze the role of Dnmt1 and Dnmt3a enzymes in ICM (using the Nanog marker) and TE (using the Cdx2 marker) differentiation, autophagy (using p62 marker), reactive oxygen species (ROS) production, and apoptosis (using TUNEL) during mouse preimplantation embryo development. Following knockdown of Dnmt1 and Dnmt3a in zygotes, expression levels of Cdx2 in the trophectoderm and Nanog in the inner cell mass were measured, as well as p62 levels, reactive oxygen species (ROS) production, and apoptosis levels after 96 hours in embryo culture. We found that knockdown of Dnmt1 or Dnmt3a significantly induced Cdx2 and Nanog expression. Similarly, p62 expression, ROS levels and apoptosis significantly increased after silencing. This study shows that Dnmt genes are highly crucial for embryonic fate determination and survival. Further studies are required to reveal the specific targets of these methylation processes related to cell differentiation, survival, autophagy, and ROS production in mouse and human preimplantation embryos.

Helicobacter pylori promotes gastric intestinal metaplasia through activation of IRF3-mediated kynurenine pathway.

BACKGROUND: Metabolic reprogramming is a critical event for cell fate and function, making it an attractive target for clinical therapy. The function of metabolic reprogramming in Helicobacter pylori (H. pylori)-infected gastric intestinal metaplasia remained to be identified. METHODS: Xanthurenic acid (XA) was measured in gastric cancer cells treated with H. pylori or H. pylori virulence factor, respectively, and qPCR and WB were performed to detect CDX2 and key metabolic enzymes expression. A subcellular fractionation approach, luciferase and ChIP combined with immunofluorescence were applied to reveal the mechanism underlying H. pylori mediated kynurenine pathway in intestinal metaplasia in vivo and in vitro. RESULTS: Herein, we, for the first time, demonstrated that H. pylori contributed to gastric intestinal metaplasia characterized by enhanced Caudal-related homeobox transcription factor-2 (CDX2) and mucin2 (MUC2) expression, which was attributed to activation of kynurenine pathway. H. pylori promoted kynurenine aminotransferase II (KAT2)-mediated kynurenine pathway of tryptophan metabolism, leading to XA production, which further induced CDX2 expression in gastric epithelial cells. Mechanically, H. pylori activated cyclic guanylate adenylate synthase (cGAS)-interferon regulatory factor 3 (IRF3) pathway in gastric epithelial cells, leading to enhance IRF3 nuclear translocation and the binding of IRF3 to KAT2 promoter. Inhibition of KAT2 could significantly reverse the effect of H. pylori on CDX2 expression. Also, the rescue phenomenon was observed in gastric epithelial cells treated with H. pylori after IRF3 inhibition in vitro and in vivo. Most importantly, phospho-IRF3 was confirmed to be a clinical positive relationship with CDX2. CONCLUSION: These finding suggested H. pylori contributed to gastric intestinal metaplasia through KAT2-mediated kynurenine pathway of tryptophan metabolism via cGAS-IRF3 signaling, targeting the kynurenine pathway could be a promising strategy to prevent gastric intestinal metaplasia caused by H. pylori infection. Video Abstract.