BRAF V600E-induced distinct DNA damage response defines the therapeutic potential of p53 activation for TP53 wild-type colorectal cancer.

BRAF V600E, one of the most frequent mutations in the MAPK pathway, confers poor prognosis to colorectal cancers (CRCs), partly because of chemotherapeutic resistance. Oncogene-induced DNA damage responses (DDRs) that primarily activate p53 are important mechanistic barriers to the malignant transformation of cells; however, the mechanism underlying this impairment in cancer remains unknown. Here, we evaluated the responses of BRAFV600E-induced DDRs in two CRC cell lines, SW48 and LIM1215, both of which harbor wild-type TP53, KRAS, and BRAF. BRAFV600E transduction exhibited distinct phenotypes in these cells: SW48 cell proliferation markedly decreased, whereas that of LIM1215 increased. BRAFV600E expression induced the activation of oncogene-induced DDR signaling in SW48 cells, but not in LIM1215 cells, whereas chemotherapeutic agents similarly activated DDRs in both cell lines. Knockdown experiments revealed that these responses in SW48 cells were mediated by p53-p21 pathway activation. Comet assay (both alkaline and neutral) revealed that BRAFV600E increased single-strand breaks to the same extent in both cell lines; however, in case of LIM1215 cells, it only facilitated double-strand breaks. Furthermore, the proliferation of LIM1215 cells, wherein no oncogene-induced DDRs occurred, was synergistically inhibited upon MDM2 inhibitor-mediated p53 activation combined with MEK inhibition. Taken together, these distinct DDR signaling responses highlight the novel characteristics of BRAFV600E-mutated CRC cells and define the therapeutic potential of p53 activation combined with MAPK inhibition against TP53 wild-type CRC harboring a BRAFV600E mutation.

RNA methylation in inflammatory bowel disease.

RNA modifications, including the renowned m6A, have recently garnered significant attention. This chemical alteration, present in mRNA, exerts a profound influence on protein expression levels by affecting splicing, nuclear export, stability, translation, and other critical processes. Although the role of RNA methylation in the pathogenesis and progression of IBD and colorectal cancer has been reported, many aspects remain unresolved. In this comprehensive review, we present recent studies on RNA methylation in IBD and colorectal cancer, with a particular focus on m6A and its regulators. We highlight the pivotal role of m6A in the pathogenesis of IBD and colorectal cancer and explore the potential applications of m6A modifications in the diagnosis and treatment of these diseases.

KLK10 derived from tumor endothelial cells accelerates colon cancer cell proliferation and hematogenous liver metastasis formation.

Tumor endothelial cells (TECs), which are thought to be structurally and functionally different from normal endothelial cells (NECs), are increasingly attracting attention as a therapeutic target in hypervascular malignancies. Although colorectal liver metastasis (CRLM) tumors are hypovascular, inhibitors of angiogenesis are a key drug in multidisciplinary therapy, and TECs might be involved in the development and progression of cancer. Here, we analyzed the function of TEC in the CRLM tumor microenvironment. We used a murine colon cancer cell line (CT26) and isolated TECs from CRLM tumors. TECs showed higher proliferation and migration than NECs. Coinjection of CT26 and TECs yielded rapid tumor formation in vivo. Immunofluorescence analysis showed that coinjection of CT26 and TECs increased vessel formation and Ki-67+ cells. Transcriptome analysis identified kallikrein-related peptide 10 (KLK10) as a candidate target. Coinjection of CT26 and TECs after KLK10 downregulation with siRNA suppressed tumor formation in vivo. TEC secretion of KLK10 decreased after KLK10 downregulation, and conditioned medium after KLK10 knockdown in TECs suppressed CT26 proliferative activity. Double immunofluorescence staining of KLK10 and CD31 in CRLM tissues revealed a significant correlation between poor prognosis and positive KLK10 expression in TECs and tumor cells. On multivariate analysis, KLK10 expression was an independent prognostic factor in disease-free survival. In conclusion, KLK10 derived from TECs accelerates colon cancer cell proliferation and hematogenous liver metastasis formation. KLK10 in TECs might offer a promising therapeutic target in CRLM.

Tumor suppressive role of the epigenetic master regulator BRD3 in colorectal cancer.

Bromodomain and extraterminal domain (BET) family proteins are epigenetic master regulators of gene expression via recognition of acetylated histones and recruitment of transcription factors and co-activators to chromatin. Hence, BET family proteins have emerged as promising therapeutic targets in cancer. In this study, we examined the functional role of bromodomain containing 3 (BRD3), a BET family protein, in colorectal cancer (CRC). In vitro and vivo analyses using BRD3-knockdown or BRD3-overexpressing CRC cells showed that BRD3 suppressed tumor growth and cell cycle G1/S transition and induced p21 expression. Clinical analysis of CRC datasets from our hospital or The Cancer Genome Atlas revealed that BET family genes, including BRD3, were overexpressed in tumor tissues. In immunohistochemical analyses, BRD3 was observed mainly in the nucleus of CRC cells. According to single-cell RNA sequencing in untreated CRC tissues, BRD3 was highly expressed in malignant epithelial cells, and cell cycle checkpoint-related pathways were enriched in the epithelial cells with high BRD3 expression. Spatial transcriptomic and single-cell RNA sequencing analyses of CRC tissues showed that BRD3 expression was positively associated with high p21 expression. Furthermore, overexpression of BRD3 combined with knockdown of, a driver gene in the BRD family, showed strong inhibition of CRC cells in vitro. In conclusion, we demonstrated a novel tumor suppressive role of BRD3 that inhibits tumor growth by cell cycle inhibition in part via induction of p21 expression. BRD3 activation might be a novel therapeutic approach for CRC.

Three-year outcomes of preoperative chemoradiotherapy plus nivolumab in microsatellite stable and microsatellite instability-high locally advanced rectal cancer.

BACKGROUND: Preoperative chemoradiotherapy (CRT) followed by surgery is the standard treatment for locally advanced rectal cancer (LARC). We reported the short-term outcomes of the VOLTAGE trial that investigated the safety and efficacy of preoperative CRT followed by nivolumab and surgery. Here, we present the 3-year outcomes of this trial. METHODS: Thirty-nine patients with microsatellite stable (MSS) LARC and five patients with microsatellite instability-high (MSI-H) LARC underwent CRT (50.4 Gy) followed by five doses of nivolumab (240 mg) and surgery. The 3-year relapse-free survival (RFS), overall survival (OS), and associations with biomarkers were evaluated. RESULTS: The 3-year RFS rates in patients with MSS and MSI-H were 79.5% and 100%, respectively, and the 3-year OS rates were 97.4% and 100%, respectively. Of the MSS patients, those with pre-CRT PD-L1 positivity, pre-CRT high CD8 + T cell/effector regulatory T cell (eTreg) ratio, pre-CRT high expression of Ki-67, CTLA-4, and PD-1 had a trend toward better 3-year RFS than those without. CONCLUSIONS: Three-year outcomes of patients with MSI-H were better than those of patients with MSS. PD-L1 positivity, elevated CD8/eTreg ratio, and high expression of Ki-67, CTLA-4, and PD-1 could be positive predictors of prognosis in patients with MSS. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02948348.

Impact of the endoscopic surgical skill qualification system on conversion to laparotomy after low anterior resection for rectal cancer in Japan (a secondary analysis of the EnSSURE study).

BACKGROUND AND AIMS: Conversion to laparotomy is among the serious intraoperative complications and carries an increased risk of postoperative complications. In this cohort study, we investigated whether or not the Endoscopic Surgical Skill Qualification System (ESSQS) affects the conversion rate among patients undergoing laparoscopic surgery for rectal cancer. METHODS: We performed a retrospective secondary analysis of data collected from patients undergoing laparoscopic surgery for cStage II and III rectal cancer from 2014 to 2016 across 56 institutions affiliated with the Japan Society of Laparoscopic Colorectal Surgery. Data from the original EnSSURE study were analyzed to investigate risk factors for conversion to laparotomy by performing univariate and multivariate analyses based on the reason for conversion. RESULTS: Data were collected for 3,168 cases, including 65 (2.1%) involving conversion to laparotomy. Indicated conversion accounted for 27 cases (0.9%), while technical conversion accounted for 35 cases (1.1%). The multivariate analysis identified the following independent risk factors for indicated conversion to laparotomy: tumor diameter [mm] (odds ratio [OR] 1.01, 95% confidence interval [CI] 1.01-1.05, p = 0.0002), combined resection of adjacent organs [+/-] (OR 7.92, 95% CI 3.14-19.97, p < 0.0001), and surgical participation of an ESSQS-certified physician [-/+] (OR 4.46, 95% CI 2.01-9.90, p = 0.0002). The multivariate analysis identified the following risk factors for technical conversion to laparotomy: registered case number of institution (OR 0.99, 95% CI 0.99-1.00, p = 0.0029), institution type [non-university/university hospital] (OR 3.52, 95% CI 1.54-8.04, p = 0.0028), combined resection of adjacent organs [+/-] (OR 5.96, 95% CI 2.15-16.53, p = 0.0006), and surgical participation of an ESSQS-certified physician [-/+] (OR 6.26, 95% CI 3.01-13.05, p < 0.0001). CONCLUSIONS: Participation of ESSQS-certified physicians may reduce the risk of both indicated and technical conversion. Referral to specialized institutions, such as high-volume centers and university hospitals, especially for patients exhibiting relevant background risk factors, may reduce the risk of conversion to laparotomy and lead to better outcomes for patients. TRIAL REGISTRATION: This study was registered with the Japanese Clinical Trials Registry as UMIN000040645.

[A Case of Sigmoid Colon Cancer with Horseshoe Kidney].

We reported a case of sigmoid colon cancer with horseshoe kidney. A 79-year-old man had lower abdominal pain and underwent colonoscopy. The results of colonoscopy revealed sigmoid cancer. Preoperative computed tomography revealed horseshoe kidney. He underwent radical laparoscopic surgery. The histopathological diagnosis was pStage Ⅱa(The 9th Edition). He has not recurred 22 months later after operation. Surgery for colorectal cancer with congenital anomalies of the urinary tract requires attention to intraoperative secondary injuries. Therefore, preoperative evaluation using 3D-CT is useful tool for safety. Operating the proper dissecting normal layer would make safe laparoscopic operation possible without unexpected injuries.

Cancer-specific tissue-resident memory T-cells express ZNF683 in colorectal cancer.

BACKGROUND: Tissue-resident memory T (Trm) cells are associated with cytotoxicity not only in viral infection and autoimmune disease pathologies but also in many cancers. Tumour-infiltrating CD103+ Trm cells predominantly comprise CD8 T cells that express cytotoxic activation and immune checkpoint molecules called exhausted markers. This study aimed to investigate the role of Trm in colorectal cancer (CRC) and characterise the cancer-specific Trm. METHODS: Immunochemical staining with anti-CD8 and anti-CD103 antibodies for resected CRC tissues was used to identify the tumour-infiltrating Trm cells. The Kaplan-Meier estimator was used to evaluate the prognostic significance. Cells immune to CRC were targeted for single-cell RNA-seq analysis to characterise cancer-specific Trm cells in CRC. RESULTS: The number of CD103+/CD8+ tumour-infiltrating lymphocytes (TILs) was a favourable prognostic and predictive factor of the overall survival and recurrence-free survival in patients with CRC. Single-cell RNA-seq analysis of 17,257 CRC-infiltrating immune cells revealed a more increased zinc finger protein 683 (ZNF683) expression in cancer Trm cells than in noncancer Trm cells and in high-infiltrating Trm cells than low-infiltrating Trm in cancer, with an upregulated T-cell receptor (TCR)- and interferon-γ (IFN-γ) signalling-related gene expression in ZNF683+ Trm cells. CONCLUSIONS: The number of CD103+/CD8+ TILs is a prognostic predictive factor in CRC. In addition, we identified the ZNF683 expression as one of the candidate markers of cancer-specific Trm cells. IFN-γ and TCR signalling and ZNF683 expression are involved in Trm cell activation in tumours and are promising targets for cancer immunity regulation.

Subclonal accumulation of immune escape mechanisms in microsatellite instability-high colorectal cancers.

BACKGROUND: Intratumor heterogeneity (ITH) in microsatellite instability-high (MSI-H) colorectal cancer (CRC) has been poorly studied. We aimed to clarify how the ITH of MSI-H CRCs is generated in cancer evolution and how immune selective pressure affects ITH. METHODS: We reanalyzed public whole-exome sequencing data on 246 MSI-H CRCs. In addition, we performed a multi-region analysis from 6 MSI-H CRCs. To verify the process of subclonal immune escape accumulation, a novel computational model of cancer evolution under immune pressure was developed. RESULTS: Our analysis presented the enrichment of functional genomic alterations in antigen-presentation machinery (APM). Associative analysis of neoantigens indicated the generation of immune escape mechanisms via HLA alterations. Multiregion analysis revealed the clonal acquisition of driver mutations and subclonal accumulation of APM defects in MSI-H CRCs. Examination of variant allele frequencies demonstrated that subclonal mutations tend to be subjected to selective sweep. Computational simulations of tumour progression with the interaction of immune cells successfully verified the subclonal accumulation of immune escape mutations and suggested the efficacy of early initiation of an immune checkpoint inhibitor (ICI) -based treatment. CONCLUSIONS: Our results demonstrate the heterogeneous acquisition of immune escape mechanisms in MSI-H CRCs by Darwinian selection, providing novel insights into ICI-based treatment strategies.

Cancer Stem Cells Persist Despite Cellular Damage, Emergence of the Refractory Cell Population.

PURPOSE: Cancer stem cells (CSCs) are responsible for chemotherapy resistance and have unique properties that protect them from chemotherapy. Investigating CSCs may help to identify the population that is more resistant to treatments, leading to recurrence. We evaluated persisting CSCs, emerging after chemotherapy that cause tumor recurrence. METHODS: Using human colorectal cancer organoids prepared from surgical specimens, we looked at changes in CSCs, the emergence and changes in the original population, which single-cell analysis identified. RESULTS: With regards to changes in cancer stem cell markers, CD44 showed low levels after 5-fluorouracil administration. Once the CD44-ve population was sorted and cultured, the CD44+ve population gradually emerged, and the CD44-ve population decreased. Compared with the CD44-ve population of an organoid parent, the CD44-ve population proliferated after chemotherapeutic agent stimulation. The CD44-ve population was derived from the CD44+ve population before chemotherapeutic agents. In addition, when the CD44 variants were evaluated, the CD44v9 population remained. In single-cell analysis, we found that POU5F1 was highly expressed in the CD44low population. Velocity analysis showed that the CD44-ve population was induced after chemotherapy and expressed POU5F1. POU5F1-EGFP-Casp9 transfected organoids resulted in the appearance of a CD44-ve population after administration of a chemotherapeutic reagent. Both in vivo and in vitro, the dimerizer administration inhibited tumor growth significantly. CONCLUSIONS: POU5F1 is involved in chemotherapy resistance in relation to stemness. For the treatment against refractory tumors, such as the recurrence after chemotherapy, the treatment should target the emerging specific population such as CD44 (or CD44v9) and proliferative cancer cells.

Clinical Significance of Preoperative and Postoperative Serum CEA and Carbohydrate Antigen 19-9 Levels in Patients Undergoing Curative Resection of Locally Recurrent Rectal Cancer.

BACKGROUND: Local recurrence is common after curative resection for rectal cancer. Although one expects radical resection of locally recurrent rectal cancer to be curative, the postoperative re-recurrence rate is relatively high. Therefore, identifying risk factors for recurrence may improve the prognosis of locally recurrent rectal cancer with early therapeutic intervention. OBJECTIVE: This study aimed to evaluate the relationship between perioperative serum CEA/carbohydrate antigen 19-9 levels and prognosis in locally recurrent rectal cancer to validate their usefulness for postoperative surveillance in locally recurrent rectal cancer. DESIGN: This was a single-center retrospective cohort study. SETTING: The study is based on data obtained from procedures at the Osaka University Hospital. PATIENTS: Ninety patients underwent radical resection for locally recurrent rectal cancer between January 2000 and January 2015. MAIN OUTCOME MEASURES: We evaluated the correlation between perioperative serum CEA/carbohydrate antigen 19-9 levels and prognosis after complete resection of locally recurrent rectal cancer and the serum CEA and carbohydrate antigen 19-9 levels at the diagnosis of postoperative re-recurrence. RESULTS: The median preoperative serum CEA level was 4 ng/mL and carbohydrate antigen 19-9 level was 12 U/mL. Of the 90 patients, 43.3% had serum CEA ≥5 ng/mL, and 15.6% had serum carbohydrate antigen 19-9 ≥37 U/mL. Preoperatively, this serum carbohydrate antigen 19-9 level strongly correlated with poorer prognoses regarding cancer-specific survival. Postoperatively, serum CEA ≥5 ng/mL significantly correlated with a worse prognosis. At the time of diagnosis of re-recurrence after resection of locally recurrent rectal cancer, 53.2% of patients had serum CEA ≥5 ng/mL, and 23.4% of patients had serum carbohydrate antigen 19-9 ≥37 U/mL. LIMITATIONS: The study was limited by its single-center retrospective design, an insufficient sample size, and a relatively long study period. CONCLUSIONS: High serum levels of carbohydrate antigen 19-9 preoperatively and CEA postoperatively are associated with poor prognosis after locally recurrent rectal cancer. Furthermore, we found a high rate of serum CEA elevation in the diagnosis of postoperative re-recurrence. See Video Abstract at http://links.lww.com/DCR/C106 . IMPORTANCIA CLNICA DE LOS NIVELES SRICOS PREOPERATORIOS Y POSOPERATORIOS DE CEA Y CA EN PACIENTES SOMETIDOS A RESECCIN CURATIVA DE CNCER DE RECTO LOCALMENTE RECURRENTE: ANTECEDENTES:La recurrencia local es común después de la resección curativa del cáncer de recto. Aunque se espera que la resección radical del cáncer rectal localmente recurrente sea curativa, la tasa de recurrencia posoperatoria es relativamente alta. Por lo tanto, la identificación de los factores de riesgo de recurrencia puede mejorar el pronóstico del cáncer de recto localmente recurrente con una intervención terapéutica temprana.OBJETIVO:Evaluamos la relación entre los niveles séricos perioperatorios de CEA/CA19-9 y el pronóstico en el cáncer de recto localmente recurrente para validar su utilidad para la vigilancia posoperatoria en el cáncer de recto localmente recurrente.DISEÑO:Este fue un estudio de cohorte retrospectivo de un solo centro.AJUSTE:El estudio se basa en datos obtenidos de procedimientos en el Hospital Universitario de Osaka.PACIENTES:Noventa pacientes fueron sometidos a resección radical por cáncer de recto localmente recurrente entre Enero de 2000 y Enero de 2015.PRINCIPALES MEDIDAS DE RESULTADOS:Evaluamos la correlación entre los niveles séricos perioperatorios de CEA/CA19-9 y el pronóstico después de la resección completa del cáncer de recto localmente recurrente y los niveles séricos de CEA y CA19-9 en el diagnóstico de recurrencia posoperatoria.RESULTADOS:La mediana de los niveles séricos preoperatorios de CEA y CA19-9 fueron de 4 ng/mL y 12 U/mL, respectivamente. De los 90 pacientes, el 43,3 % tenía CEA sérico ≥5 ng/mL y el 15,6 % tenía CA19-9 sérico ≥37 U/mL. Antes de la operación, este nivel sérico de CA19-9 se correlacionó fuertemente con peores pronósticos con respecto a la supervivencia específica del cáncer. Después de la operación, el CEA sérico ≥5 ng/mL se correlacionó significativamente con un peor pronóstico. En el momento del diagnóstico de recurrencia después de la resección del cáncer de recto localmente recurrente, el 53,2 % de los pacientes tenían CEA sérico ≥5 ng/mL y el 23,4 % de los pacientes tenían CA19-9 sérico ≥37 U/mL.LIMITACIONES:El estudio estuvo limitado por su diseño retrospectivo de un solo centro, un tamaño de muestra insuficiente y un período de estudio relativamente largo.CONCLUSIONES:Los niveles séricos altos de CA19-9 antes de la operación y de CEA después de la operación están asociados con un mal pronóstico después del cáncer de recto localmente recurrente. Además, encontramos una alta tasa de elevación del CEA sérico en el diagnóstico de recurrencia posoperatoria. Consulte el Video Resumen en http://links.lww.com/DCR/C106 . (Traducción-Dr. Yesenia Rojas-Khalil ).

Simple Detection and Culture of Circulating Tumor Cells from Colorectal Cancer Patients Using Poly(2-Methoxyethyl Acrylate)-Coated Plates.

Here we aimed to establish a simple detection method for detecting circulating tumor cells (CTCs) in the blood sample of colorectal cancer (CRC) patients using poly(2-methoxyethyl acrylate) (PMEA)-coated plates. Adhesion test and spike test using CRC cell lines assured efficacy of PMEA coating. A total of 41 patients with pathological stage II-IV CRC were enrolled between January 2018 and September 2022. Blood samples were concentrated by centrifugation by the OncoQuick tube, and then incubated overnight on PMEA-coated chamber slides. The next day, cell culture and immunocytochemistry with anti-EpCAM antibody were performed. Adhesion tests revealed good attachment of CRCs to PMEA-coated plates. Spike tests indicated that ~75% of CRCs from a 10-mL blood sample were recovered on the slides. By cytological examination, CTCs were identified in 18/41 CRC cases (43.9%). In cell cultures, spheroid-like structures or tumor-cell clusters were found in 18/33 tested cases (54.5%). Overall, CTCs and/or growing circulating tumor cells were found in 23/41 CRC cases (56.0%). History of chemotherapy or radiation was significantly negatively correlated with CTC detection (p = 0.02). In summary, we successfully captured CTCs from CRC patients using the unique biomaterial PMEA. Cultured tumor cells will provide important and timely information regarding the molecular basis of CTCs.

[A Case of Squamous Cell Carcinoma of the Lower Rectum].

A 61-year-old man presented with dyschezia, and further examination revealed squamous cell carcinoma of the lower rectum invading the bladder and seminal vesicles. The clinical diagnosis was squamous cell carcinoma of the lower rectum, cT4b(bladder and seminal vesicle)N0M0, cStage Ⅱc. Neoadjuvant chemoradiotherapy was administered with external irradiation of the entire pelvis(50.4 Gy/28 Fr)and chemotherapy with 5-fluorouracil, Leucovorin, and oxaliplatin(FOLFOX). Once tumor shrinkage was observed 3 months after chemoradiotherapy, laparoscopic total pelvic exenteration with TaTME approach was performed. The patient was discharged on the 26th postoperative day without any postoperative complications. Histopathological examination showed only squamous cell carcinoma component with Grade 1a histological treatment effect. The pathological diagnosis was ypT4b(bladder, seminal vesicle)ypN0cM0, ypStage Ⅱc. The patient was alive without any recurrence 6 months after surgery.

Low G9a expression is a tumor progression factor of colorectal cancer via IL-8 promotion.

The histone methyltransferase G9a is expressed in various types of cancer cells, including colorectal cancer (CRC) cells. Interleukin 8 (IL)-8, also known as C-X-C motif chemokine ligand 8 (CXCL8), is a chemokine that plays a pleiotropic function in the regulation of inflammatory responses and cancer development. Here, we examined the relationship between G9a and IL-8 and the clinical relevance of this association. We immunohistochemically analyzed 235 resected CRC samples to correlate clinical features. Samples with high G9a expression had better overall survival and relapse-free survival than those with low G9a expression. Univariate and multivariate analyses demonstrated that low G9a expression remained a significant independent prognostic factor for increased disease recurrence and decreased survival (P < 0.05). G9a was expressed at high levels in commercially available CRC cell lines HCT116 and HT29. Knockdown of G9a by siRNA, shRNA or the G9a-specific inhibitor BIX01294 upregulated IL-8 expression. The number of spheroids was significantly increased in HCT116 cells with stably suppressed G9a expression, and the number of spheroids was significantly decreased in HCT116 cells with stably suppressed IL-8 expression. Thus, the suppression of IL-8 by G9a may result in a better prognosis in CRC cases with high G9a expression. Furthermore, G9a may suppress cancer stemness and increase chemosensitivity by controlling IL-8. Therefore, G9a is a potential novel marker for predicting CRC prognosis, and therapeutic targeting of G9a in CRC should be controversial.

A stem cell marker KLF5 regulates CCAT1 via three-dimensional genome structure in colorectal cancer cells.

BACKGROUND: KLF5 plays a crucial role in stem cells of colorectum in cooperation with Lgr5 gene. In this study, we aimed to explicate a regulatory mechanism of the KLF5 gene product from a view of three-dimensional genome structure in colorectal cancer (CRC). METHODS: In vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)-seq method was used to identify the regions that bind to the KLF5 promoter. RESULTS: We revealed that the KLF5 promoter region interacted with the KLF5 enhancer region as well as the transcription start site (TSS) region of the Colon Cancer Associated Transcript 1 (CCAT1) gene. Notably, the heterodeletion mutants of KLF5 enhancer impaired the cancer stem-like properties of CRC cells. The KLF5 protein participated in the core-regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which constructs the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. In vitro analysis indicated that KLF5 regulated CCAT1 expression and we found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples. CONCLUSIONS: Our data propose the mechanistic insight that the KLF5 protein constructs the core-regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC.

PLXND1/SEMA3E Promotes Epithelial-Mesenchymal Transition Partly via the PI3K/AKT-Signaling Pathway and Induces Heterogenity in Colorectal Cancer.

Colorectal cancer (CRC) is a major cause of cancer-related deaths. Metastasis is enhanced through epithelial-mesenchymal transition (EMT), a process primarily induced by the transforming growth factor beta (TGF-ß)-mediated canonical Smad pathway. This study focused on plexin D1 (PLXND1), a chemoreceptor for the ligand SEMA3E to mechanosensory, showing that PLXND1 induces EMT via activation of the PI3K/AKT pathway in CRC cells. The findings showed that PLXND1-knockdown decreases cell migration and invasion significantly, and that the binding of p61-SEMA3E to the PLXND1 enhances the invasiveness and migration through EMT. Furin inhibitor suppresses EMT, decreasing cell migration and invasion. Furin cleaves full-length SEMA3E and converts it to p61-SEMA3E, suggesting that furin inhibitors block PLXND1 and p61-SEMA3E binding. Furin is a potential therapeutic target for the purpose of suppressing EMT by inhibiting the binding of p61-SEMA3E to PLXND1. In vivo experiments have shown that PLXND1-knockdown suppresses EMT. Mesenchymal cells labeled with ZEB1 showed heterogeneity depending on PLXND1 expression status. The high-expression group of PLXND1 in 182 CRC samples was significantly associated with poor overall survival compared with the low-expression group (P = 0.0352, median follow-up period of 60.7 months) using quantitative real-time polymerase chain reaction analysis. Further research is needed to determine whether cell fractions with a different expression of PLXND1 have different functions.

Development and evaluation of a Japanese prediction model for low anterior resection syndrome after rectal cancer surgery.

BACKGROUND: Low anterior resection syndrome (LARS) is the most common complication after rectal cancer resection. We aimed to identify LARS' predictive factors and construct and evaluate a predictive model for LARS. METHODS: This retrospective study included patients with rectal cancer more than 1 year after laparoscopic or robotic-assisted surgery. We administered a questionnaire to evaluate the degree of LARS. In addition, we examined clinical characteristics with univariate and multivariate analysis to identify predictive factors for major LARS. Finally, we divided the obtained data into a learning set and a validation set. We constructed a predictive model for major LARS using the learning set and assessed the predictive accuracy of the validation set. RESULTS: We reviewed 160 patients with rectal cancer and divided them into a learning set (n = 115) and a validation set (n = 45). Univariate and multivariate analyses in the learning set showed that male (odds ratio [OR]: 2.88, 95% confidence interval [95%CI] 1.11-8.09, p = 0.03), age < 75 years (OR: 5.87, 95%CI 1.14-47.25, p = 0.03) and tumors located < 8.5 cm from the AV (OR: 7.20, 95%CI 2.86-19.49, p < 0.01) were significantly related to major LARS. A prediction model based on the patients in the learning set was well-calibrated. CONCLUSIONS: We found that sex, age, and tumor location were independent predictors of major LARS in Japanese patients that underwent rectal cancer surgery. Our predictive model for major LARS could aid medical staff in educating and treating patients with rectal cancer before and after surgery.

TRESBIEN (OGSG 2101): encorafenib, binimetinib and cetuximab for early recurrent stage II/III BRAF V600E-mutated colorectal cancer.

The BRAF V600E mutation accounts for approximately 5% of colorectal cancer (CRC) cases and is an extremely poor prognostic factor. However, there are no clear recommendations regarding first-line therapy for patients with early recurrent BRAF V600E-mutated CRC, during or after adjuvant chemotherapy. Recently, a novel combination of encorafenib, binimetinib and cetuximab, showed a higher response rate than standard chemotherapy in patients with BRAF V600E-mutated CRC. Here we describe our plan for the TRESBIEN study (OGSG 2101), which is an open-label, multicenter, single-arm, phase II study designed to evaluate whether encorafenib, binimetinib and cetuximab are effective for patients with early recurrent BRAF V600E-mutated colorectal cancer, during or after adjuvant chemotherapy. The planned number of subjects is 25.

An ongoing study to evaluate encorafenib, binimetinib and cetuximab for people with early recurrent BRAF V600E-mutated colorectal cancer. BRAF V600E-mutated colorectal cancer (CRC) is a type of cancer caused by change (mutation) in a gene called BRAF. It is one of the most difficult types of CRC to treat because currently available drugs do not effectively treat the disease. Recently, two novel treatments, encorafenib and cetuximab, have been approved for use together in several countries for the treatment of advanced or metastatic BRAF V600E-mutated CRC. In Japan, these drugs are also approved to be given with another treatment called binimetinib, an approach called triplet therapy. This article describes the ongoing TRESBIEN study that is looking at how effective and how safe triplet therapy is for the treatment of people with early recurrent BRAF V600E-mutated CRC, during or after they have additional (adjuvant) chemotherapy. This study is ongoing, and the researchers are currently recruiting new participants. TRESBIEN will evaluate the percentage of participants whose tumors shrink with triplet therapy. The study will also look at any side effects. Clinical Trial Registration: jRCTs051210152 (ClinicalTrials.gov) (Japan Registry of Clinical Trials https://jrct.niph.go.jp/search?language=en&page=1).

Fecal Stream Diversion Changes Intestinal Environment, Modulates Mucosal Barrier, and Attenuates Inflammatory Cells in Crohn's Disease.

BACKGROUND: The intestinal environment plays important roles in mucosal barrier homeostasis and intestinal inflammation, as clarified in studies using experimental animals but not in humans. AIMS: We investigated whether environmental changes in the fecal stream cause phenotypic changes in the human mucosal barrier. METHODS: We obtained human ileal samples after fecal stream diversions in patients with rectal cancer or Crohn's disease. We investigated the bacterial load and diversity in the human defunctioned ileum, defined as the anal side of the ileum relative to the ileostomy. We also examined the epithelium and lamina propria cell phenotypes in the defunctioned ileum. RESULTS: After fecal stream diversion, bacterial loads decreased significantly in the defunctioned ileum. Based on the Chao1, Shannon, and observed species indices, the diversity of mucosa-associated microbiota was lower in the defunctioned ileum than in the functional ileum. Moreover, the healthy defunctioned ileum showed reductions in villous height, goblet cell numbers, and Ki-67+ cell numbers. Additionally, interferon-γ+, interleukin-17+, and immunoglobulin A+ cell abundance in the lamina propria decreased. After the intestinal environment was restored with an ileostomy closure, the impaired ileal homeostasis recovered. The defunctioned ileum samples from patients with Crohn's disease also showed reductions in interferon-γ+ and interleukin-17+ cell numbers. CONCLUSIONS: Fecal stream diversion reduced the abundance and diversity of intestinal bacteria. It also altered the intestinal mucosal barrier, similar to the alterations observed in germ-free animals. In patients with Crohn's disease, Th1 and Th17 cell numbers were attenuated, which suggests that the host-microbiome interaction is important in disease pathogenesis.

Locally advanced rectal cancer receiving total neoadjuvant therapy combined with nivolumab: a case report and literature review.

BACKGROUND: The standard treatment for locally advanced rectal cancer (LARC) is preoperative chemoradiotherapy (CRT) followed by surgery and adjuvant chemotherapy. However, it has been suggested that intensification of neoadjuvant treatment with polychemotherapy in addition to CRT instead of as an adjuvant chemotherapy is better tolerated and associated with a higher pathological complete response (pCR) rate. This concept is known as total neoadjuvant therapy (TNT). Recently, the addition of immunotherapy to preoperative CRT has been reported to be useful in LARC patients with mismatch-repair-deficiency and high levels of microsatellite instability (MSI-H), but there are no reports showing the therapeutic effect of nivolumab in combination with TNT. CASE PRESENTATION: A 23-year-old man had frequent diarrhea. Preoperative examination revealed two adenocarcinomas in the rectum. His maternal grandmother had a rectal cancer patient who developed the disease at age 70s. The larger tumor was located at the peritoneal reflection, and its anterior border close to the prostate (<1 mm); there were eight enlarged pararectal lymph nodes. Considering the size and depth of the tumor, it was judged that radical resection with sufficient margins would be difficult. Therefore, it was decided that TNT would be performed. At first, CAPOX (capecitabine and L-OHP) was administered, followed by preoperative CRT (RT:50.4 Gy and capecitabine). During this period, genetic testing diagnosed this patient as MSI-H, so additional nivolumab was administered after CRT. Colonoscopy revealed that the larger tumor was no longer detectable, so robot-assisted intersphincteric resection and bilateral lateral lymph node dissection was performed. The diagnosis of pCR was made for the larger tumor and partial response was achieved for the smaller tumor, and no lymph node metastasis was found. Major complications were not observed and the patient was discharged on the 14th day after surgery. He was followed up without adjuvant chemotherapy and is alive and recurrence-free after 9 months. CONCLUSION: A case of LARC with MSI-H was treated with TNT with nivolumab, resulting in pCR and complete radical resection. This result suggests that nivolumab in addition to TNT can be an option as a preoperative strategy for LARC with MSI-H.