The orphan nuclear receptor SHP acts as a negative regulator in inflammatory signaling triggered by Toll-like receptors.

The orphan nuclear receptor SHP (small heterodimer partner) is a transcriptional corepressor that regulates hepatic metabolic pathways. Here we identified a role for SHP as an intrinsic negative regulator of Toll-like receptor (TLR)-triggered inflammatory responses. SHP-deficient mice were more susceptible to endotoxin-induced sepsis. SHP had dual regulatory functions in a canonical transcription factor NF-κB signaling pathway, acting as both a repressor of transactivation of the NF-κB subunit p65 and an inhibitor of polyubiquitination of the adaptor TRAF6. SHP-mediated inhibition of signaling via the TLR was mimicked by macrophage-stimulating protein (MSP), a strong inducer of SHP expression, via an AMP-activated protein kinase-dependent signaling pathway. Our data identify a previously unrecognized role for SHP in the regulation of TLR signaling.

Dominant role of peroxiredoxin/JNK axis in stemness regulation during neurogenesis from embryonic stem cells.

Redox balance has been suggested as an important determinant of "stemness" in embryonic stem cells (ESCs). In this study, we demonstrate that peroxiredoxin (Prx) plays a pivotal role in maintenance of ESC stemness during neurogenesis through suppression of reactive oxygen species (ROS)-sensitive signaling. During neurogenesis, Prx I and Oct4 are expressed in a mutually dependent manner and their expression is abruptly downregulated by an excess of ROS. Thus, in Prx I(-/-) or Prx II(-/-) ESCs, rapid loss of stemness can occur due to spontaneous ROS overload, leading to their active commitment into neurons; however, stemness is restored by the addition of an antioxidant or an inhibitor of c-Jun N-terminal kinase (JNK). In addition, Prx I and Prx II appear to have a tight association with the mechanism underlying the protection of ESC stemness in developing teratomas. These results suggest that Prx functions as a protector of ESC stemness by opposing ROS/JNK cascades during neurogenesis. Therefore, our findings have important implications for understanding of maintenance of ESC stemness through involvement of antioxidant enzymes and may lead to development of an alternative stem cell-based therapeutic strategy for production of high-quality neurons in large quantity.

Testicular hyperthermia induces Unfolded Protein Response signaling activation in spermatocyte.

The testes of most mammals are sensitive to temperature. To survive and adapt under conditions that promote endoplasmic reticulum (ER) stress such as heat shock, cells have a self-protective mechanism against ER stress that has been termed the "Unfolded Protein Response" (UPR). However, the cellular and molecular events underlying spermatogenesis with testicular hyperthermia involved in the UPR signaling pathway under ER stress remain poorly understood. In the present study, we verified that UPR signaling via phospho-eIF2α/ATF4/GADD34, p90ATF6, and phospho-IRE1α/XBP-1 is activated with testicular hyperthermia (43 °C, 15 min/day) and induced ER stress-mediated apoptosis associated with CHOP, phospho-JNK, and caspase-3 after repetitive periods of hyperthermia. Levels of phospho-eIF2α protein of mouse spermatocytes in the testis were rapidly increased by one cycle of testicular hyperthermia. ATF4/GADD34 and p90ATF6 expression gradually increased and decreased, respectively, with repetitive cycles of hyperthermia. Spliced XBP1 mRNA as a marker of IRE1 activity was increased after one, three cycles of hyperthermia and decreased by five cycles of hyperthermia. Although the levels of anti-apoptotic phospho-JNK (p54) were gradually decreased after three cycles of hyperthermia, CHOP expression was rapidly increased. After five cycles of testicular hyperthermia, the levels of cleaved caspase-3 and TUNEL-positive apoptotic spermatocytes cells were significantly increased. Our data demonstrated that testicular hyperthermia induces UPR signaling and repetitive cycles of hyperthermia lead to apoptosis of spermatocytes in mouse testis. These results suggest a link between the UPR signaling pathway and testicular hyperthermia.

Autophagy negatively regulates keratinocyte inflammatory responses via scaffolding protein p62/SQSTM1.

The scaffolding adaptor protein p62/SQSTM1 (p62) has been shown to be an autophagy receptor that acts as a link between the ubiquitination and autophagy machineries. However, the roles of autophagy and p62 in human keratinocytes are not well understood. In this study, we show that keratinocyte autophagy negatively regulates p62 expression, which is essential for the prevention of excessive inflammation and the induction of cathelicidin in human keratinocytes. Stimulation of TLR2/6 or TLR4 in primary human keratinocytes robustly activated autophagy pathways and up-regulated p62 expression through induction of NADPH oxidases 2 and 4 and the generation of reactive oxygen species. MyD88 and TNFR-associated factor 6, key signaling molecules that mediate TLR activation, played an essential role in the induction of autophagy and p62 expression. Additionally, blockade of autophagy significantly increased the generation of inflammatory cytokines and expression of p62 in primary human keratinocytes. Notably, silencing hp62 through RNA interference resulted in a significant decrease in NF-κB activation, inflammatory cytokine production, cathelicidin expression, and cell proliferation (as well as cyclin D1 expression) in keratinocytes. Epidermal expression of p62 was further found to be significantly higher in psoriatic skin than in skin affected by atopic dermatitis or from healthy controls. Collectively, our data provide new insights into the roles of autophagy and p62 in controlling cutaneous inflammation.

NDRG2 is involved in the oncogenic properties of renal cell carcinoma and its loss is a novel independent poor prognostic factor after nephrectomy.

BACKGROUND: Although NDRG2 is a candidate tumor suppressor, its exact role in renal cell carcinoma (RCC) is not fully understood. We investigated the functional role of NDRG2 and its clinical relevance in RCC tumorigenesis. METHODS: NDRG2 expression and its clinical implications in clear cell RCC were evaluated. Biological function was assessed by a proliferation assay, anchorage-independent growth assay, and wound healing and transwell migration assays in RCC cell lines overexpressing NDRG2 coupled with an investigation of the effects of NDRG2 expression on the epithelial-mesenchymal transition (EMT). RESULTS: NDRG2 was differentially expressed in patients with RCC. A loss of NDRG2 was significantly associated with a higher proportion of tumors >10 cm and a high nuclear grade. Furthermore, multivariate analyses indicated that a loss of NDRG2 was an independent poor prognostic factor for patient survival (recurrence-free survival, hazard ratio 7.901; disease-specific survival, hazard ratio 15.395; overall survival, hazard ratio 11.339; P < 0.001 for all parameters). NDRG2 expression inhibited the anchorage-independent growth and migration of RCC cells. NDRG2 expression also modulated the expression of EMT-related genes such as Snail, Slug, and SIP1, and it decreased EMT signaling in RCC cells. Finally, NDRG2 recovered E-cadherin expression in E-cadherin-negative RCC cells. CONCLUSIONS: These results indicate that a lack of NDRG2 is associated with oncogenic properties through the loss of its role as a tumor suppressor, and that NDRG2 is an independent poor prognostic factor predicting survival in clear cell RCC, suggesting that it can serve as a novel prognostic biomarker.

Microglial peroxiredoxin V acts as an inducible anti-inflammatory antioxidant through cooperation with redox signaling cascades.

Reactive oxygen species (ROS) actively participate in microglia-mediated pathogenesis as pro-inflammatory molecules. However, little is known about the involvement of specific antioxidants in maintaining the microglial oxidative balance. We demonstrate that microglial peroxiredoxin (Prx) 5 expression is up-regulated by lipopolysaccharide (LPS) through activation of the ROS-sensitive signaling pathway and is involved in attenuation of both microglial activation and nitric oxide (NO) generation. Unlike in stimulation of oxidative insults with paraquat and hydrogen peroxide, Prx V expression is highly sensitive to LPS-stimulation in microglia. Reduction of ROS level by treatment with either NADPH oxidase inhibitor or antioxidant ablates LPS-mediated Prx V up-regulation in BV-2 microglial cells and is closely associated with the activation of the c-jun N-terminal kinase (JNK) signaling pathway. This suggests the involvement of ROS/JNK signaling in LPS-mediated Prx V induction. Furthermore, NO induces Prx V up-regulation that is ablated by the addition of inducible nitric oxide synthase inhibitor or deleted mutation of inducible nitric oxide synthase in LPS-stimulated microglia. Therefore, these results suggest that Prx V is induced by cooperative action among the ROS, RNS, and JNK signaling cascades. Interestingly, knockdown of Prx V expression causes the acceleration of microglia activation, including augmented ROS generation and JNK-dependent NO production. In summary, we demonstrate that Prx V plays a key role in the microglial activation process through modulation of the balance between ROS/NO generation and the corresponding JNK cascade activation.

Peroxiredoxin I contributes to TRAIL resistance through suppression of redox-sensitive caspase activation in human hepatoma cells.

Reactive oxygen species (ROS) have been implicated in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) resistance of many cancers. We evaluated the role of peroxiredoxin (Prx) I in TRAIL resistance governed by coupling of nicotinamide adenosine dinucleotide phosphate oxidase (Nox)-derived ROS signaling with the p38 mitogen-activated protein kinase (MAPK)/caspase-signaling cascade in liver cancer cells. Upregulated Prx I expression was found in neoplastic regions of human patient liver, and Prx I knockdown resulted in accelerated TRAIL-induced cell death in SK-Hep-1 human hepatoma cells. The TRAIL cytotoxicity by Prx I knockdown was dependent on activation of caspase-8/3 cascades, which was ablated by addition of inhibitors for p38 MAPK, ROS or Nox, suggesting the association with Nox-driven redox signaling. Furthermore, we found that Nox4 was constitutively expressed in both SK-Hep-1 cells and tumor regions of patient livers, knockdown of Nox4 expression could alleviate ROS generation and TRAIL-mediated cytotoxicity. In accordance with previous findings, increased activation of both p38 MAPK and caspase cascades by Prx I knockdown was inhibited by either Nox4 knockdown or SB203580 addition. Collectively, these data suggest that Prx I functions to block propagation of Nox-derived ROS signaling to the p38 MAPK/caspase/cell death cascade during TRAIL treatment and also provides a molecular mechanism by which Prx I contributes to TRAIL resistance in liver cancers.

Podoplanin is involved in the prognosis of head and neck squamous cell carcinoma through interaction with VEGF-C.

We investigated the clinical significance of podoplanin expression in relation to clinicopathological variables in head and neck squamous cell carcinoma (HNSCC), to determine its effectiveness as a marker for high-risk HNSCC patients. Upregulation of podoplanin in HNSCC tissues was examined using immunohistochemistry and clinicopathological analyses. Wound healing and invasion assays were performed using HNSCC cells that were transfected with podoplanin siRNA, podoplanin vector and cotransfection with both the podoplanin vector and VEGF-C siRNA. High podoplanin expression was significantly associated with ~3- and 5-fold increases in the presence of positive lymph node metastasis and poor histological grade, respectively (P<0.05). High levels of podoplanin expression were significantly associated with decreased overall and disease-specific survival rates (P<0.05). Furthermore, upregulation of podoplanin induced cell wound repair activity and invasiveness in the FaDu and SNU-1041 cells, respectively, while downregulation of podoplanin expression through VEGF-C silencing using co-transfection of both the podoplanin vector and VEGF-C siRNA suppressed cell wound healing and invasion abilities in the HNSCC cells. Our findings suggest that high podoplanin expression is associated with aggressive tumor behavior, poor prognosis and metastatic regulation through interaction with VEGF-C, suggesting that podoplanin may be used as a potential prognostic biomarker for HNSCC.

The prognostic significance of Smad3, Smad4, Smad3 phosphoisoform expression in esophageal squamous cell carcinoma.

Smad3 functions as an integrator of diverse signaling, including transforming growth factor ß signaling and the function of Smad3 is complexly regulated by differential phosphorylation at various sites of Smad3. Despite the importance of Smad3 and its various phosphoisoforms, their prognostic significance has rarely been studied. In this study, we demonstrated the prognostic significance of Smad3, its phosphoisoforms, and Smad4 expression by immunohistochemistry in 126 esophageal squamous cell carcinomas. The phosphoisoforms of Smad3 studied in this article included phosphorylation at C-terminal (pSmad3C)(Ser(423/425)) and phosphorylation at the linker region (pSmad3L)(Ser(213)). High expression of Smad3 was associated with shorter overall survival. Co-existence of high expression of pSmad3L(S213) and low expression of pSmad3C(S423/425) were associated with advanced N stage and an independent prognostic factor for overall [hazard ratio (HR) 2.03, 95 % confidence interval (CI) (1.10-3.75), p = 0.023] and disease-free survival [HR 2.41, 95 % CI (1.32-4.39), p = 0.004]. In conclusion, co-existence of high pSmad3L(Ser(213)) expression and low pSmad3C(Ser(423/425)) expression can be considered as immunohistochemical biomarkers for predicting prognosis as well as future therapeutic targets. In addition, our results of combinatory effect of differential phosphorylation of Smad3 on prognosis suggest the mode of action of Smad3 might be logically determined by its phosphorylation pattern.

Expression of carboxyl terminus of Hsp70-interacting protein (CHIP) indicates poor prognosis in human gallbladder carcinoma.

Gallbladder carcinoma (GBC) is a lethal neoplasm, and new prognostic markers are required. Deregulation of E3 ligases contributes to cancer development and is associated with poor prognosis. Carboxyl terminus of heat shock protein 70-interacting protein (CHIP) is a U-box-type E3 ubiquitin ligase, the role of which has not been evaluated in GBC. Therefore, the present study investigated CHIP expression in GBC and its prognostic significance. In the present study, CHIP expression was measured in 78 tumor specimens of GBC by immunohistochemistry and the correlation between CHIP expression and clinicopathological factors was analyzed. Of the tumor specimens, 26.9% showed high staining intensity [the CHIP high expression group (HEG)]. The CHIP-HEG was not associated with other common clinicopathological parameters, including T stage, and lymph node and distant metastases. CHIP-HEG patients had a significantly worse prognosis than patients with low CHIP expression with median cancer-specific survival times of 8.0 months (range, 1-34 months) and 13.0 months (range, 1-110 months), respectively (P=0.023). Multivariate analyses showed that CHIP expression was close to being an independent risk factor for predicting patient survival. CHIP expression may be associated with a poor prognosis in GBC. Since CHIP is not associated with other clinicopathological prognostic factors, it may serve as an ideal molecular marker for predicting patient outcomes.

Differential expression of Yes-associated protein and phosphorylated Yes-associated protein is correlated with expression of Ki-67 and phospho-ERK in colorectal adenocarcinoma.

Yes-associated protein (YAP) is a transcriptional co-activator and functions as a nuclear downstream effector of the Hippo pathway. Differential expression of YAP and phosphorylated Yes-associated protein (pYAP), which are involved in the expression of Ki-67 and phosphorylated extracellular signal-regulated kinase (pERK) in colorectal adenocarcinoma (CRAC), is not clear. Herein, we hypothesized that nuclear expression of YAP could predict cell proliferation and poor prognosis, while cytoplasmic expression of pYAP would show a reverse correlation with cell proliferation. Paraffin-embedded samples from 144 CRAC patients were studied using immunohistochemistry for YAP, pYAP, Ki-67 and pERK. Frozen samples from 20 CRAC patients were examined for YAP mRNA in tumor and non-tumor tissues, using quantitative real-time PCR. High nuclear YAP expression coincided with high Ki-67 expression (P=0.002). The high nuclear YAP expression group tended to display a poor overall and disease-free survival (P=0.089 and P=0.089, respectively), but YAP mRNA levels in the 20 CRAC tissues were not significantly different in comparison with the 20 non-tumor tissues (P=0.929). We observed an inverse correlation between high cytoplasmic pYAP expression and high Ki-67 expression (P=0.001). Nuclear pERK expression was positively correlated with nuclear YAP expression, but negatively correlated with cytoplasmic pYAP expression (P=0.017 and P=0.020, respectively). Activated nuclear YAP and inactivated cytoplasmic pYAP in CRAC showed a positive correlation with Ki-67 and nuclear pERK expression, suggesting that the expression of YAP and pYAP is a possible predictor of tumor cell proliferation and prognosis in CRAC.

PHF20 regulates NF-κB signalling by disrupting recruitment of PP2A to p65.

Constitutive NF-κB activation in cancer cells is caused by defects in the signalling network responsible for terminating the NF-κB response. Here we report that plant homeodomain finger protein 20 (PHF20) maintains NF-κB in an active state in the nucleus by inhibiting the interaction between PP2A and p65. We show that PHF20 induces canonical NF-κB signalling by increasing the DNA-binding activity of NF-κB subunit p65. In PHF20 overexpressing cells, the termination of tumour necrosis factor-induced p65 phosphorylation is impaired whereas upstream signalling events triggered by tumour necrosis factor are unaffected. This effect strictly depends on the interaction between PHF20 and methylated lysine residues of p65, which hinders recruitment of PP2A to p65, thereby maintaining p65 in a phosphorylated state. We further show that PHF20 levels correlate with p65 phosphorylation levels in human glioma specimens. Our work identifies PHF20 as a novel regulator of NF-κB activation and suggests that elevated expression of PHF20 may drive constitutive NF-κB activation in some cancers.

Correlation of expression of phosphorylated and non-phosphorylated Yes-associated protein with clinicopathological parameters in esophageal squamous cell carcinoma in a Korean population.

Yes-associated protein (YAP) is a nuclear downstream effector of the Hippo pathway, a regulator of cell growth. The phosphorylated form of YAP (pYAP), located in the cytoplasm, prevents cellular proliferation by spatially segregating YAP from the nucleus. This study aimed at investigating the relationships of pYAP and YAP with clinicopathological variables in esophageal squamous cell carcinoma (ESCC). Samples of ESCC from 142 patients were studied using immunohistochemistry for YAP, pYAP and Ki-67. In all cases of ESCC, higher nuclear expression of YAP was correlated with Ki-67 expression, tumor diameter, histological grade (1-2 versus 3), and pathological TNM stage (I versus II-IV) in univariate analyses (p=0.036, p=0.025, p=0.021, and p=0.033, respectively). Higher nuclear expression of YAP was associated with worse overall and disease-free survival (p=0.006 and p=0.008, respectively). Multivariate analysis showed higher nuclear expression of YAP to be an independent prognostic marker for overall survival (p=0.034). We observed a trend towards inverse correlation of cytoplasmic pYAP expression and histological grade (1-2 versus 3) (p=0.087). Our results suggest that YAP shifts from the nucleus to the cytoplasm as a consequence of phosphorylation, which occurs in the presence of high tumor cell density in the case of ESCC, and may be a potential indication of histological differentiation. Nuclear expression of YAP is correlated with tumor cell proliferation and is an independent predictor of worse prognosis of ESCC.

CD44 is associated with tumor recurrence and is an independent poor prognostic factor for patients with localized clear cell renal cell carcinoma after nephrectomy.

CD44 has been implicated in tumor development and progression in several types of cancer. CD44 expression is altered in renal cell carcinoma (RCC) and has been suggested as a useful prognostic marker, but its prognostic role in RCC remains controversial. We investigated the expression of CD44 in a large homogeneous set of localized clear cell RCC to determine its potential prognostic value. We examined 110 patients with localized clear cell RCC who underwent nephrectomy. The clinicopathological data were obtained retrospectively and the expression level of CD44 was studied by immunohistochemistry. Correlations between CD44 expression and clinical parameters as well as survival were determined. The CD44-high expression group (HEG) was significantly associated with a higher nuclear grade (P=0.014) and tumor recurrence (P<0.001) when compared with the CD44-low expression group (LEG). Concerning survival, the 5-year recurrence-free survival (RFS) rates for the CD44-HEG and CD44-LEG groups were 38.9 and 91.3%, respectively (P<0.001), and the 5-year disease-specific survival (DSS) rates for the CD44-HEG and CD44-LEG groups were 55.6 and 94.6%, respectively (P<0.001). Multivariate analyses showed that CD44 expression [hazard ratio (HR), 9.204; P<0.001] was an independent risk factor predicting RFS in patients with clear cell RCC. CD44 expression remained an independent prognostic factor for DSS (P=0.002). In conclusion, these data indicate that CD44 expression is associated with the progression of clear cell RCC and is an independent poor prognostic factor for tumor recurrence and survival, suggesting that CD44 may serve as a useful molecular marker.

Overexpression of IL-32 is a novel prognostic factor in patients with localized clear cell renal cell carcinoma.

Interleukin-32 (IL-32) is a proinflammatory cytokine that acts as a significant pathogenetic factor in various diseases and malignancies. However, the clinical effect of IL-32 expression in renal cell carcinoma (RCC) has not previously been investigated. The aim of the present study was to examine the significance of IL-32 overexpression in localized clear cell RCC (CCRCC). We examined 112 patients with localized CCRCC who underwent nephrectomy. The clinicopathological data were obtained by retrospective review and the expression levels of IL-32 were studied by immunohistochemistry. Tumors were classified according to staining intensity (0, no staining intensity; 1, weak; 2, intermediate; 3, strong). The cases with staining intensities from 0 to 2 comprised the IL-32 low-expression group (LEG), whereas those with a staining intensity of 3 comprised the IL-32 high-expression group (HEG). Correlations between IL-32 expression and clinicopathological parameters were determined. Staining intensities were determined for all cases as follows: 26 cases (23.2%) (score 0), 43 cases (38.4%) (score 1), 31 cases (27.7%) (score 2) and 12 cases (10.7%) (score 3). IL-32 HEG exhibited a higher recurrence rate compared to the IL-32 LEG (50 vs. 13%, P=0.001). For survival rates, the 5-year recurrence-free survival (RFS), disease-specific survival (DSS) and overall survival (OS) rates were lower in the IL-32 HEG group compared with the IL-32 LEG group (RFS, P=0.001; DSS, P<0.001; OS, P=0.026, respectively). Univariate analyses revealed that Fuhrman nuclear grade and a high IL-32 expression were significant prognostic factors for predicting RFS, DSS and OS in CCRCC, whereas multivariate analyses indicated that Fuhrman nuclear grade and high IL-32 expression were still independent risk factors. In conclusion, IL-32 overexpression was associated with high recurrence rates and low RFS, DSS and OS, indicating that it may be a novel prognostic factor for predicting outcomes in patients with CCRCC.

Correlation of epithelial-mesenchymal transition markers with clinicopathologic parameters in adenocarcinomas and squamous cell carcinoma of the lung.

Epithelial-mesenchymal transition (EMT) is characterized by the loss of epithelial cell junction proteins and the gain of mesenchymal markers. The aim of this study was to analyze the associations between the EMT-related markers vimentin, E-cadherin, ß-catenin, slug, snail, and twist1 and clinicopathologic parameters as well as epidermal growth factor receptor (EGFR) gene copy number and protein expression in non-small cell lung carcinoma (NSCLC). Fifty-nine squamous cell carcinomas (SCC) and 43 adenocarcinomas (AD) were immunohistochemically examined for respective EMT markers and for EGFR, using the EGFR PharmDx kit (Dako) for protein expression and automated silver enhanced in situ hybridization (SISH) for copy number. Vimentin expression in tumor epithelia was significantly higher in AD samples than in SCC samples (P=0.015). Among AD samples, vimentin expression was positively correlated with histologic grade (2 vs. 3; P=0.021) and exhibited a tendency toward a positive correlation with pTNM stage (I vs. II-IV; P=0.052). EGFR gene copy number was positively correlated with EGFR protein expression among both AD samples (P=0.008) and SCC samples (P=0.042), with EGFR protein expression being significantly higher in SCC samples compared with AD (P=0.038). Among AD samples, EGFR protein expression was associated with higher cytoplasmic expression of ß-catenin (P=0.031). Among SCC samples, EGFR protein expression was negatively correlated with nuclear expression of ß-catenin (P=0.033) but positively with nuclear slug (P=0.021). The expression pattern of EMT markers in AD suggests that vimentin is a possible immunohistochemical predictor of tumor progression.

Chemokine (C-X-C motif) ligand 12 is associated with gallbladder carcinoma progression and is a novel independent poor prognostic factor.

PURPOSE: Although recent studies have suggested that chemokine (C-X-C motif) ligand 12 (CXCL12) is important in the progression of various malignancies, its role in gallbladder carcinoma (GBC) remains unknown. We investigated CXCL12 expression in GBC and its biologic and prognostic role in GBC tumorigenesis. EXPERIMENTAL DESIGN: We examined CXCL12 expression in tumor specimens from 72 patients with GBC by immunohistochemistry and analyzed the correlation between CXCL12 expression and clinicopathologic factors or survival. The functional significance of CXCL12 expression was investigated by CXCL12 treatment and suppression of CXCR4, a major receptor of CXCL12, as well as by CXCL12 overexpression in in vitro and in vivo studies. RESULTS: CXCL12 was differentially expressed in GBC tissues. CXCL12 expression was significantly associated with a high histologic grade (P = 0.042) and nodal metastasis (P = 0.015). Multivariate analyses showed that CXCL12 expression (HR, 8.675; P = 0.014) was an independent risk factor for patient survival. CXCL12 significantly increased anchorage-dependent and -independent growth, migration, invasion, adhesiveness, and survival of GBC cells in vitro, and these effects were dependent on CXCR4. Consistent with these results, overexpression of CXCL12 significantly promoted GBC tumorigenicity in a xenograft model. CONCLUSIONS: Our results indicate that GBC cells express both CXCL12 and its receptor CXCR4, and CXCL12 may have a role in GBC progression through an autocrine mechanism. In addition, CXCL12 is a novel independent poor prognostic factor in patients with GBCs. Thus, targeting CXCL12 and CXCR4 may provide a novel therapeutic strategy for GBC treatment.

Differential expression of Yes-associated protein is correlated with expression of cell cycle markers and pathologic TNM staging in non-small-cell lung carcinoma.

Yes-associated protein, a downstream effector of the Hippo signaling pathway, has been linked to progression of non-small-cell lung carcinoma. The aim of this study was to investigate expression of Yes-associated protein in lung adenocarcinoma and squamous cell carcinoma. Associations of Yes-associated protein expression with clinicopathologic parameters, expression of cell cycle-specific markers, and epidermal growth factor receptor gene amplification were also analyzed. In a univariate analysis of the 66 adenocarcinomas, high nuclear expression of Yes-associated protein was significantly correlated with expression of cyclin A and mitogen-activated protein kinase. Multivariate analysis, including age and sex, showed that cyclin A expression was independently correlated with nuclear expression of Yes-associated protein in adenocarcinomas. Furthermore, high nuclear expression of Yes-associated protein was also a significant predictor of epidermal growth factor receptor gene amplification for adenocarcinoma. For the 102 squamous cell carcinomas, univariate analysis revealed that high cytoplasmic expression of Yes-associated protein was correlated with the low pathologic TNM staging (stage I) and histologic grading. Multivariate analysis, including age and sex, showed that cytoplasmic expression of Yes-associated protein was an independent predictor of low pathologic TNM staging. These results indicate that nuclear overexpression of Yes-associated protein contributes to pulmonary adenocarcinoma growth and that high cytoplasmic expression of Yes-associated protein is an independent predictor of low pathologic TNM staging and histologic grading. The differential effects of Yes-associated protein expression patterns in adenocarcinomas and squamous cell carcinomas suggest that Yes-associated protein may play important roles in different pathways in distinct tumor subtypes. These observations may, therefore, lead to new perspectives on therapeutic targeting of these tumor types.

L1 cell adhesion molecule as a novel independent poor prognostic factor in gallbladder carcinoma.

Gallbladder carcinoma is a lethal malignancy and is hard to cure by current treatment. Thus, identification of molecular prognostic markers to predict gallbladder carcinoma as therapeutic targets is urgently needed. Recent studies have demonstrated that L1 cell adhesion molecule is associated with the prognosis of variable malignancy. Here, we investigated L1 cell adhesion molecule expression in gallbladder carcinoma and its prognostic significance. In this study, we examined L1 cell adhesion molecule expression in tumor specimens from 69 patients with gallbladder carcinoma by immunohistochemistry and analyzed the correlation between L1 cell adhesion molecule expression and clinicopathologic factors or survival. L1 cell adhesion molecule was not expressed in the normal epithelium of the gallbladder but in 63.8% of gallbladder carcinomas, remarkably at the invasive front of the tumors. In addition, L1 cell adhesion molecule expression was significantly associated with high histologic grade, advanced pathologic T stage and clinical stage, and positive venous/lymphatic invasion. Multivariate analyses showed that L1 cell adhesion molecule expression (hazard ratio, 3.503; P = .028) and clinical stage (hazard ratio, 3.091; P = .042) were independent risk factor for disease-free survival. L1 cell adhesion molecule expression in gallbladder carcinoma was significantly correlated with tumor progression and unfavorable clinicopathologic features. L1 cell adhesion molecule expression was an independent poor prognostic factor for disease-free survival in patients with gallbladder carcinoma. Taken together, our findings suggest that L1 cell adhesion molecule expression could be used as a novel prognostic factor for patient survival and might be a potential therapeutic target in gallbladder carcinomas.

Differential expression of DUSP6 with expression of ERK and Ki-67 in non-small cell lung carcinoma.

Dual specificity phosphatase 6 (DUSP6) is a member of the MAP kinase phophatase family. DUSP6 inactivates extracellular signal-regulated kinase (ERK), belonging to the MAP kinase family, and can act in tumor suppressive pathways. The aim of this study was to investigate associations of DUSP6 expression with expression of ERK and Ki-67 and with clinicopathological parameters in lung adenocarcinoma and squamous cell carcinoma. A total of 102 squamous cell carcinomas and 66 adenocarcinomas were studied using immunohistochemistry for DUSP6, ERK1/2, and Ki-67. In 66 adenocarcinomas, high DUSP6 expression was positively correlated with ERK1/2 expression. High DUSP6 expression was correlated with lower histological grade and lower Ki-67 index in the adenocarcinomas. In 102 squamous cell carcinomas, high DUSP6 expression was correlated with lower ERK expression, with greater smoking pack-years, but not with the Ki-67 index. These results indicate that DUSP6 acts as a negative feedback regulator of ERK in adenocarcinoma progression, but that DUSP6 does not play a role in the downregulation of ERK in squamous cell carcinoma. The differential expression of DUSP6 correlated with Ki-67 index, suggesting that DUSP6 plays an important role in cancer resistance in different subtypes of non-small cell lung carcinoma.