Involvement of dual‐strand of the miR‐144 duplex and their targets in the pathogenesis of lung squamous cell carcinoma

The prognosis of patients with advanced‐stage lung squamous cell carcinoma (LUSQ) is poor, and effective treatment protocols are limited. Our continuous analyses of antitumor microRNAs (miRNAs) and their oncogenic targets have revealed novel oncogenic pathways in LUSQ. Analyses of our original miRNA expression signatures indicated that both strands of miR‐144 (miR‐144‐5p, the passenger strand; miR‐144‐3p, the guide strand) showed decreased expression in cancer tissues. Additionally, low expression of miR‐144‐5p significantly predicted a poor prognosis in patients with LUSQ by The Cancer Genome Atlas database analyses (overall survival, P = 0.026; disease‐free survival, P = 0.023). Functional assays revealed that ectopic expression of miR‐144‐5p and miR‐144‐3p significantly blocked the malignant abilities of LUSQ cells, eg, cancer cell proliferation, migration, and invasion. In LUSQ cells, 13 and 15 genes were identified as possible oncogenic targets that might be regulated by miR‐144‐5p and miR‐144‐3p, respectively. Among these targets, we identified 3 genes (SLC44A5, MARCKS, and NCS1) that might be regulated by both strands of miR‐144. Interestingly, high expression of NCS1 predicted a significantly poorer prognosis in patients with LUSQ (overall survival, P = 0.013; disease‐free survival, P = 0.048). By multivariate analysis, NCS1 expression was found to be an independent prognostic factor for patients with LUSQ patients. Overexpression of NCS1 was detected in LUSQ clinical specimens, and its aberrant expression enhanced malignant transformation of LUSQ cells. Our approach, involving identification of antitumor miRNAs and their targets, will contribute to improving our understanding of the molecular pathogenesis of LUSQ.     

Immune infiltration in renal cell carcinoma

Immune infiltration of tumors is closely associated with clinical outcome in renal cell carcinoma (RCC). Tumor‐infiltrating immune cells (TIICs) regulate cancer progression and are appealing therapeutic targets. The purpose of this study was to determine the composition of TIICs in RCC and further reveal the independent prognostic values of TIICs. CIBERSORT, an established algorithm, was applied to estimate the proportions of 22 immune cell types based on gene expression profiles of 891 tumors. Cox regression was used to evaluate the association of TIICs and immune checkpoint modulators with overall survival (OS). We found that CD8+ T cells were associated with prolonged OS (hazard ratio [HR] = 0.09, 95% confidence interval [CI].01‐.53; P = 0.03) in chromophobe carcinoma (KICH). A higher proportion of regulatory T cells was associated with a worse outcome (HR = 1.59, 95% CI 1.23‐.06; P < 0.01) in renal clear cell carcinoma (KIRC). In renal papillary cell carcinoma (KIRP), M1 macrophages were associated with a favorable outcome (HR = .43, 95% CI .25‐.72; P < 0.01), while M2 macrophages indicated a worse outcome (HR = 2.55, 95% CI 1.45‐4.47; P < 0.01). Moreover, the immunomodulator molecules CTLA4 and LAG3 were associated with a poor prognosis in KIRC, and IDO1 and PD‐L2 were associated with a poor prognosis in KIRP. This study indicates TIICs are important determinants of prognosis in RCC meanwhile reveals potential targets and biomarkers for immunotherapy development.     

Oxysterol binding protein‐related protein‐5 is related to invasion and poor prognosis in pancreatic cancer

In previous studies, the gene expression profiles of two hamster pancreatic cancer cells with different potentials for invasion and metastasis were analyzed. In the present study, we identified that one of the genes expressed strongly in the highly metastatic cell line is hamster oxysterol binding protein‐related protein (ORP)‐5. The aim of the present study was to clarify the relationship between ORP5 and invasion and poor prognosis of human pancreatic cancer. Invasion assays were carried out in both hamster and human pancreatic cancer cells by suppressing the ORP5 gene with short interfering RNA or inducing its expression by introducing an expression vector. To evaluate the relationship between ORP5 and the characteristics of human pancreatic cancer, 56 pancreatic cancer tissue specimens were analyzed and the ORP5 expression in each pancreatic cancer tissue specimen was analyzed by immunohistochemistry. In both the hamster and human pancreatic cancer cells, suppression of ORP5 significantly reduced the invasion rate of the cells and induction of ORP5 significantly enhanced the invasion rate of the cells. In the clinical sample, the median survival times of the patients with ORP5‐positive (n = 33) and ORP5‐negative (n = 23) cancer were 8.3 and 17.2 months, respectively (P = 0.02). Also, the 1‐year survival rates of patients with ORP5‐positive and ORP5‐negative cancer were 36.4 and 73.9%, respectively (P = 0.005). The ORP5 expression level was related to both invasion and poor prognosis in human pancreatic cancer. These findings suggest that the expression of ORP5 may induce cancer cell invasion, resulting in the poor prognosis of pancreatic cancer. (Cancer Sci 2008; 99: 2387–2394)     

High expression of KIBRA in low atypical protein kinase C‐expressing gastric cancer correlates with lymphatic invasion and poor prognosis

Overexpression of atypical protein kinase Cλ/ι (aPKCλ/ι), a regulator of cell polarity, is frequently associated with the poor prognoses of several cancers, including gastric cancer. Recent studies revealed a molecular link between aPKC and KIBRA, an upstream regulator of tumor suppressor Hippo pathway that regulates cell proliferation and apoptosis. Further, KIBRA directly inhibits the kinase activity of aPKC to regulate epithelial cell polarity. These observations suggest that the KIBRA‐aPKC connection plays a role in cancer progression; however, clinical significance of the correlation between these factors remains unclear. Here we examined the correlation between KIBRA/aPKCλ/ι expression, as detected by immunohistochemistry, and clinicopathological outcomes in 164 gastric cancer patients using Fisher's exact test and Kaplan–Meier log‐rank test. We found an intimate correlation between the expression level of KIBRA and aPKCλ/ι (P = 0.012). Furthermore, high expression of KIBRA is correlated with lymphatic (P = 0.046) and venous invasion (P = 0.039). The expression level of KIBRA by itself did not correlate with the prognosis; however, high expression of KIBRA in low aPKCλ/ι‐expressing gastric cancer correlated with disease‐specific (P = 0.037) and relapse‐free survival (P = 0.041) by Kaplan–Meier with log‐rank test and higher lymphatic invasion cases by Fisher's exact test (P = 0.042). Furthermore, overexpression of the aPKC‐binding region of KIBRA disrupted tight junctions in epithelial cells. These results suggest that high expression of KIBRA in low aPKC‐expressing cells causes massive loss of aPKC activity, leading to loss of polarity and invasiveness of gastric cancer cells.     

Expression of L‐type amino acid transporter 1 (LAT1) as a prognostic and therapeutic indicator in multiple myeloma

L‐type amino‐acid transporter 1 (LAT1) plays a key role in cell growth and survival. To determine the prognostic significance of LAT1 in multiple myeloma (MM), we investigated the expression of LAT1 and its functional subunit, 4Fc heavy chain (CD98), on myeloma cells by immunohistochemistry in 100 newly diagnosed MM patients. High expression (moderate or strong staining intensity) of LAT1 and CD98 was detected in 56% and 45% of patients, respectively. The LAT1 expression score was positively correlated with Ki‐67 index (r = 0.631, P < 0.001), and there was a statistically significant difference in Durie–Salmon stage between patients with high and low LAT1 expression (P = 0.03). In 43 patients treated with melphalan and prednisolone, the overall response rate was significantly higher in the high LAT1 expression group (60.0%) than in the low LAT1 expression group (17.6%) (P = 0.03). Multivariate analysis confirmed that high expression of LAT1 was a significant prognostic factor for predicting poor overall survival independently from the International Staging System (both P = 0.01). Here, we show that the overexpression of LAT1 is significantly associated with high proliferation and poor prognosis in newly diagnosed MM patients. Thus, LAT1 may be a promising pathological marker for identifying high‐risk MM.     

MET4 expression predicts poor prognosis of gastric cancers with Helicobacter pylori infection

The role of HGF/SF‐MET signaling is important in cancer progression, but its relation with Helicobacter pylori‐positive gastric cancers remains to be elucidated. In total, 201 patients with primary gastric carcinoma who underwent curative or debulking resection without preoperative chemotherapy were studied. MET4 and anti‐HGF/SF mAbs were used for immunohistochemical analysis. Survival of gastric cancer patients was estimated by Kaplan–Meier method and compared with log‐rank. Cox proportional hazards models were fit to determine the independent association of MET‐staining status with outcome. The effect of live H. pylori bacteria on cell signaling and biological behaviors was evaluated using gastric cancer cell lines. MET4‐positive gastric cancers showed poorer prognosis than MET4‐negative cases (overall survival, P = 0.02; relapse‐free survival, P = 0.06). Positive staining for MET4 was also a statistically significant factor to predict poor prognosis in H. pylori‐positive cases (overall survival, P < 0.01; relapse‐free survival, P = 0.01) but not in H. pylori‐negative cases. Gastric cancers positively stained with both HGF/SF and MET4 showed a tendency of the worst prognosis. Stimulation of MET‐positive gastric cancer cells with live H. pylori bacteria directly upregulated MET phosphorylation and activated MET downstream signals such as p44/42MAPK and Akt, conferring cell proliferation and anti‐apoptotic activity. In conclusion, positive staining for MET4 was useful for predicting poor prognosis of gastric cancers with H. pylori infection. Helicobacter pylori stimulated MET‐positive gastric cancers and activated downstream signaling, thereby promoting cancer proliferation and anti‐apoptotic activity. These results support the importance of H. pylori elimination from gastric epithelial surface in clinical therapy.     

KIAA0247 inhibits growth,migration, invasion of non‐small‐cell lung cancer through regulating the Notch pathway

Lung cancer remains the leading cause of cancer‐related death worldwide. Previous studies have shown that the novel KIAA0247 gene potentially targeted by the tumor suppressor p53 may inhibit the development of several cancers. However, the exact function of KIAA0247 in non‐small‐cell lung cancer (NSCLC) is unknown. The purpose of the present study was to clarify the role of KIAA0247 in NSCLC. KIAA0247 expression was evaluated in tumors and adjacent normal tissues of 197 NSCLC patients by immunohistochemistry and real‐time PCR and analyzed for association with clinicopathological parameters. Results indicated that KIAA0247 levels positively correlated with cell differentiation (P < .001) and patient survival (P < .0001) and negatively correlated with lymph node metastasis (P < .001) and advanced p‐TNM stage (P < .001). In cultured NSCLC cell lines, KIAA0247 overexpression inhibited cell migration, invasion, and proliferation and downregulated the expression of Jagged1, Notch1 intracellular domain (NICD), Snail, cyclin D1, RhoA, RhoC, and MMP9, while upregulating that of E‐cadherin and p21. The Notch inhibitor DAPT reduced the biological effects of KIAA0247 knockdown, suggesting that KIAA0247 decreased the carcinogenic activity of NSCLC cells through downregulation of Notch signaling. Our results indicate that KIAA0247 inhibits NSCLC progression by reducing the metastatic potential of cancer cells through downregulation of the Notch pathway, which may underlie the association of KIAA0247 expression with favorable clinicopathological characteristics of NSCLC patients. These findings suggest that KIAA0247 is a candidate prognostic biomarker and potential therapeutic target in NSCLC.     

Upregulation of transgelin is an independent factor predictive of poor prognosis in patients with advanced pancreatic cancer

Transgelin is a known actin‐binding protein, which plays a role in regulating the functions of smooth muscle cells or fibroblasts. Recent evidence indicates that transgelin is involved in diverse human cancers, yet its role in pancreatic cancer remains unclear. We therefore evaluated the expression characteristics and function of transgelin in pancreatic cancer. Immunohistochemical analysis of benign (n = 30 patients) and malignant (n = 114 patients) pancreatic ductal cells showed significantly higher transgelin staining in malignant cells. Lymph node metastasis (P = 0.026) and diabetes (P = 0.041) were shown to significantly correlate with transgelin protein expression. Patients with high transgelin expression showed a shorter 5‐year overall survival and a lower tumor‐specific survival than those with low transgelin expression. Multivariate analysis revealed that transgelin was an independent factor affecting pancreatic tumor‐specific survival (P = 0.025). In vitro, RNA interference‐mediated transgelin knockdown resulted in inhibition of pancreatic cancer cell proliferation, migration and invasion. Depletion of transgelin expression could suppress pancreatic tumorigenicity and tumor growth in vivo, and produce enhanced cytotoxic effects of gemcitabine on pancreatic cancer cells both in vitro and in vivo. Our results indicate that transgelin plays a promoting role in tumor progression, and appears to be a novel prognostic marker for advanced pancreatic cancer.     

Potential role of Jun activation domain-binding protein 1 and phosphorylated p27 expression in prognosis of glioma

To investigate whether Jun activation domain-binding protein 1 (Jab1) expression is correlated with p27 protein and its phosphorylation status as well as how it might be clinically relevant in glioma, we carried out an immunohistochemical study of Jab1, Ser10-phosphorylated p27 (pSer10p27), and p27 using biopsies from 192 patients with primary glioma. Kaplan–Meier survival and Cox regression analyses were performed to evaluate the prognosis of patients. Immunostaining revealed a positive correlation between Jab1 and cytoplasmic p27 as well as pSer10p27 in all glioma cases. In addition, patients displaying the overexpression of Jab1, cytoplasmic p27, and pSer10p27 were significantly associated with unfavorable clinicopathological variables. Statistical analysis showed that patients expressing Jab1, cytoplasmic p27, and pSer10p27 have poor overall survival rates relative to those not expressing these proteins. Cox multifactor analysis showed that Jab1 (P = 0.006), cytoplasmic p27 (P = 0.01), and pSer10p27 (P = 0.009) were independent prognosis factors for human glioma. In conclusion, the current results showed convincing evidence that the overexpression of Jab1, cytoplasmic p27, and pSer10p27 proteins is correlated with poor outcome in patients with glioma and that these three proteins may be useful markers to predict the prognosis of this tumor.     

Human endoplasmic reticulum oxidoreductin 1‐α is a novel predictor for poor prognosis of breast cancer

Human endoplasmic reticulum oxidoreductin 1‐α (hERO1‐α) is an oxidizing enzyme that exists in the endoplasmic reticulum and its expression is augmented under hypoxia. It regulates a redox state of various kinds of protein through reoxidation of “client” protein disulfide isomerase. Interestingly, although the expression of hERO1‐α in normal tissues was comparatively limited, various types of cancer cells expressed it in large amounts. Therefore, we examined the role of ERO1‐α in tumor growth using murine breast cancer line 4T1 and found that knockdown of murine ERO1‐α inhibited in vivo tumor growth and decreased lung metastasis compared with wild‐type 4T1. Moreover, we investigated the relationship between expression of hERO1‐α and prognosis in breast cancer patients. Seventy‐one patients with breast cancer who underwent surgery between 2005 and 2006 in Sapporo Medical University Hospital (Sapporo, Japan) were analyzed in this study. Significant differences were found between the hERO1‐α‐positive group (n = 33) and hERO1‐α‐negative group (n = 38) in nuclear grade (P < 0.001) and intrinsic subtype (P = 0.021) in univariate analysis. More importantly, in multivariate analysis of disease‐free survival by Cox regression, expression of hERO1‐α was the only independent prognosis factor (P = 0.035). Finally, in univariate survival analysis, patients positive for hERO1‐α had significantly shorter disease‐free survival and overall survival than those patients negative for hERO1‐α. These findings indicate that the expression of hERO1‐α in cancer cells is associated with poorer prognosis and thus can be a prognostic factor for patients with breast cancer.     

Combined evaluation of hexokinase 2 and phosphorylated pyruvate dehydrogenase‐E1α in invasive front lesions of colorectal tumors predicts cancer metabolism and patient prognosis

Although numerous studies have shown the significance of cancer‐specific aerobic glycolysis, how glycolysis contributes to tumor invasion, a critical phenomenon in metastasis, remains unclear. With regard to colorectal cancer (CRC), we studied two critical gate enzymes, hexokinase 2 (HK2), which is involved in glycolysis, and phosphorylated pyruvate dehydrogenase‐E1α (p‐PDH), which is involved in oxidative phosphorylation (OxPhos). Immunohistochemical analyses using anti‐HK2 and p‐PDH antibodies were performed on surgically resected CRC samples (n = 104), and the expression in invasive front lesions of tumors was assessed. Positive HK2 expression correlated with extensive tumor diameter (P = 0.0460), advanced tumor depth (P = 0.0395), and presence of lymph node metastasis (P = 0.0409). Expression of p‐PDH tended to be higher in right‐sided CRCs than in left‐sided CRCs (P = 0.0883). In survival analysis, the combined evaluation of positive HK2 and negative p‐PDH was associated with reduced recurrence‐free survival (RFS) (P = 0.0169 in all stages and P = 0.0238 in Stage II and III patients, respectively). This evaluation could predict RFS more precisely than the independent evaluation. The present study indicated that high HK2 expression combined with low p‐PDH expression in the invasive front lesions of CRC tumors is predictive of tumor aggressiveness and survival of CRC cases.     

Epidermal growth factor receptor mRNA expression: A potential molecular escape mechanism from regorafenib

Regorafenib has improved the survival of patients with refractory metastatic colorectal cancer (mCRC), yet the mechanisms of inherited or acquired resistance are not well understood. A total of 50 patients with refractory mCRC were enrolled. Circulating tumor cell (CTC) enumeration was carried out at baseline, day 21 after initiation of regorafenib, and at the time of progression of disease (PD) using the CellSearch System (Veridex LLC, NJ, USA). Poly(A) mRNA was extracted from CTCs, and gene expression of epithelial and epithelial‐mesenchymal transition markers was analyzed by a multiplex‐PCR based DNA Chip. Patients with fewer than 3 CTCs at baseline and day 21 had a longer progression‐free survival than those with 3 or more CTCs (3.3 vs 2.0 months, P = .008 and 3.3 vs 2.0 months, P = .004, respectively). Patients with fewer than 3 CTCs at baseline and day 21 had a longer overall survival (OS) than those with 3 or more CTCs (10.0 vs 4.6 months, P < .001 and 8.7 vs 3.8 months, P = .003, respectively). In multivariable analysis, CTC counts remained significantly associated with OS at baseline and day 21 (P = .019 and P = .028). Circulating tumor cell EGFR gene expression was upregulated at day 21 and/or PD in 64% of patients. Patients had significantly increased EGFR expression at PD compared to baseline (P = .041) and at day 21 and/or PD compared to baseline (P = .004). Our findings suggest that CTC count and EGFR expression could be useful markers of regorafenib efficacy and outcomes. Upregulation of CTC EGFR expression might be a molecular escape mechanism under regorafenib therapy.     

Prognostic value of programmed death‐ligand 1 expression in patients with stage III colorectal cancer

The programmed death‐1/programmed death‐ligand 1 (PD‐L1) pathway is a negative feedback pathway that suppresses the activity of T cells. Previous studies reported that high PD‐L1 expression on tumor cells (TC) was associated with poor survival in patients with colorectal cancer; however, the prognostic evaluation of these studies was limited because they included patients at various disease stages. The purpose of the present study was to evaluate the relationship between PD‐L1 status in the immune microenvironment and the clinicopathological features of stage III colorectal cancer. Two hundred and thirty‐five patients were included in the analysis. PD‐L1 expression on TC and tumor‐infiltrating mononuclear cells (TIMC) was evaluated by immunohistochemistry. The median follow‐up of thisi study was 52.9 months. A total of 8.1% of stage III colorectal cancer showed high PD‐L1 expression on TC and 15.3% showed high PD‐L1 expression on TIMC. Patients with high PD‐L1 expression on TC had significantly shorter disease‐free survival (DFS) than patients with low expression (hazard ratio [HR] 2.36; 95% confidence interval [CI], 1.21–4.62; P = 0.012). In addition, patients with high PD‐L1 expression on TIMC were associated with longer DFS than patients with low expression (HR 0.40; 95% CI, 0.16–0.98; P = 0.046). These findings suggest that PD‐L1 expression status may be a new predictor of recurrence for stage III colorectal cancer patients and highlight the necessity of evaluating PD‐L1 expression on TC and TIMC separately in the tumor microenvironment.     

Long non‐coding RNA HOTAIR is an independent prognostic marker for nasopharyngeal carcinoma progression and survival

Identification of new nasopharyngeal carcinoma (NPC) biomarkers is of great clinical value for the diagnosis and treatment of NPC. HOTAIR, a cancer‐related long non‐coding RNA, was tested and its prognostic value for NPC was evaluated. As determined using in situ hybridization (ISH), 91 of 160 (56.87%) paraffin‐embedded NPC biopsies showed high expression levels of HOTAIR (staining index score ≥ 6). HOTAIR was upregulated in tumors with a large size (P = 0.021), more advanced clinical stage (P = 0.012) and increased lymph node tumor burden (P = 0.005). Quantified using real‐time PCR, HOTAIR expression levels in fresh tissue and paraffin‐embedded samples were 5.2 ~ 48.4‐fold higher compared with non‐cancer tissue samples. Moreover, HOTAIR expression levels increased with clinical stage progression, which was consistent with ISH findings in the paraffin‐embedded tissue. Most importantly, NPC patients with higher HOTAIR levels had a poor prognosis for overall survival using univariate and multivariate analysis. In addition, HOTAIR mediated the migration, invasion and proliferation of NPC cells in vitro. HOTAIR is a potential biomarker for the prognosis of NPC, and dysregulation of HOTAIR might play an important role in NPC progression.     

Tumor necrosis factor receptor modulator spermatogenesis‐associated protein 2 is a novel predictor of outcome in ovarian cancer

Inflammation plays a crucial role in the pathogenesis of cancer with tumor necrosis factor‐α (TNF‐α) as a key mediator. Recently, spermatogenesis‐associated protein 2 (SPATA2) was identified as a TNF receptor modulator which is required for TNF‐induced inflammation and apoptosis. The available data on TNF‐α in ovarian cancer (OC) are inconsistent, and SPATA2 is completely uncharacterized in tumorigenesis. We analyzed expression of SPATA2 and TNFA by quantitative real‐time polymerase chain reaction in tissues of 171 patients with low‐grade serous (LGSOC), high‐grade serous (HGSOC), endometrioid and clear cell OC compared with 28 non‐malignant control tissues. We stimulated OC cells (OVCAR3) with pro‐inflammatory (TNF‐α, interleukin [IL]‐1β) and mitogenic stimuli (IL‐6, lysophosphatidic acid) to establish a direct effect between inflammatory signaling and SPATA2. Pro‐inflammatory, but not mitogenic stimuli, potently induced SPATA2 expression in OC cells. Expression of TNFA and SPATA2 was higher in OC compared with control tissues (P = 0.010 and P = 0.001, respectively) and correlated with each other (P = 0.034, r_s = 0.198). When compared with grade 1 cancers, SPATA2 was expressed higher in grade 2 and 3 tumors (P = 0.011) as well as in HGSOC compared with LGSOC (P = 0.024). Multivariate survival analyses revealed that OC with high SPATA2 expression were associated with reduced progression‐free survival (P = 0.048) and overall survival (P < 0.001). In conclusion, SPATA2 expression is regulated by TNF‐α and IL‐1β and is found to independently affect clinical outcome in OC patients. These data implicate a role of SPATA2 in tumorigenesis which warrants further investigation in gynecological malignancies.     

High levels of CD20 expression predict good prognosis in chronic lymphocytic leukemia

Heterogeneity of CD20 expression exists in chronic lymphocytic leukemia (CLL), therefore, we explored the prognostic significance of CD20 expression in Chinese patients with CLL. Multiparameter flow cytometry was used to detect the expression of CD20 in CD5⁺CD19⁺ cells. In 172 CLL patients, the median expression percent of CD20 was 97.82% (range, 0–100), and the median mean fluorescence intensity (MFI) of CD20 in CLL cells was 731.45 (range, 0.00–9071.90). The percentage of CD20⁺ cells in the patient group with mutated variable region of immunoglobulin genes (IGHV) was higher than in the non‐mutant IGHV group (mean, 92.1% vs 80.4%, P < 0.001). There were no differences in the MFI of CD20⁺ cells in all prognostic factor groups. Representation of the data using a receiver operating characteristic plot reflected separation between the two IGHV groups, with an area under the curve of 0.661 (95% confidence interval, 0.569–0.753). At the cut‐off value of 60.3% for percentage of CD20, the sensitivity and specificity were 90.00% and 38.46%, respectively. Patients whose percentage of CD20 antigen was above 60.3% had longer treatment‐free survival (hazard ratio, 0.452; 95% confidence interval, 0.232–0.884, P = 0.020). Percentage and MFI of CD20 were the variables not associated with treatment‐free survival by multivariate Cox regression analysis (P < 0.05). High level of CD20 expression in de novo CLL appears to be associated with a good prognosis.