Naoya Yoshida

MicroRNA-21 Regulates the Proliferation and Invasion in Esophageal Squamous Cell Carcinoma

Purpose: MicroRNAs are ∼22 nucleotide noncoding RNA molecules that posttranscriptionally regulate gene expression. The aim of this study was (a) to determine a role of microRNA-21 in esophageal squamous cell carcinoma and (b) to elucidate the regulation of the programmed cell death 4 (PDCD4) gene by microRNA-21. Experimental Design: MicroRNA-21 expression was investigated in 20 matched normal esophageal epitheliums and esophageal squamous cell carcinomas and seven esophageal squamous cell carcinoma cell lines (TE6, TE8, TE10, TE11, TE12, TE14, KYSE30) by TaqMan quantitative real-time PCR and in situ hybridization. To evaluate the role of microRNA-21, cell proliferation and invasion were analyzed with anti–microRNA-21–transfected cells. In addition, the regulation of PDCD4 by microRNA-21 was elucidated to identify the mechanisms of this regulation. Results: Of 20 paired samples, 18 cancer tissues overexpressed microRNA-21 in comparison with matched normal epitheliums. Specifically, patients with lymph node metastasis or venous invasion showed significantly high expression of microRNA-21. In situ hybridization for microRNA-21 showed strong positive staining in paraffin-embedded esophageal squamous cell carcinoma tissues. All seven esophageal squamous cell carcinoma cell lines also overexpressed microRNA-21, and anti–microRNA-21–transfected cells showed significant reduction in cellular proliferation and invasion. The PDCD4 protein levels in esophageal squamous cell carcinoma cells have an inverse correlation with microRNA-21 expression. Anti–microRNA-21–transfected cells increased PDCD4 protein expression without changing the PDCD4 mRNA level and increased a luciferase-reporter activity containing the PDCD4-3′ untranslated region construct. Conclusions: MicroRNA-21 targets PDCD4 at the posttranscriptional level and regulates cell proliferation and invasion in esophageal squamous cell carcinoma. It may serve as a novel therapeutic target in esophageal squamous cell carcinoma.

Negative Impact of Skeletal Muscle Loss after Systemic Chemotherapy in Patients with Unresectable Colorectal Cancer

Background Skeletal muscle depletion (sarcopenia) is closely associated with limited physical ability and high mortality. This study evaluated the prognostic significance of skeletal muscle status before and after chemotherapy in patients with unresectable colorectal cancer (CRC). Methods We conducted a retrospective analysis of 215 consecutive patients with unresectable CRC who underwent systemic chemotherapy. Skeletal muscle cross-sectional area was measured by computed tomography. We evaluated the prognostic value of skeletal muscle mass before chemotherapy and the rate of skeletal muscle change in cross-sectional area after chemotherapy. Results One-hundred-eighty-two patients met our inclusion criteria. There were no significant differences in progression-free survival (PFS) or overall survival (OS) associated with skeletal muscle mass before chemotherapy. However, 22 patients with skeletal muscle loss (>5%) after chemotherapy showed significantly shorter PFS and OS compared with those without skeletal muscle loss (PFS, log-rank p = 0.029; OS, log-rank p = 0.009). Multivariate Cox regression analysis revealed that skeletal muscle loss after chemotherapy (hazard ratio, 2.079; 95% confidence interval, 1.194–3.619; p = 0.010) was independently associated with OS. Conclusions Skeletal muscle loss after chemotherapy was an independent, negative prognostic factor in unresectable CRC.

The microRNA-21/PTEN pathway regulates the sensitivity of HER2-positive gastric cancer cells to trastuzumab.

AbstractBackground The ToGA trial demonstrated the significant efficacy of trastuzumab in addition to chemotherapy in patients with HER2-positive gastric cancer (GC). Although trastuzumab has become a key drug in breast cancer treatment, resistance to trastuzumab is a major problem in clinical practice. The aim of the current study was to identify the micro-RNA (miR)/gene pathway regulating the sensitivity of HER2-positive GC cells to trastuzumab.Methods Correlations between the expression levels of miR-21, PTEN, and p-AKT were analyzed by real-time PCR and Western blot test in HER2-positive GC cell lines. The effects of overexpression or suppression of miR-21 on the sensitivity of GC cells to trastuzumab were also analyzed in vitro.ResultsOverexpression of miR-21 down-regulated PTEN expression, increased AKT phosphorylation, and did not affect HER2 expression. Inversely, suppression of miR-21 increased PTEN expression and down-regulated AKT phosphorylation, but still did not affect HER2 expression. Overexpression of miR-21 decreased the sensitivity of GC cells to trastuzumab, while suppression of miR-21 expression restored the resistance of GC cells to trastuzumab. Overexpression of miR-21 significantly suppressed trastuzumab-induced apoptosis.ConclusionsTo our knowledge, this study was the first reveal the miR-21/PTEN pathway regulated the sensitivity of HER2-positive GC cell lines to trastuzumab through modulation apoptosis. These findings suggest that this pathway may be crucial to the mechanism of resistance to trastuzumab in GC, which may lead to the development of individualized treatment in clinical practice.

Prognostic Impact of Postoperative Complications in 502 Patients With Surgically Resected Esophageal Squamous Cell Carcinoma: A Retrospective Single Institution Study

Objective: To investigate the relationship between postoperative complications and long-term survival in patients with surgically resected esophageal squamous cell carcinoma (ESCC). Summary Background Data: Esophagectomy is the mainstay of curative treatment for ESCC; however, this complex procedure has high risks of postoperative morbidity and mortality. The impact of postoperative complications on long-term survival of such patients remains controversial. Methods: This retrospective single institution study included 502 consecutive patients who had undergone resection of ESCC. The Cox proportional hazard model was used to compute the hazard ratio (HR) for mortality. Results: Postoperative complications (≥Clavien-Dindo classification grade 2) occurred in 217 patients (43%). Overall, postoperative complications did not affect long-term clinical outcomes of these patients. However, patients with pulmonary complications had worse overall survival than those without pulmonary complications [log rank P = 0.0002; univariate HR = 1.51, 95% confidence interval (CI) 1.20–1.88, P = 0.0006; multivariate HR = 1.60, 95% CI 1.05–2.38, P = 0.029]. The effect of pulmonary complications was not significantly modified by clinical or pathological features (P for all assessed interactions >0.05). In addition, postoperative chylothorax was also associated with poor overall survival (log rank P = 0.0021), whereas surgical site infection, recurrent nerve paralysis, cardiovascular complication, and anastomotic leakage were not. Conclusions: Postoperative pulmonary complications may be an independent predictor of poorer long-term survival in patients undergoing resection of ESCCs.

Prognostic Nutritional Index Predicts Severe Complications, Recurrence, and Poor Prognosis in Patients With Colorectal Cancer Undergoing Primary Tumor Resection.

BACKGROUND: The prognostic nutritional index is reportedly related to postoperative outcomes. OBJECTIVE: The aim of this study was to elucidate the clinical importance of the prognostic nutritional index in patients with colorectal cancer who were undergoing primary tumor resection. DESIGN: This is a retrospective study from a single institution. SETTINGS: This study was conducted at a colorectal surgery service in an academic teaching hospital. PATIENTS: The 556 patients with colorectal cancer who were undergoing surgery between March 2005 and August 2014 were eligible for this study. MAIN OUTCOME MEASURES: The preoperative prognostic nutritional index was calculated. Classification and regression tree analysis was performed to determine the prognostic nutritional index cutoff value. The associations of the prognostic nutritional index status with clinicopathological factors and postoperative outcomes were examined using univariate and multivariate analyses. RESULTS: Classification and regression tree analysis demonstrated that 45.5 was the optimal cutoff value. The low status (⩽45.5) was correlated with older age, low BMI, low estimated glomerular filtration rate, CEA positivity, carbohydrate antigen 19-9 positivity, preoperative chemotherapy, tumors invading muscular or deeper layers, distant metastasis, poor differentiation, severe postoperative complications, tumor recurrence, and poor survival. In multivariate analysis, the low status was an independent risk factor for severe postoperative complications (OR = 2.06 [95% CI, 1.22–3.50]; p = 0.007) and low overall survival (HR =3.98 [95% CI, 2.38–6.89]; p < 0.001). LIMITATIONS: Our data set was collected retrospectively from a single institution. In addition, our study was only for preoperative prognostic nutritional index status, not considering the postoperative host status. CONCLUSIONS: The preoperative prognostic nutritional index predicts severe complications, recurrence, and poor prognosis in patients with colorectal cancer who are undergoing primary tumor resection. Investigation of the nutritional and immunologic statuses using the prognostic nutritional index could be a useful clinical approach.

The sensitivity of gastric cancer to trastuzumab is regulated by the miR-223/FBXW7 pathway

A recent large‐scale phase III study (the ToGA trial) demonstrated the significant efficacy of trastuzumab combined with chemotherapy in patients with HER2‐positive gastric cancer. Although trastuzumab has become a key drug in cancer treatment, the resistance of breast cancer to trastuzumab is a major problem in clinical practice. However, it is unclear whether similar mechanisms of trastuzumab resistance are involved in gastric cancer (GC). The aim of the current study was to identify a novel micro‐RNA (miR)/gene pathway that regulates the sensitivity of HER2‐positive GC cells to trastuzumab. We focused on F‐box and WD repeat domain‐containing 7 (FBXW7), which is one of the major causes of drug resistance. We also identified miR‐223, which can regulate FBXW7, using miR quantitative reverse transcription‐PCR array analysis using by resistance cell line, which we established. Overexpression of miR‐223 decreased FBXW7 expression and the sensitivity of GC cells to trastuzumab, while suppression of miR‐223 restored FBXW7 expression and the sensitivity of GC cells to trastuzumab. Moreover, overexpression of miR‐223 significantly suppressed trastuzumab‐induced apoptosis. This study is the first report to reveal that the miR‐223/FBXW7 pathway regulates the sensitivity of a HER2‐positive GC cell line to trastuzumab through the modulation of apoptosis. These findings suggest that this pathway can be crucial for the mechanism of trastuzumab resistance in GC, which may lead to the development of individualized treatment in clinical practice.

Methylation levels of LINE-1 in primary lesion and matched metastatic lesions of colorectal cancer

Background:LINE-1 methylation level is a surrogate marker of global DNA methylation. LINE-1 methylation in primary colorectal cancers (CRCs) is highly variable and strongly associated with a poor prognosis. However, no study has examined LINE-1 methylation levels of metastatic CRCs in relation to prognosis or assessed the heterogeneity of LINE-1 methylation level within the primary CRCs.Methods:Pyrosequencing was used to quantify LINE-1 methylation level in 42 liver metastases, 26 matched primary tumours, and 6 matched lymph node (LN) metastases. KRAS, BRAF, and PIK3CA mutation status and microsatellite instability (MSI) status were also examined.Results:The distribution of LINE-1 methylation level in liver metastases was as follows: mean, 67.3; range, 37.1–90.1. Primary tumours showed LINE-1 methylation levels similar to those of matched liver and LN metastases. The difference in LINE-1 methylation level between superficial areas and invasive front areas was within 7.0 in all six cases evaluated. Prognostic impact of LINE-1 hypomethylation in liver metastases on overall survival was not observed. The concordance rate was 94% for KRAS, 100% for BRAF, 88% for PIK3CA, and 97% for MSI.Conclusion:Alteration of LINE-1 methylation level may occur in early CRC tumorigenesis, and the LINE-1 methylation level is relatively stable during CRC progression.

CXCL12/CXCR4 activation by cancer‐associated fibroblasts promotes integrin β1 clustering and invasiveness in gastric cancer

Cancer‐associated fibroblasts (CAFs) are reportedly involved in invasion and metastasis in several types of cancer, including gastric cancer (GC), through the stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying these tumor‐promoting effects are not well understood, which limits the potential to develop therapeutic targets against CAF‐mediated CXCL12/CXCR4 signaling. CXCL12 expression was analyzed in resected GC tissues from 110 patients by immunohistochemistry (IHC). We established primary cultures of normal fibroblasts (NFs) and CAFs from the GC tissues and examined the functional differences between these primary fibroblasts using co‐culture assays with GC cell lines. We evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF‐573,228) on the invasive ability of GC cells. High CXCL12 expression levels were significantly associated with larger tumor size, increased tumor depth, lymphatic invasion and poor prognosis in GC. CXCL12/CXCR4 activation by CAFs mediated integrin β1 clustering at the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient than PF‐573,228 at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling. These results suggest that CXCL12 derived from CAFs promotes GC cell invasion by enhancing the clustering of integrin β1 in GC cells, resulting in GC progression. Taken together, the inhibition of CXCL12/CXCR4 signaling in GC cells may be a promising therapeutic strategy against GC cell invasion.

Neoadjuvant treatment for esophageal squamous cell carcinoma.

Squamous cell carcinoma and adenocarcinoma are types of esophageal cancer, one of the most aggressive malignant diseases. Since both histological types present entirely different diseases with different epidemiology, pathogenesis and tumor biology, separate therapeutic strategies should be developed against each type. While surgical resection remains the dominant therapeutic intervention for patients with operable esophageal squamous cell carcinoma (ESCC), alternative strategies are actively sought to reduce the frequency of post-operative local or distant disease recurrence. Such strategies are particularly sought in the preoperative setting. Currently, the optimal management of resectable ESCC differs widely between Western and Asian countries (such as Japan). While Western countries focus on neoadjuvant or definitive chemoradiotherapy, neoadjuvant chemotherapy followed by surgery is the standard treatment in Japan. Importantly, each country and region has established its own therapeutic strategy from the results of local randomized control trials. This review discusses the current knowledge, available data and information regarding neoadjuvant treatment for operable ESCC.

Prognostic and clinical impact of sarcopenia in esophageal squamous cell carcinoma

Recently, depletion of skeletal muscle mass (sarcopenia) has been linked to poor prognosis in several types of cancers, but has not been investigated in esophageal squamous cell carcinoma (ESCC). This retrospective study investigates the relationship between sarcopenia and clinical outcome in ESCC patients treated by surgical resection or definitive chemoradiation therapy (dCRT). The study was retrospectively conducted in a single academic hospital in Kumamoto, Japan, and involved 325 ESCC patients (256 surgical cases and 69 dCRT cases) treated between April 2005 and April 2011. Skeletal muscle mass was quantified by radiologic measures using standard computed tomography scans. The skeletal muscle tissue in the 325 ESCC patients was distributed as follows: mean: 47.10; median: 46.88; standard deviation (SD): 7.39; range: 31.48-71.11; interquartile range, 46.29-47.90. Skeletal muscle tissue was greater in male patients than in female patients (P < 0.0001), but was independent of other clinical and tumor features. Sarcopenia was not significantly associated with overall survival (log rank P = 0.54). Lymph node involvement significantly altered the relationship between sarcopenia and survival rate (P for interaction = 0.026). Sarcopenia significantly reduced the overall survival of patients without lymph node involvement (log rank P = 0.035), but was uncorrelated with overall survival in patients with lymph involvement (log rank, P = 0.31). The anastomosis leakage rate was significantly higher in the sarcopenia group than in the non-sarcopenia group (P = 0.032), but other surgical complications did not significantly differ between the two groups. Sarcopenia in ESCC patients without lymph node involvement is associated with poor prognosis, indicating sarcopenia as a potential biomarker for identifying patients likely to experience an inferior outcome. Moreover, sarcopenia was associated with anastomosis leakage but no other short-term surgical outcome.