Mikio Kawamura

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.

Cancer-associated fibroblasts correlate with poor prognosis in rectal cancer after chemoradiotherapy

Cancer-associated fibroblasts (CAFs) in the stroma play an important role in influencing the proliferation, invasion and metastasis of cancer cells. Fibroblast activation protein-α (FAP-α) is known as a marker of CAFs, while stromal cell-derived factor-1 (SDF-1) is primarily expressed by CAFs. Herein, we investigated whether the expression levels of these genes are associated with clinical outcome after pre-operative chemoradiotherapy (CRT) in rectal cancer patients. We obtained total RNA from residual cancer stroma using microdissection from a total of 52 rectal cancer specimens from patients who underwent pre-operative CRT, we performed transcriptional analyses, and the serum protein concentrations in 40 matched microdissected specimens were measured by enzyme-linked immunosorbent assay. Additionally, we sought to clarify the location of FAP-α and SDF-1 expression using immunohistochemical staining. Of the 52 patients, 15.6 and 36.8% showed detectable FAP-α and SDF-1 mRNA expression, respectively. A significant correlation was observed between stromal FAP-α and SDF-1 mRNA levels. Moreover, there was a significant correlation between stromal SDF-1 gene expression levels and serum protein levels. Patients who developed distant recurrences after CRT had positive expression of both genes (P<0.05). The positive expression of both genes was also associated with poor probability of recurrence-free and overall survival (P<0.05). Patients with elevated serum SDF-1 levels had equally poor overall survival as those with positive stromal SDF-1 gene expression (P<0.05). In immunohistochemistry, both FAP-α and SDF-1 expression was observed in certain activated fibroblasts. In conclusion, FAP-α and SDF-1 expression was shown to be involved in tumor re-growth and recurrence in rectal cancer patients treated with pre-operative CRT.

Brain-derived neurotrophic factor/tropomyosin-related kinase B pathway in gastric cancer

Background:Brain-derived neutrophic factor (BDNF) is a member of the neutrophin family that is known to activate the high-affinity tropomyosin-related receptor kinase B (TrkB). This study aimed to clarify the clinical and biological significance of the BDNF/TrkB pathway in gastric cancer.Methods:We analysed BDNF and TrkB expression in gastric cancer samples by real-time reverse transcription PCR and immunohistochemistry. To investigate the biological role of BDNF/TrkB axis, recombinant human BDNF (rhBDNF) and the Trk antagonist K252a were used for in vitro and in vivo analysis.Results:The BDNF expression at the invasive front of primary tumours was significantly elevated compared with that in the tumour core and adjacent normal mucosa. Increased BDNF expression at the invasive front was significantly correlated with factors reflecting disease progression, and poor prognosis. Increased co-expression of the BDNF/TrkB axis was significantly correlated with poor prognosis. Gastric cancer cells expressed BDNF, and administration of rhBDNF promoted proliferation, migration, invasion, and inhibition of anoikis. These effects were generally inhibited by K252a. In an in vivo assay, BDNF(+)/TrkB(+) gastric cancer cells injected into nude mice established peritoneal dissemination, whereas K252a inhibited tumour growth.Conclusion:The BDNF/TrkB pathway might be deeply involved in gastric cancer disease progression.

Evaluation of CXCL10 as a novel serum marker for predicting liver metastasis and prognosis in colorectal cancer

The aim of this study was to identify novel and reliable serum markers related to the prognosis of colorectal cancer (CRC) patients and to assess the association between selected markers and clinical outcome. We performed experiments using cytokine arrays to investigate the cytokine profiles in serum from stage IV CRC patients, compared with those of stage I patients. Serum CXCL10 was measured using an ELISA in 218 CRC patients and 17 normal volunteers to clarify the association of CXCL10 with clinical outcome. The mean serum CXCL10 concentration in CRC patients was significantly higher compared to that in normal volunteers. Serum CXCL10 levels increased significantly in accordance with the progression of UICC stage classification. Serum CXCL10 was significantly associated with high pathological T stage, the presence of vascular invasion and distant metastasis. Elevated serum CXCL10 levels were significantly associated with poor survival in all stages or in stage I-III with curative patients, respectively, and were an independent marker in predicting liver metastasis. Immunohistochemical analysis showed that CXCL10 was expressed in cancer cells at primary tumor and liver metastases sites, and in normal liver tissue surrounding metastatic cancer cells. Comprehensive analysis using cytokine arrays identified the novel serum prognosis marker CXCL10. Preoperative high serum levels of CXCL10 were associated with poor prognosis and liver metastasis in CRC.

Brain-derived neurotrophic factor (BDNF)-induced tropomyosin-related kinase B (Trk B) signaling is a potential therapeutic target for peritoneal carcinomatosis arising from colorectal cancer.

Tropomyosin-related receptor kinase B (TrkB) signaling, stimulated by brain-derived neurotrophic factor (BDNF) ligand, promotes tumor progression, and is related to the poor prognosis of various malignancies. We sought to examine the clinical relevance of BDNF/TrkB expression in colorectal cancer (CRC) tissues, its prognostic value for CRC patients, and its therapeutic potential in vitro and in vivo. Two hundred and twenty-three CRC patient specimens were used to determine both BDNF and TrkB mRNA levels. The expression of these proteins in their primary and metastatic tumors was investigated by immunohistochemistry. CRC cell lines and recombinant BDNF and K252a (a selective pharmacological pan-Trk inhibitor) were used for in vitro cell viability, migration, invasion, anoikis resistance and in vivo peritoneal metastasis assays. Tissue BDNF mRNA was associated with liver and peritoneal metastasis. Tissue TrkB mRNA was also associated with lymph node metastasis. The co-expression of BDNF and TrkB was associated with liver and peritoneal metastasis. Patients with higher BDNF, TrkB, and co-expression of BDNF and TrkB had a significantly poor prognosis. BDNF increased tumor cell viability, migration, invasion and inhibited anoikis in the TrkB-expressing CRC cell lines. These effects were suppressed by K252a. In mice injected with DLD1 co-expressing BDNF and TrkB, and subsequently treated with K252a, peritoneal metastatic nodules was found to be reduced, as compared with control mice. BDNF/TrkB signaling may thus be a potential target for treating peritoneal carcinomatosis arising from colorectal cancer.

In Vivo Time-Course Imaging of Tumor Angiogenesis in Colorectal Liver Metastases in the Same Living Mice Using Two-Photon Laser Scanning Microscopy

In vivo real-time visualization of the process of angiogenesis in secondary tumors in the same living animals presents a major challenge in metastasis research. We developed a technique for intravital imaging of colorectal liver metastasis development in live mice using two-photon laser scanning microscopy (TPLSM). We also developed time-series TPLSM in which intravital TPLSM procedures were performed several times over periods of days to months. Red fluorescent protein-expressing colorectal cancer cells were inoculated into the spleens of green fluorescent protein-expressing mice. First- and second-round intravital TPLSM allowed visualization of viable cancer cells (red) in hepatic sinusoids or the space of Disse. Third-round intravital TPLSM demonstrated liver metastatic colonies consisting of viable cancer cells and surrounding stroma with tumor vessels (green). In vivo time-course imaging of tumor angiogenesis in the same living mice using time-series TPLSM could be an ideal tool for antiangiogenic drug evaluation, reducing the effects of interindividual variation.

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.

Elevated serum angiopoietin-like protein 2 correlates with the metastatic properties of colorectal cancer: a serum biomarker for early diagnosis and recurrence

Purpose: Angiopoietin-like protein 2 (ANGPTL2) is a mediator of chronic inflammation and inflammatory carcinogenesis. The biologic and clinical significance of ANGPTL2 remains unknown in human cancer. Therefore, we investigated the function of ANGPTL2 and evaluated its clinical significance in both primary tumors and matched sera in patients with colorectal cancer. Experimental Design: A colorectal cancer cell line was transfected with siRNA against ANGPTL2 for the assessment of its function. We examined ANGPTL2 expression in colorectal cancer tissues (n = 195) by immunohistochemistry. Finally, we screened serum ANGPTL2 levels from 32 colorectal cancers and 23 normal controls (NC), and validated these results in serum samples obtained from 195 colorectal cancers and 45 NCs by ELISA. Results: Knockdown of ANGPTL2 in vitro significantly inhibited cell proliferation, migration, and invasion, whereas it enhanced anoikis. ANGPTL2 was overexpressed in colorectal cancer tissues, and was significantly associated with advanced T stage, lymph node, and liver metastasis. Likewise, serum ANGPTL2 levels in colorectal cancers were significantly higher than NCs (P < 0.01), and allowed distinguishing of colorectal cancers from NCs with high accuracy (AUC = 0.837). The subsequent validation step confirmed that serum ANGPTL2 levels in colorectal cancers were significantly higher than in NCs (P < 0.0001), and had a high AUC value (0.885) for distinguishing colorectal cancers from NCs. High serum ANGPTL2 was significantly associated with advanced T stage, lymph node and liver metastasis, early relapse, and poor prognosis in colorectal cancers. Conclusion: Serum ANGPTL2 is a novel diagnostic and recurrence-predictive biomarker in patients with colorectal cancer. Clin Cancer Res; 20(23); 6175–86. ©2014 AACR.

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.

Clinical significance of LGR5 and CD44 expression in locally advanced rectal cancer after preoperative chemoradiotherapy

LGR5, known as a target of Wnt signaling, has been reported as an intestinal stem cell marker. Recent reports showed that LGR5 was associated with carcinogenesis and tumor invasion in colorectal cancer. CD44 is a ubiquitously expressed cell adhesion molecule and also a potential cell surface marker on colon cancer stem cells. Both LGR5 and CD44 have been also reported to be Wnt signal targets. The aim of this study was to investigate the association of these markers with clinical outcome in rectal cancer after preoperative chemoradiotherapy (CRT). A total of 52 rectal cancer specimens were obtained from patients who underwent preoperative CRT. We performed transcriptional and immunohistochemical analyses, and retrospectively studied the association of LGR5 and CD44 expression levels with clinical outcomes. For CD44, its expression in cancer stroma was also evaluated. The levels of cancer LGR5 and CD44 gene expression were significantly and positively correlated. LGR5 gene expression level in cancer and positivity of CD44 gene expression in cancer stroma were significantly correlated with disease recurrence. Elevated cancer LGR5 gene expression and positive CD44 gene expression in cancer stroma were significantly associated with poor recurrence-free and overall survival. Multivariate analysis indicated that positivity of stromal CD44 gene expression was an independent prognostic factor for the recurrence and overall survival of patients with rectal cancer after preoperative CRT. In conclusion, LGR5 and CD44 expression may be coordinately associated with tumor relapse in locally advanced rectal cancer after preoperative CRT.