Takeshi Yokoe

Correlation of CD133, OCT4, and SOX2 in Rectal Cancer and Their Association with Distant Recurrence After Chemoradiotherapy

BackgroundCancer stem cells are associated with metastatic potential, treatment resistance, and poor patient prognosis. Distant recurrence remains the major cause of mortality in rectal cancer patients with preoperative chemoradiotherapy (CRT). We investigated the role of three stem cell markers (CD133, OCT4, and SOX2) in rectal cancer and evaluated the association between these gene levels and clinical outcome in rectal cancer patients with preoperative CRT.MethodsThirty-three patients with rectal cancer underwent preoperative CRT. Total RNAs of rectal cancer cells before and after CRT were isolated. Residual cancer cells after CRT were obtained from formalin-fixed paraffin-embedded (FFPE) specimens using microdissection. The expression levels of three stem cell genes were measured using real-time reverse-transcription polymerase chain reaction (RT-PCR). The association between these gene levels and radiation was evaluated using colon cancer cell lines. Immunohistochemical staining of these markers after CRT was also investigated.ResultsThere were significant positive correlations among the three genes after CRT. Patients who developed distant recurrence had higher levels of the three genes compared with those without recurrence in residual cancer after CRT. These elevated gene levels were significantly associated with poor disease-free survival. The radiation caused upregulation of these gene levels in LoVo and SW480 in vitro. Immunohistochemically, CD133 staining was observed in not only luminal surface but also cytoplasm.ConclusionsExpression of CD133, OCT4, and SOX2 may predict distant recurrence and poor prognosis of rectal cancer patients treated with preoperative CRT. Correlations among these genes may be associated with tumor regrowth and metastatic relapse after CRT.

Preoperative C‐reactive protein as a prognostic and therapeutic marker for colorectal cancer

This study aimed to evaluate the significance of preoperative C‐reactive protein (CRP) as a prognostic marker for carcinoembryonic antigen (CEA)‐independent stage I or II colorectal cancer (CRC) patients.

Radiation induces epithelial-mesenchymal transition in colorectal cancer cells

Radiotherapy remains a major approach to adjuvant therapy for patients with advanced rectal cancer. Nevertheless, the effects of radiation on malignant processes have yet to be clarified. The aim of this study was to assess the biological effects of radiation on colorectal cancer (CRC) cells with special reference to epithelial-mesenchymal transition (EMT), a key developmental program often activated during cancer invasion and metastasis. We investigated the effect of radiation on two colorectal cancer cell lines, CaR1 and DLD1, assessing cell morphology, motility, migration and invasive ability. Expression of molecules associated with EMT was determined using RT-PCR, Western blotting, and immunofluorescence staining in control and irradiated cells. We also used real-time RT-PCR to examine the expression of molecules associated with EMT before and after chemoradiotherapy. Thus, we studied 26 rectal cancer patients who received preoperative chemoradiotherapy followed by radical surgery. In addition, we examined the relationship between disease recurrence and the expression of a number of proteins. Irradiation caused CRC cells to undergo phenotypic changes characteristic of EMT: spindle-cell shape, loss of polarity, intercellular separation and pseudopodia formation. Irradiation enhanced cell migration and invasiveness. In irradiated CRC cells, molecular changes consistent with EMT were observed. In clinical samples, we observed molecular changes consistent with EMT, and those changes were significantly enhanced in patients with recurring disease. These results indicate that irradiation induces an alteration to a malignant phenotype consistent with EMT in colorectal cancer cells.

Tripartite Motif-Containing 29 (TRIM29) Is a Novel Marker for Lymph Node Metastasis in Gastric Cancer

BackgroundTripartite motif-containing 29 (TRIM29) belongs to the TRIM protein family, which has unique structural characteristics, including multiple zinc finger motifs and a leucine zipper motif. TRIM29, also known as ataxia telangiectasia group D complementing gene, possesses radiosensitivity suppressor functions. Although TRIM29 has been reported to be underexpressed in prostate and breast cancer, its expression in gastrointestinal cancer has not been studied.MethodsBy use of real-time reverse transcriptase–polymerase chain reaction, we analyzed TRIM29 mRNA expression status with respect to various clinicopathological parameters in 124 patients with gastric cancer. An immunohistochemical study was also conducted.ResultsThe expression of TRIM29 was far higher in gastric cancer tumor tissue. Increased TRIM29 mRNA expression was markedly associated with such parameters as histological grade, large tumor size, extent of tumor invasion, and lymph node metastasis. In the TRIM29 high-expression group, it was an independent predictor for lymph node metastasis. Furthermore, patients with high TRIM29 mRNA expression showed a far poorer survival rate than those with low TRIM29 mRNA expression.ConclusionsTRIM29 expression may serve as a good marker of lymph node metastasis in gastric cancer.

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.

Gene expression profiles of epidermal growth factor receptor, vascular endothelial growth factor and hypoxia-inducible factor-1 with special reference to local responsiveness to neoadjuvant chemoradiotherapy and disease recurrence after rectal cancer surgery.

AIMS To establish a causal relationship between the gene expression profiles of angiogenetic molecular markers, including epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1 (HIF-1), in rectal cancer and the local responsiveness to neoadjuvant chemoradiotherapy and subsequent disease recurrence. MATERIALS AND METHODS We examined the pre-treatment tumour biopsies (n=40) obtained from patients with rectal adenocarcinoma (clinical International Union Against Cancer stage ll/III) who were scheduled to receive neoadjuvant 5-fluorouracil-based chemoradiotherapy for EGFR, VEGF and HIF-1 expression by quantitative real-time polymerase chain reaction. RESULTS Responders (patients with significant tumour regression, i.e. pathological grades 2/3) showed significantly lower VEGF, HIF-1 and EGFR gene expression levels than the non-responders (patients with insignificant tumour regression, i.e. pathological grades 0/1) in the pre-treatment tumour biopsies. The elevated expression level of each gene could predict patients with a low response to chemoradiation. During the median follow-up of all patients (41 months; 95% confidence interval 28-60 months), 6/40 (15%) developed disease recurrence. Although local responsiveness to neoadjuvant chemoradiotherapy was associated with neither local nor systemic disease recurrence, lymph node metastasis and an elevated VEGF gene expression level were independent predictors of systemic disease recurrence. The 3-year disease-free survival rates of the patients with lower VEGF or EGFR expression levels were significantly lower than those of patients with higher VEGF or EGFR expression levels. CONCLUSIONS Analysing VEGF expression levels in rectal cancer may be of benefit in estimating the effects of neoadjuvant chemoradiotherapy and in predicting systemic recurrence after rectal cancer surgery.

Efficient Identification of a Novel Cancer/Testis Antigen for Immunotherapy Using Three-Step Microarray Analysis

Advanced technology in molecular biology has provided us powerful tools for the diagnosis and treatment for cancer. We herein adopted a new methodology to identify a novel cancer/testis (CT) antigen with high frequency of expression in colorectal cancer as follows: (a) combining laser microdissection and cDNA microarray was used to analyze the gene expression profile of colorectal cancer cells; (b) genes overexpressed in testis and underexpressed in normal colon epithelium were analyzed using cDNA microarray; and (c) the gene expression profile of colorectal cancer cells was compared with that of normal testis. Using this methodology, we selected 38 candidates for CT antigen. Among these genes, we identified a novel CT antigen, serine/threonine kinase 31 (STK31), which was previously reported as a gene expressed in spermatogonia. Reverse transcription-PCR analysis showed that STK31 gene expression levels in cancer samples were significantly higher (P < 0.0001) than those in normal samples. The STK31 gene was frequently expressed not only in colorectal cancer but also in gastric and esophageal cancer. Moreover, STK31 peptide was able to elicit specific CTLs and induced CTLs lysed either peptide-loading or endogenously STK31-expressing target cells. These results showed that the new methodology in this study facilitated identification of CT antigens and that STK31 may be a candidate for cancer immunotherapy against gastrointestinal cancer.

Clinical significance of CD133 and hypoxia inducible factor-1α gene expression in rectal cancer after preoperative chemoradiotherapy.

AIMS The mechanism of distant recurrence in rectal cancer after preoperative chemoradiotherapy (CRT) has yet to be fully elucidated. Further improvements in survival rates cannot be achieved without decreasing distant recurrence after preoperative CRT. Recently, it was reported that hypoxic conditions were correlated with cancer stem cell generation. Therefore, we investigated the correlation between the expression of CD133 and hypoxia inducible factor-1α (HIF-1α), and their association with clinical outcome. MATERIALS AND METHODS Fifty-two patients with rectal cancer underwent preoperative CRT. Residual cancer cells after CRT were obtained from formalin-fixed paraffin-embedded specimens using micro-dissection. The expression levels of CD133 (PROM1) and HIF-1α genes were measured using real-time reverse transcription polymerase chain reaction. The correlation between expression and irradiation was evaluated using colon cancer cell lines. Immunohistochemical staining of these proteins after CRT was also investigated. RESULTS We observed a significant inverse correlation between the gene expression of CD133 (PROM1) and HIF-1α genes in residual cancer cells after CRT. Elevated CD133 gene expression was associated with distant recurrence and poor recurrence-free survival. Elevated HIF-1α gene expression was associated with poor overall survival. In vitro, the change in gene expression levels after irradiation showed inverse patterns. Immunohistochemical analyses showed that residual cancer cells strongly expressed CD133 and lacked HIF-1α expression. CONCLUSION Our results suggest that CD133 and HIF-1α expression is associated with tumour re-growth and distant recurrence after CRT. These results may assist in clarifying the development of future cancer therapeutics in rectal cancer patients undergoing preoperative CRT.

DPEP1, expressed in the early stages of colon carcinogenesis, affects cancer cell invasiveness.

BackgroundWe investigated changes in the gene expression profile in colon cancer in order to identify gene markers that may be useful in the management of this disease.MethodsThe Cancer Genome Anatomy Project was used to detect differences in gene expression between normal and cancer tissue. The overexpression of dipeptidase-1 (DPEP1) in cancer tissue was confirmed in a sample of 76 patients by real-time PCR. To identify the function of DPEP1, RNA interference (RNAi) was used to inactivate this gene in the colon cancer cell line. Immunohistochemical analysis was performed to characterize the pattern of DPEP1 expression in colon cancer.ResultsDPEP1 expression in cancer was significantly higher than that in normal tissue. However, DPEP1 expression decreased with pathological differentiation, lymph-node and distant metastasis. Patients with tumors with decreased DPEP1 expression showed a poorer prognosis, and this was also true of patients with tumors who are treated with curative intent. RNAi-mediated DPEP1 reduction in the colon cancer cell line did not result in cell proliferation or apoptosis, but was associated with an increased invasive ability. DPEP1 protein was observed on the apical side of the cancer cells, and is expressed in the early stages of carcinogenesis, even in adenomas of both sporadic colorectal cancer and familial adenomatous polyposis patients.ConclusionsDPEP1 expression in normal colonic mucosa is very low, but it is highly expressed in colorectal adenoma and cancer specimens and is negatively correlated with parameters of pathological aggressiveness and poor prognosis. DPEP1 is expressed in the early stages of colon carcinogenesis and affects cancer cell invasiveness.

Soluble intercellular adhesion molecule-1 as a prognostic marker for stage II colorectal cancer patients.

BackgroundSoluble intercellular adhesion molecule-1 (sICAM-1) represents a circulating form of ICAM-1 that is constitutively expressed or is inducible, which localizes to the cell surfaces of different cell lines and is related to the metastatic potential of cancer cells. The aim of the present study was to determine the relationships between the preoperative serum concentration of sICAM-1 and clinicopathological features, established tumor markers and prognosis, in colorectal cancer patients.MethodsOne hundred and thirty-eight patients with histologically proven colorectal cancer and 40 normal volunteers were included in this trial. Preoperative serum was collected, and sICAM-1 levels were assayed using a commercially available enzyme-linked immunosorbent assay kit.ResultsThe mean sICAM-1 level in patients was significantly higher than that in controls, and increased with disease progression. The prognosis of patients with an elevated sICAM-1 level was significantly worse than that of patients with a normal sICAM-1 level. In a Cox multivariate analysis, the strongest prognostic factor in all patients was distant metastasis followed by sICAM-1 level, while in patients with stage II classification, the strongest prognostic factor was serum level of sICAM-1. The prognosis of stage II patients positive for sICAM-1 was comparable to that of stage III patients.ConclusionsPreoperative sICAM-1 level is an independent prognostic marker for stage II colorectal cancer. Measuring serum sICAM-1 may provide valuable information, especially for stage II patients, when selecting appropriate candidates for adjuvant chemotherapy.