Junichiro Hiro

CXCL5, a promoter of cell proliferation, migration and invasion, is a novel serum prognostic marker in patients with colorectal cancer

PURPOSE Serum CXCL5 levels in patients with colorectal cancer (CRC) were assessed to evaluate correlation with clinicopathologic features and prognosis. The effects of CXCL5 on CRC cells were also investigated in vitro. METHODS Based on cytokine array analysis, CXCL5 was identified as a novel prognostic serum marker. Serum levels of CXCL5 were assessed in 250 CRC patients and 33 normal volunteers by enzyme-linked immunosorbent assay (ELISA), and their relation to clinicopathologic findings and survival investigated. CXCL5 levels in CRC cell lines were also measured by ELISA, and CXCL5 and CXCR2 expression was evaluated by immunohistochemistry. To investigate the biological role of the CXCL5/CXCR2 axis, recombinant human CXCL5 and CXCR2 neutralisation antibodies were used for proliferation, migration and invasion assays. RESULTS Preoperative serum CXCL5 was significantly elevated in patients with CRC compared with healthy volunteers (p=0.013). High serum CXCL5 was significantly associated with female sex (p=0.0098) and liver metastasis (p=0.0040). Univariate analysis correlated elevated CXCL5 with poor overall survival (p=0.0002). Multivariate analysis showed that elevated CXCL5 was a significant and independent prognostic factor of survival in all CRC patients (p=0.038). CRC cells secreted CXCL5, and administration of recombinant human CXCL5 promoted proliferation, migration and partial invasion. These effects were generally inhibited by CXCR2 neutralisation antibody. CONCLUSIONS Preoperative serum CXCL5 could serve as a novel predictive marker for prognosis determination of CRC patients. CXCL5/CXCR2 axis might be associated with colorectal cancer progression.

The expression patterns of Toll-like receptors in the ileal pouch mucosa of postoperative ulcerative colitis patients.

The aim of this study was to evaluate the expression pattern of Toll-like receptors (TLRs) in the pouch mucosa of ulcerative colitis patients in comparison with that in the ileum mucosa of noninflammatory bowel disease patients. Pouch mucosal biopsy specimens were collected from postoperative patients who had undergone surgery for ulcerative colitis. Normal ileum specimens were collected from colon cancer patients. The specimens were assessed by immunofluorescence histochemistry using TLR2, TLR3, TLR4, and TLR5 polyclonal antibodies. The normal ileal mucosa constitutively expressed TLR3 and TLR5, whereas TLR2 and TLR4 were barely detectable. In the mucosa of active pouchitis, TLR2 and TLR4 was strongly upregulated, and TLR4 was upregulated even in a noninflamed pouch. No TLR3 or TLR5 expression was detectable. These data suggest that pouchitis may be associated with distinctive changes in selective TLR expression in the pouch mucosa, and that TLR4 alterations in the innate response system may contribute to the pathogenesis of these disorders in particular.

Soluble CXCL16 in preoperative serum is a novel prognostic marker and predicts recurrence of liver metastases in colorectal cancer patients.

PurposeThis study was designed to identify novel and reliable serum prognostic markers in patients with colorectal cancer (CRC).MethodsBased on cytokine array analysis, we identified soluble CXCL16 as a novel prognostic serum marker. Serum levels of CXCL16 were assessed in 314 CRC patients and 20 normal volunteers by enzyme-linked immunosorbent assay, and their relationships with clinicopathologic findings, including survival, were investigated. Proliferation, invasion, and wound healing assays were used to investigate the biological role of soluble CXCL16 in CRC cells, by exposure of HT-29 cells to recombinant CXCL16.ResultsThe median serum CXCL16 concentration in CRC patients was significantly higher than that in normal volunteers. In addition, serum CXCL16 levels increased significantly in accordance with the progression of UICC stage classification. Elevated serum CXCL16 level was significantly associated with poor survival and was an independent prognostic marker in CRC patients. Furthermore, in stage I–III CRC patients who underwent curative intent surgery, elevated serum CXCL16 levels were significantly associated with metachronous liver recurrence and poor survival. Recombinant soluble CXCL16 promoted the epithelial–mesenchymal transition (EMT) phenotype characterized by impaired E-cadherin production and induction of vimentin in vitro. In addition, recombinant soluble CXCL16 promoted cell growth, migration, and invasion in a CRC cell line.ConclusionsIn this study, we identified CXCL16 as a novel prognostic marker. Preoperative high serum levels of CXCL16 were associated with metachronous liver recurrence and poor prognosis in CRC patients. Soluble CXCL16 may play an important role in liver metastases through the induction of EMT.

Prognostic Significance of a Systemic Inflammatory Response in Patients Undergoing Multimodality Therapy for Advanced Colorectal Cancer

Objectives: The inflammation-based Glasgow Prognostic Score (GPS) is associated with outcome in a variety of cancers. This study investigated whether a modified GPS (mGPS) could predict survival in patients undergoing multimodality therapy for advanced colorectal cancer (CRC). Methods: We enrolled 245 patients with advanced CRC who received chemotherapy. The mGPS was recorded prior to first-line chemotherapy and to cytoreductive therapy including secondary surgery and/or radiofrequency ablation. The prognostic significance of the mGPS was analyzed using Kaplan-Meier, univariate, and multivariate analyses. Results: In patients who received chemotherapy alone (n = 163), the mGPS prior to chemotherapy was an independent prognostic indicator of survival [odds ratio (OR) 1.858; 95% confidence interval (CI) 1.213–2.846; p = 0.0044]. In patients who also underwent cytoreductive therapy (n = 82), the mGPS decreased after chemotherapy in 22 patients (27%) and increased in 5 (6%). In these patients, the mGPS prior to cytoreductive therapy was an independent prognostic indicator of survival (OR 3.412; 95% CI 1.198–9.720; p = 0.0216), but the mGPS prior to chemotherapy was not. Conclusions: The mGPS is an independent prognostic indicator of survival in patients undergoing multimodality therapy for advanced CRC, if recorded at a relevant time point.

RacGAP1 expression, increasing tumor malignant potential, as a predictive biomarker for lymph node metastasis and poor prognosis in colorectal cancer

Rac GTPase-activating protein (RacGAP) 1 plays a key role in controlling various cellular phenomena including cytokinesis, transformation, invasive migration and metastasis. This study investigated the function and clinical significance of RacGAP1 expression in colorectal cancer (CRC). The intrinsic functions of RacGAP1 in CRC cells were analyzed using small interfering RNA (siRNA). We analyzed RacGAP1 mRNA expression in surgical specimens from 193 CRC patients (Cohort 1) by real-time PCR. Finally, we validated RacGAP1 protein expression using formalin-fixed paraffin-embedded samples from 298 CRC patients (Cohort 2) by immunohistochemistry. Reduced RacGAP1 expression by siRNA in CRC cell lines showed significantly decreased cellular proliferation, migration and invasion. In Cohort 1, RacGAP1 expression in CRC was significantly higher than in adjacent normal mucosa and increased according to tumor node metastasis stage progression. High RacGAP1 expression in tumors was significantly associated with progression and prognosis. In Cohort 2, RacGAP1 protein was overexpressed mainly in the nuclei of CRC cells; however, its expression was scarcely observed in normal colorectal mucosa. RacGAP1 protein expression was significantly higher in CRC patients with higher T stage, vessel invasion and lymph node and distant metastasis. Increased expression of RacGAP1 protein was significantly associated with poor disease-free and overall survival. Multivariate analyses revealed that high RacGAP1 expression was an independent predictive marker for lymph node metastasis, recurrence and poor prognosis in CRC. Our data provide novel evidence for the biological and clinical significance of RacGAP1 as a potential biomarker for identifying patients with lymph node metastasis and poor prognosis in CRC.

Systemic Analysis of Predictive Biomarkers for Recurrence in Colorectal Cancer Patients Treated with Curative Surgery

AbstractBackgroundPreoperative serum systemic inflammatory response (SIR) in patients with colorectal cancer (CRC) has been reported to be a predictive biomarker of early recurrence. The molecular status of CRC, including microsatellite instability (MSI), BRAF and KRAS mutations, and tumor-infiltrating lymphocytes (TILs), has also been associated with recurrence in CRC patients treated with curative surgery.AimWe investigated the impacts of SIR status, TILs, and MSI on recurrence in curative CRC patients.MethodsIn this retrospective study, we enrolled 157 patients with stage I–III CRC undergoing curative surgery, for whom preoperative neutrophil/lymphocyte ratio (NLR), platelet/lymphocyte ratio (PLR), and C-reactive protein (CRP) data were available as indicators of SIR status. Molecular status was evaluated by counting TILs as the numbers of intratumoral Foxp3- and CD8-positive T cells by immunohistochemistry. MSI status was determined using five mononucleotide repeat microsatellite markers.ResultsKaplan–Meier analysis of SIR indicators revealed that higher CRP, NLR, and PLR were associated with significantly poorer disease-free survival (DFS). Low levels of infiltrating CD8-positive T cells in CRC tissue was a significant predictor of poor DFS. Multivariate analysis showed that few infiltrating CD8-positive T cells and high serum CRP levels were independent predictive factors for recurrence. Furthermore, the combination of high CRP and few infiltrating CD8-positive T cells increased the predictive accuracy in these patients.ConclusionsThe results of this study suggest that both CRP levels in preoperative serum and CD8 T cells in CRC tissue are useful biomarkers for predicting early relapse in CRC patients treated with curative surgery.

Gene expression profiles of tumor regression grade in locally advanced rectal cancer after neoadjuvant chemoradiotherapy

Tumor regression grading (TRG) reportedly has prognostic value in rectal cancer patients after pre-operative chemoradiotherapy (CRT). The aim of this retrospective study was to differentiate gene expression profiles based on TRG in residual cancer cells after CRT. We evaluated pathological response using the criteria of four TRG systems: the Japanese Society for the Cancer of Colon and Rectum (JSCCR), Mandard, Dworak and Rödel. Total RNA was obtained using microdissection from 52 locally advanced rectal cancer specimens from patients who underwent pre-operative CRT to examine the expression levels of 20 genes [PCNA, MKI67, CDKN1A (p21Cip1), CDK2, CHEK1, PDRG1, LGR5, PROM1 (CD133), CD44, SOX2, POU5F1 (OCT4), LKB1, VEGF, EGFR, HGF, MET, HIF1, GLUT1, BAX and BCL2] using real-time quantitative RT-PCR. Gene expression was compared across the four TRG systems. LGR5 gene expression levels in CRT non-responders were significantly higher than in responders in all four grading systems. Patients with elevated PDRG1 and GLUT1 gene expression had poor pathological response in three TRG systems (JSCCR, Dworak and Rödel). MKI67 gene expression in non-responders was significantly higher than in responders in two grading systems (JSCCR and Rödel). While, BAX gene expression in responders was significantly higher than in non-responders in the Mandard TRG system. The results of this study suggest that TRG may reflect characteristics, such as proliferative activity, stemness potency and resistance to hypoxia, of residual cancer cells following pre-operative CRT.

Macrophage inflammatory protein-3 alpha (MIP-3a) is a novel serum prognostic marker in patients with colorectal cancer.

A significant prognostic difference exists among metastatic colorectal cancer (CRC) patients despite of any treatments. We identify the specific cytokines related to prognosis of metastatic CRC and assess their prognostic significance.

Mechanism of resistance to chemoradiation in p53 mutant human colon cancer

To understand one of the mechanisms of resistance to chemoradiation in colon cancer cells, we investigated whether 5-fluorouracil (5-FU) mediated the expression of epidermal growth factor receptor (EGFR) and modified repair of radiation-induced DNA damage, especially in a p53 independent pathway. Cytotoxicity was determined for 5-FU combined with radiation for three colon cancer cell lines that contain mutant p53 (SW480, HT29 and WiDr), using the WST-8 colorimetric assay. EGFR and the excision repair cross complementation group 1 (ERCC1) proteins during chemoradiation were measured by Western blot analysis. SW480 cells were significantly resistant to chemoradiation compared to the other mutant p53 cell lines. The alteration of EGFR and ERCC1 proteins during chemoradiation in SW480 was apparently inversely related to that of the other radiosensitive cell lines. 5-FU-induced activation of EGFR followed by radiation in SW480 cells resulted in up-regulation of ERCC1. In contrast, 5-FU-induced degradation of EGFR followed by radiation in the other radiosensitive cell lines resulted in down-regulation of ERCC1. This suggested a complementary interaction between EGFR and ERCC1, and that 5-FU-induced EGFR activation conferred protection against radiation, through activation of DNA repair. Interaction of EGFR and ERCC1 might correlate with radiation-induced DNA damage when p53 mutant colon cancer cell lines are exposed to 5-FU followed by radiation.

Possibility of paclitaxel as an alternative radiosensitizer to 5-fluorouracil for colon cancer

To evaluate the feasibility of paclitaxel (PTX) radiosensitization for colon cancer, we investigated the cytotoxic and G2/M checkpoint protein (Chk1, Wee1, Bub1, MAD2) effects of 5-fluorouracil (5-FU) or PTX combined with radiation in the human colon cancer cell line LoVo. Cytotoxicity and radiocytotoxicity were evaluated for each drug by the WST-8 colorimetric assay. The IC20 for each drug was determined as a cytotoxic concentration from a survival curve. LoVo cells were exposed to the IC20 of each drug for 24 h and then irradiated. Expressions of the G2/M checkpoint proteins were confirmed by Western blot analysis. Cytotoxicity was induced by 5-FU or PTX alone in a time- and dose-dependent manner. The IC20 of PTX caused higher radiosensitivity than the IC20 of 5-FU (P<0.05). Western blot analysis revealed different expression patterns of the G2/M checkpoint proteins between 5-FU and PTX pre-treatments. 5-FU combined with radiation tended to decrease the expressions of all G2/M checkpoint proteins in a time-dependent manner. PTX combined with radiation maintained high expressions of Chk1 and MAD2 proteins for 24 h post-radiation and, in particular, MAD2 protein was strongly induced by PTX with high-dose radiation. PTX showed higher radio-sensitization than 5-FU for the colon cancer cell line LoVo and may be an alternative radiosensitizer to 5-FU in the clinical setting.