Ascitic autotaxin as a potential prognostic, diagnostic, and therapeutic target for epithelial ovarian cancer.

BACKGROUND: Malignant ascites contributes to the metastatic process by facilitating the multifocal dissemination of ovarian tumour cells onto the peritoneal surface. However, the prognostic and diagnostic relevance of ascitic fluid remains largely unknown. Herein, we investigated the potential clinical value and therapeutic utility of ascitic autotaxin (ATX) in epithelial ovarian cancer (EOC). METHODS: ATX expression was assessed in clinical samples. Spheroid-forming assay, real-time PCR, western blot analysis, invadopodia assay, and adhesion assays were performed. RESULTS: Ascitic ATX expression was highly elevated in patients with ovarian cancer compared to those with benign ascites and was associated with advanced stage, high grade, and a short disease-free period in patients with EOC. Combining the diagnostic ability of ascitic ATX and serum CA-125 levels significantly improved the area under the curve (AUC) value for EOC compared to serum CA125 level alone. This marker combination showed a large odds ratio for short disease-free period in high-risk EOC groups. Functional studies revealed that ascitic ATX was required for maintaining cancer stem cell-like characteristics and invadopodia formation. CONCLUSION: Ascitic ATX levels may serve as a useful prognostic indicator for predicting aggressive behaviour in EOC. ATX-linked invadopodia are a potential target to prevent peritoneal dissemination in ovarian cancer.

MEK/ERK signaling is a critical regulator of high-risk human papillomavirus oncogene expression revealing therapeutic targets for HPV-induced tumors.

Intracellular pathogens have evolved to utilize normal cellular processes to complete their replicative cycles. Pathogens that interface with proliferative cell signaling pathways risk infections that can lead to cancers, but the factors that influence malignant outcomes are incompletely understood. Human papillomaviruses (HPVs) predominantly cause benign hyperplasia in stratifying epithelial tissues. However, a subset of carcinogenic or "high-risk" HPV (hr-HPV) genotypes are etiologically linked to nearly 5% of all human cancers. Progression of hr-HPV-induced lesions to malignancies is characterized by increased expression of the E6 and E7 oncogenes and the oncogenic functions of these viral proteins have been widely studied. Yet, the mechanisms that regulate hr-HPV oncogene transcription and suppress their expression in benign lesions remain poorly understood. Here, we demonstrate that EGFR/MEK/ERK signaling, influenced by epithelial contact inhibition and tissue differentiation cues, regulates hr-HPV oncogene expression. Using monolayer cells, epithelial organotypic tissue models, and neoplastic tissue biopsy materials, we show that cell-extrinsic activation of ERK overrides cellular control to promote HPV oncogene expression and the neoplastic phenotype. Our data suggest that HPVs are adapted to use the EGFR/MEK/ERK signaling pathway to regulate their productive replicative cycles. Mechanistic studies show that EGFR/MEK/ERK signaling influences AP-1 transcription factor activity and AP-1 factor knockdown reduces oncogene transcription. Furthermore, pharmacological inhibitors of EGFR, MEK, and ERK signaling quash HPV oncogene expression and the neoplastic phenotype, revealing a potential clinical strategy to suppress uncontrolled cell proliferation, reduce oncogene expression and treat HPV neoplasia.

High-Dose Selenium Induces Ferroptotic Cell Death in Ovarian Cancer.

Selenium is a promising multi-target chemotherapeutic agent with controversial clinical results. Hence, reassessing the anticancer effects of Se is necessary to clearly understand the potential of high-dose selenium in cancer treatment. Here, we observed that high-dose sodium selenite (SS) significantly decreased the proliferation and increased the death of ovarian cancer cells, mediated by an increased generation of reactive oxygen species. Notably, high-dose SS decreased the levels of glutathione peroxidase (GPx), a selenoprotein with antioxidant properties, without altering other selenoproteins. Furthermore, high-dose SS triggered lipid peroxidation and ferroptosis, a type of iron-dependent cell death, due to dysregulated GPx4 pathways. We demonstrated that intravenous high-dose SS significantly reduced the tumor growth and weight in SKOV3-bearing mice. Consistent with our in vitro results, mice with SKOV3 cells treated with high-dose SS showed decreased GPx4 expression in tumors. Therefore, we highlight the significance of high-dose SS as a potential chemotherapeutic agent for ovarian cancer. High-dose SS-mediated ferroptotic therapy integrating glutathione depletion and ROS generation is a promising strategy for cancer therapy.

NANOG regulates epithelial-mesenchymal transition via AMPK/mTOR signalling pathway in ovarian cancer SKOV-3 and A2780 cells.

NANOG engages with tumour initiation and metastasis by regulating the epithelial-mesenchymal transition (EMT) in epithelial ovarian cancer (EOC). However, its role in association with pAMPKα, and its clinical significance in EOC have not been elucidated even though AMPK is known to degrade NANOG in various human cancers. Hence, we investigated the role of pAMPKα and its association with NANOG as potential prognostic biomarkers in EOC. Both NANOG and pAMPKα expression were significantly overexpressed in EOCs comparing nonadjacent normal epithelial tissues, benign tissues, and borderline tumours. NANOG overexpression was significantly associated with poor disease-free survival (DFS) and overall survival (OS), whereas pAMPKα overexpression was associated with good DFS and OS. Importantly, multivariate analysis revealed that the combination of high NANOG and low pAMPKα expression was a poor independent prognostic factor for DFS and was associated with platinum resistance. In ovarian cancer cell lines, siRNA-mediated NANOG knockdown diminished migration and invasion properties by regulating the EMT process via the AMPK/mTOR signalling pathway. Furthermore, treatment with AMPK activator suppressed expression of stemness factors such as NANOG, Oct4 and Sox2. Collectively, these findings established that the combination of high NANOG and low pAMPKα expression was associated with EOC progression and platinum resistance, suggesting a potential prognostic biomarker for clinical management in EOC patients.

RNA sequencing and bioinformatics analysis revealed PACSIN3 as a potential novel biomarker for platinum resistance in epithelial ovarian cancer.

BACKGROUND: Failure to respond to treatment in epithelial ovarian cancer can often be attributed to platinum-based chemotherapy resistance. However, the possible mechanisms or candidate biomarkers associated with platinum resistance are yet to be elucidated, even though many researchers have performed related studies. METHODS: We performed RNA sequencing of clinical specimens obtained from patients with platinum-sensitive or resistant epithelial ovarian cancer (EOC). Furthermore, various bioinformatics approaches, including spatial analysis of functional enrichment, were used to identify key regulators and associated underlying mechanisms of platinum resistance in EOC. RESULTS: Through RNA-sequencing, we identified 263 differentially expressed genes (98 upregulated and 165 downregulated) and subjected them to Gene Oncology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses, which were characterized to the traditional platinum-resistant characteristics. Subsequently, the gene interaction network and module analysis by spatial analysis of functional enrichment software demonstrated protein kinase C and casein kinase substrate in neurons 3 (PACSIN3) as the only upregulated hub gene, and neurotensin (NTS) and KIAA0319 as downregulated hub genes in platinum-resistant EOC. We selected PACSIN3 for further analysis because it has not been studied in relation to response to platinum-based chemotherapy. PACSIN3 was significantly upregulated in ovarian cancer cells compared to immortalized human ovarian surface epithelial cells. In addition, cisplatin-induced apoptosis was measured in PACSIN3 knockout OVCA433 and BRCA-mutated EOC cell line, SNU251, by a fluorescence-activated cell sorting-based Annexin-V/propium iodide double staining assay, which revealed a significant increase in apoptosis. CONCLUSIONS: Taken together, the present study presents PACSIN3 as a promising predictive biomarker associated with platinum resistance, especially in BRCA-mutated epithelial ovarian cancers.

The Combination of Transient Receptor Potential Vanilloid Type 1 (TRPV1) and Phosphatase and Tension Homolog (PTEN) is an Effective Prognostic Biomarker in Cervical Cancer.

Transient receptor potential vanilloid type 1 (TRPV1) has been reported to play an important role in human cancers. However, the knowledge about TRPV1 in cervical cancer is sparse. Therefore, we evaluated the expression and clinical significance of TRPV1 in cervical cancer. Immunohistochemical analyses were performed for TRPV1 and phosphatase and tension homolog (PTEN) to delineate clinical significance using 150 cervical cancers, 230 cervical intraepithelial neoplasias, and 312 normal cervical epithelial tissues in a tissue microarray. Furthermore, the role of TRPV1 in cell growth was assessed in a cervical cancer cell line. The TRPV1 expression was significantly higher in cervical cancer tissues than in cervical intraepithelial neoplasias, and normal epithelial tissues (P<0.001). In cervical cancer tissues, TRPV1 expression negatively correlated with PTEN expression (Spearman ρ=-0.121, P=0.009). Multivariate survival analysis revealed high TRPV1 expression (hazard ratio=3.41, 95% confidence interval: 1.25-9.27, P=0.016) as an independent prognostic factor for overall survival. Notably. the high TRPV1/low PTEN expression showed the highest hazard ratio (5.87; 95% confidence interval: 2.18-15.82, P<0.001) for overall survival. In vitro results demonstrated that the overexpression of TRPV1 was associated with increased cell viability and colony formation. Overexpression of TRPV1 could be a good biomarker for the prediction of chemoradiation response. Our result suggested promising potential of high TRPV1/low PTEN as prognostic and survival makers. The possible link between the biologic function of TRPV1 and PTEN in cervical cancer warrants further studies.

Clinical Significance of Tumor Infiltrating Lymphocytes in Association with Hormone Receptor Expression Patterns in Epithelial Ovarian Cancer.

Hormone receptor expression patterns often correlate with infiltration of specific lymphocytes in tumors. Specifically, the presence of specific tumor-infiltrating lymphocytes (TILs) with particular hormone receptor expression is reportedly associated with breast cancer, however, this has not been revealed in epithelial ovarian cancer (EOC). Therefore, we investigated the association between hormone receptor expression and TILs in EOC. Here we found that ERα, AR, and GR expression increased in EOC, while PR was significantly reduced and ERß expression showed a reduced trend compared to normal epithelium. Cluster analysis indicated poor disease-free survival (DFS) in AR+/GR+/PR+ subgroup (triple dominant group); while the Cox proportional-hazards model highlighted the triple dominant group as an independent prognostic factor for DFS. In addition, significant upregulation of FoxP3+ TILs, PD-1, and PD-L1 was observed in the triple dominant group compared to other groups. NanoString analyses further suggested that tumor necrosis factor (TNF) and/or NF-κB signaling pathways were activated with significant upregulation of RELA, MAP3K5, TNFAIP3, BCL2L1, RIPK1, TRAF2, PARP1, and AKT1 in the triple dominant EOC group. The triple dominant subgroup correlates with poor prognosis in EOC. Moreover, the TNF and/or NF-κB signaling pathways may be responsible for hormone-mediated inhibition of the immune microenvironment.

CDK7 is a reliable prognostic factor and novel therapeutic target in epithelial ovarian cancer.

OBJECTIVE: Cyclin-dependent kinase 7 (CDK7) engages tumor growth by acting as a direct link between the regulation of transcription and the cell cycle. Here, we investigated the clinical significance of CDK7 expression and its potential as a therapeutic target in epithelial ovarian cancer (EOC). METHODS: CDK7 expression was examined in 436 ovarian tissues including normal to metastatic ovarian tumors using immunohistochemistry, and its clinical implications were analyzed. Furthermore, we performed in vitro and in vivo experiments using CDK7 siRNA or a covalent CDK7 inhibitor (THZ1) to elucidate the effect of CDK7 inhibition on tumorigenesis in EOC cells. RESULTS: The patient incidence of high CDK7 expression (CDK7High) gradually increased from normal ovarian epithelium to EOC (P < 0.001). Moreover, CDK7High was associated with an advanced stage and high-grade histology (P = 0.035 and P = 0.011, respectively) in EOC patients and had an independent prognostic significance in EOC recurrence (P = 0.034). CDK7 inhibition with siRNA or THZ1 decreased cell proliferation and migration, and increased apoptosis in EOC cells, and this anti-cancer mechanism is caused by G0/G1 cell cycle arrest. In in vivo therapeutic experiments using cell-line xenograft and PDX models, CDK7 inhibition significantly decreased the tumor weight, which was mediated by cell proliferation and apoptosis. CONCLUSION: Mechanistic interrogation of CDK7 revealed that it is significantly associated with an aggressive phenotype of EOC, and it has independent prognostic power for EOC recurrence. Furthermore, CDK7 may be a potential therapeutic target for patients with EOC, whether platinum sensitive or resistant.

Prognostic implications of forkhead box protein O1 (FOXO1) and paired box 3 (PAX3) in epithelial ovarian cancer.

BACKGROUND: Transcription factors forkhead box protein O1 (FOXO1) and paired box 3 (PAX3) have been reported to play important roles in various cancers. However, their role in epithelial ovarian cancer (EOC) has not been elucidated yet. Therefore, we evaluated the expression and clinical significance of FOXO1 and PAX3 in EOC. METHODS: Immunohistochemical analyses of FOXO1 and PAX3 in 212 EOCs, 57 borderline ovarian tumors, 153 benign epithelial ovarian tumors, and 79 nonadjacent normal epithelial tissues were performed using tissue microarray. Various clinicopathological variables, including the survival of EOC patients, were compared. In addition, the effect of FOXO1 on cell growth was assessed in EOC cell lines. RESULTS: FOXO1 and PAX3 protein expression levels were significantly higher in EOC tissues than in nonadjacent normal epithelial tissues, benign tissues, and borderline tumors (all p < 0.001). In EOC tissues, FOXO1 expression was positively correlated with PAX3 expression (Spearman's rho = 0.118, p = 0.149). Multivariate survival analysis revealed that high FOXO1 expression (hazard ratio = 2.77 [95% CI, 1.48-5.18], p = 0.001) could be an independent prognostic factor for overall survival. Most importantly, high expression of both FOXO1 and PAX3 showed a high hazard ratio (4.60 [95% CI, 2.00-10.55], p < 0.001) for overall survival. Also in vitro results demonstrated that knockdown of FOXO1 was associated with decreased cell viability, migration, and colony formation. CONCLUSIONS: This study revealed that high expression of FOXO1/PAX3 is an indicator of poor prognosis in EOC. Our results suggest the promising potential of FOXO1 and PAX3 as prognostic and therapeutic markers. The possible link between biological functions of FOXO1 and PAX3 in EOC warrants further studies.

Combined treatment with modulated electro-hyperthermia and an autophagy inhibitor effectively inhibit ovarian and cervical cancer growth.

PURPOSE: Modulated electro-hyperthermia (mEHT), known as oncothermia, is an anticancer therapy that induces radiofrequency thermal damage to the cancer tissues. This study aimed to evaluate the potential effectiveness of mEHT as a therapeutic tool in ovarian and cervical cancer. MATERIALS AND METHODS: We used both tumor-bearing mice and ovarian and cervical OVCAR-3, SK-OV-3, HeLa and SNU-17 cancer cell lines to investigate the effects of mEHT in vivo and in vitro, respectively, and determine whether it was enhanced by cotreatment with an autophagy inhibitor. RESULTS: We discovered that phosphorylation of p38, a stress-dependent kinase, was induced at the Thr180/Tyr182 residue in cancer cells exposed to mEHT. Apoptotic markers such as cleaved caspase-3 and poly-ADP ribose polymerase (PARP) were increased in OVCAR-3 and SNU-17 cells. Fluorescence-activated cell sorting (FACS) analysis showed a significant increase in the population of sub-G1 mEHT-exposed cells, which are dying and apoptotic cells. mEHT also reduced both weight and volume of xenograft tumors in mice transplanted with ovarian and cervical cancer cells and patient-derived cancer tissues. We determined that mEHT-induced cellular damage recovery was mediated by autophagy and, therefore, expectedly, cotreatment with mEHT and 3-methyladenine (3-MA), an autophagy inhibitor, more effectively inhibited cancer cell growth than individual treatment did. CONCLUSIONS: mEHT treatment alone was sufficient to inhibit cancer growth, while a combined treatment with mEHT and an autophagy inhibitor amplified this inhibition effect.

Forkhead box protein O1 (FOXO1) and paired box gene 3 (PAX3) overexpression is associated with poor prognosis in patients with cervical cancer.

PURPOSE: Forkhead box protein O1 (FOXO1) and paired box gene 3 (PAX3) have been reported to play an imported role in human cancers, but their role in cervical cancer has not yet been clarified. In this study, we evaluated the functional role of FOXO1 in cervical cancer cells and investigated the expression and clinical significance of FOXO1 and PAX3 in cervical lesions. METHODS: In vitro assessment of cell function by cell viability, migration, and invasion assays were performed on FOXO1-knockdown cervical cancer cells. Immunohistochemical (IHC) staining analyses of FOXO1 and PAX3 were performed with a tissue microarray (TMA). The clinical significance was evaluated by comparing the data with various clinicopathologic characteristics, including survival of patients with cervical cancer. RESULTS: In vitro results revealed that knockdown of FOXO1 is associated with decreased cell viability (p < 0.001), migration (p < 0.001), and invasion (p < 0.05), supporting the oncogenic role of FOXO1 in cervical cancer. FOXO1 and PAX3 expression was significantly higher in CIN (both p < 0.001) and cancer tissue (both p < 0.001) than in normal tissue. Multivariate analysis indicated that FOXO1 expression (hazard ratio 4.01 [95% CI 1.22-13.10], p = 0.021) and an advanced FIGO stage (hazard ratio 3.89 [95% CI 1.35-11.19], p = 0.012) were independent prognostic factors for overall survival. CONCLUSIONS: This study reveals increased FOXO1 and PAX3 expression in cervical cancers and indicates an oncogenic role of FOXO1 in cervical cancer cells that correlates with poor patient survival.

Dual loss of USP10 and p14ARF protein expression is associated with poor prognosis in patients with small intestinal adenocarcinoma.

Oncogene-induced senescence occurs following oncogene activation in normal cells and is considered as a critical tumor-suppressing mechanism. Ubiquitin-specific protease 10 (USP10) has been reported to play a vital role in oncogene-induced senescence via the deubiquitination-dependent stabilization of p14ARF. However, knowledge of the clinical significance of USP10 and p14ARF expression in patients with small intestinal adenocarcinoma is limited. To study the clinical significance of USP10 and p14ARF expression, we performed immunohistochemistry for USP10 and p14ARF on 195 surgically resected small intestinal adenocarcinoma specimens. Furthermore, we performed methylation analysis on five small intestinal adenocarcinoma samples and matched adjacent normal intestinal tissue samples. UPS10 ( p = 0.023) and p14ARF ( p = 0.007) expression were significantly decreased in adenocarcinoma in comparison with normal tissue. The loss of USP10 was observed in 124/194 (63.9%) of small intestinal adenocarcinoma samples and was correlated with a higher pT stage ( p = 0.044), lymphatic invasion ( p = 0.033), and the absence of sporadic adenoma ( p = 0.024) and peritumoral dysplasia ( p = 0.019). p14ARF expression was downregulated in 75/195 (38.5%) of small intestinal adenocarcinoma samples and was associated with vascular ( p = 0.011) and lymphatic ( p = 0.013) invasions. The loss of USP10 expression was associated with the loss of p14ARF expression ( r = 0.342, p < 0.001). Multivariate survival analysis revealed that the combined loss of USP10 and p14ARF expression could be an independent prognostic factor for overall survival in small intestinal adenocarcinoma. Furthermore, the aberrant hypermethylation of the USP10 and p14ARF promoter could be a key mechanism for the downregulation of USP10 and p14ARF proteins in small intestinal adenocarcinoma. These findings suggest that the dual loss of USP10 and p14ARF could be used as a prognostic indicator of small intestinal adenocarcinoma.

Prognostic implication of programmed cell death 1 protein and its ligand expressions in endometrial cancer.

OBJECTIVE: Monoclonal antibodies targeting programmed cell death-1 (PD-1)/programmed death ligand 1 (PD-L1) demonstrated promising clinical response. The predictive/prognostic value of PD-1/PD-L1 immunohistochemistry (IHC) has been evaluated in many cancer types. However, the prognostic value of PD-1/PD-L1 IHC has not been evaluated in endometrial cancer. METHODS: We conducted a retrospective study to quantify the IHC CD8, PD-1, and PD-L1 expressions in immune cells at center of tumor (CT), invasive margin (IM), and/or tumor cell in 183 primary endometrial cancer samples from a single cohort, followed by their reciprocal combinations, including compartmental differences, and correlated them with overall survival (OS) and progression-free survival (PFS). RESULTS: In repeated Cox multivariable models adjusted by clinicoimmunopathologic factors, high CT-PD-L1 was an independent adverse prognostic factor for PFS in all patients and in the microsatellite-stable subgroup. Immune marker ratios revealed independently shorter PFS for high CT-PD-L1/CT-CD8 and CT-PD-L1/CT-PD-1 ratios. Classification of endometrial cancer into four groups based on CT-CD8 and CT-PD-L1 revealed significantly different survival among groups. CONCLUSIONS: The high PD-L1/CD8 ratio and the high expression of PD-L1 on immune cells were independent poor prognostic factors for PFS in endometrial cancer, providing insights into the tumor microenvironment.

A melanin-bleaching methodology for molecular and histopathological analysis of formalin-fixed paraffin-embedded tissue.

Removal of excessive melanin from heavily pigmented formalin-fixed paraffin-embedded (FFPE) melanoma tissues is essential for histomorphological and molecular diagnostic assessments. Although there have been efforts to address this issue, current methodologies remain complex and time-consuming, and are not suitable for multiple molecular applications. Herein, we have developed a robust and rapid melanin-bleaching methodology for FFPE tissue specimens. Our approach is based on quick bleaching (15 min) at high temperature (80 °C) with 0.5% diluted hydrogen peroxide (H2O2) in Tris-HCl, PBS, or Tris/Tricine/SDS buffer. Immunostaining for Ki-67 and HMB45 was enhanced by bleaching with 0.5% H2O2 in Tris/Tricine/SDS and Tris-HCl, respectively. In addition to histopathological applications, our approach also facilitates recovery of protein and nucleic acid from archival melanin-rich FFPE tissue sections. Protein extracted from bleached FFPE tissues was compatible with western blotting using anti-human GAPDH and AKT antibodies. Our bleaching condition significantly improved RNA quality compared with unbleached tissues without compromising the yield. Notably, the RNA/DNA obtained from bleached tissues was suitable for end point PCR and real-time quantitative RT-PCR. In conclusion, this improved melanin-bleaching method enhances and simplifies immunostaining procedures, and facilitates the use of melanin-rich FFPE tissues for histomorphological and PCR amplification-based molecular assays.

Clinical significance of OCT4 and SOX2 protein expression in cervical cancer.

BACKGROUND: Cancer stem cell markers have become a major research focus because of their relationship with radiation or chemotherapy resistance in cancer therapy. Cancer stem cell markers including OCT4 and SOX2 have been found in various solid tumors. Here, we investigate the expression and clinical significance of OCT4 and SOX2 in cervical cancer. METHODS: To define the clinical significance of OCT4 and SOX2 expression, we performed immunohistochemistry for OCT4 and SOX2 on 305 normal cervical epithelium samples, 289 cervical intraepithelial neoplasia samples, and 161 cervical cancer cases and compared the data with clinicopathologic factors, including survival rates of patients with cervical cancer. RESULTS: OCT4 and SOX2 expression was higher in cervical cancer than normal cervix (both p < 0.001). OCT4 overexpression was associated with lymphovascular space invasion (p = 0.045), whereas loss of SOX2 expression was correlated with large tumor size (p = 0.015). Notably, OCT4 and SOX2 were significantly co-expressed in premalignant cervical lesions, but not in malignant cervical tumor. OCT4 overexpression showed worse 5-year disease-free and overall survival rates (p = 0.012 and p = 0.021, respectively) when compared to the low-expression group, while SOX2 expression showed favorable overall survival (p = 0.025). Cox regression analysis showed that OCT4 was an independent risk factor (hazard ratio = 11.23, 95 % CI, 1.31 - 95.6; p = 0.027) for overall survival while SOX2 overexpression showed low hazard ratio for death (hazard ratio = 0.220, 95 % CI, 0.06-0.72; p = 0.013). CONCLUSIONS: These results suggest that OCT4 overexpression and loss of SOX2 expression are strongly associated with poor prognosis in patients with cervical cancer.

Prognostic Significance of AMP-Dependent Kinase Alpha Expression in Cervical Cancer.

OBJECTIVES: Cervical cancer is one of the most common gynecological malignancies worldwide, and its association with the AMP-activated protein kinase (AMPK) is still unknown. We aimed to investigate the clinical correlation between AMPK expression and cervical cancer. METHODS: The expression of AMPKα1, AMPKα2 and phosphorylated AMPKα (p-AMPKα) was determined immunohistochemically in 524 formalin-fixed, paraffin-embedded malignant and premalignant cervical tissues. Subsequently, associations with clinicopathological characteristics and patient survival were assessed. RESULTS: AMPKα2 expression was observed in the cytoplasm and nucleus, while expression of AMPKα1 and p-AMPKα was mainly observed in the cytoplasm. p-AMPKα expression increased during the normal-to-tumor transition of cervical carcinoma (p < 0.001), but, once cancer developed, the expression of AMPKα2 and p-AMPKα decreased in large-sized tumors when compared to smaller tumors (36 vs. 68%, p = 0.004 and 39 vs. 64%, p = 0.029, respectively). Notably, AMPKα2 expression was significantly associated with better disease-free survival (HR 0.29, 95% CI 0.10-0.86, p = 0.026). CONCLUSION: The AMPKα2 isoform showed potential as a favorable prognostic marker in cervical cancer. Therefore, additional studies are necessary to further clarify the complex contribution of AMPK isoforms and of phosphorylation status to cervical cancer progression and prognosis.

Expression of stress-induced phosphoprotein1 (STIP1) is associated with tumor progression and poor prognosis in epithelial ovarian cancer.

Stress-induced phosphoprotein1 (STIP1) is a candidate biomarker in epithelial ovarian cancer (EOC). In this study, we investigated in detail the expression of STIP1, as well as its functions, in EOC. STIP1 expression was assessed by immunohistochemistry (IHC) and the results were compared with clinicopathologic factors, including survival data. The effects of STIP1 gene silencing via small interfering RNA (siRNA) were examined in EOC cells and a xenograft model. The expression of STIP1 protein in EOC was significantly higher than in the other study groups (P < 0.001), and this increase of expression was significantly associated with tumor stage (P = 0.005), tumor grade (P = 0.029), and lymph node metastasis (P = 0.020). In multivariate analysis, overall survival in EOC was significantly shorter in cases with high STIP1 expression (HR = 2.78 [1.01-7.63], P = 0.047). STIP1 silencing in EOC cells resulted in inhibition of cell proliferation and invasion. In addition, in vivo experiments using STIP1 siRNA clearly showed a strong inhibition of tumor growth and a modulation of expression of prosurvival and apoptotic genes, further suggesting that STIP1 silencing can prevent cell proliferation and invasion. In conclusion, increased STIP1 expression is associated with poor survival outcome in EOC, and STIP1 may represent a useful therapeutic target in EOC patients.

HER-2 assessment in formalin-fixed paraffin-embedded breast cancer tissue by well-based reverse phase protein array.

BACKGROUND: The human epidermal growth factor receptor-2 (HER-2) expression level is a critical element for determining the prognosis and management of breast cancer. HER-2 targeted therapy in breast cancer depends on the reliable assessment of HER-2 expression status but current standard methods are lacking a rigorous quantitative assay. To address this challenge, we developed an assessment of HER-2 expression method by well-based reverse phase protein array (RPPA). RESULTS: Well-based RPPA is based on a robust protein isolation methodology paired with a novel electrochemiluminescence detection system. HER-2 value of well-based RPPA significantly correlated with dot blotting results (R(2) = 0.939). By well-based RPPA, we successfully detected HER-2 expression in 76 human breast formalin-fixed paraffin-embedded tissue samples. We observed 93.4% (71/76) concordance between well-based RPPA and current HER-2 immunohistochemical assessment guideline. When the cutoff level of HER-2 value was set to 0.689 (HER-2/GAPDH) on the basis of receiver-operating characteristic curve, the area under the curve was 0.975 (95% CI, 0.941-1.000). Sensitivity and specificity of well-based RPPA was 92.1% and 94.7%, respectively. CONCLUSIONS: HER-2 value by well-based RPPA was correlated with the current HER-2 status guideline, suggesting that this normalized HER-2 assessment may offer advantages over unnormalized current immunohistochemical assessment methods.

MICA/B and ULBP1 NKG2D ligands are independent predictors of good prognosis in cervical cancer.

BACKGROUND: NKG2D (natural killer group 2, member D) is thought to play an important role in mediating the activation of anticancer immune response. Expression of NKG2D ligands (NKG2DLs) is pronounced in malignancies and the heterogeneity of NKG2DL expression remains unclear. Here, we investigate the expression and clinical significance of NKG2DLs in cervical cancer. METHODS: Immunohistochemical analyses of MICA/B, ULBP1, ULBP2, ULBP3, RAET1E, and RAET1G were performed using tissue microarray analysis of 200 cervical cancers, 327 high-grade cervical intraepithelial neoplasias (CINs), 99 low-grade CINs, and 541 matched nonadjacent normal cervical epithelial tissues and compared the data with clinicopathologic variables, including the survival of cervical cancer patients. RESULTS: MICA/B, ULBP1, and RAET1E expression was higher in cervical cancer than in low-grade CIN (p<0.001, p=0.012, p=0.013, respectively) and normal cervix (all p<0.001). Among these markers, expression of ULBP1 was significantly different depending on patient tumor stage (p=0.010) and tumor size (p=0.045). ULBP1 expression was correlated with MICA/B (p<0.001) and ULBP2 (p=0.002) expression in cervical cancer. While MICA/B+ or ULBP1+ patients had improved disease-free survival time (p=0.027 and p=0.009, respectively) relative to that of the low expression group, RAET1E+ or RAET1G+ was correlated with shorter survival time (p=0.018 and p=0.029, respectively). However, in terms of overall survival, the ULBP1+ group had significantly longer survival time than the low expression group (p=0.009). Multivariate analysis indicated that MICA/B+/ULBP1+ (HR=0.16, p=0.015) and ULBP1+ (HR=0.31, p=0.024) are independent prognostic factors of disease-free survival in cervical cancer. CONCLUSIONS: High expression of either ULBP1 or MICA/B and ULBP1 combined is an indicator of good prognosis in cervical cancer, suggesting their potential utility as prognostic tests in clinical assessment.

Apoptosis inhibitor-5 overexpression is associated with tumor progression and poor prognosis in patients with cervical cancer.

BACKGROUND: The apoptosis inhibitor-5 (API5), anti-apoptosis protein, is considered a key molecule in the tumor progression and malignant phenotype of tumor cells. Here, we investigated API5 expression in cervical cancer, its clinical significance, and its relationship with phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) in development and progression of cervical cancer. METHODS: API5 effects on cell growth were assessed in cervical cancer cell lines. API5 and pERK1/2 immunohistochemical staining were performed on a cervical cancer tissue microarray consisting of 173 primary cervical cancers, 306 cervical intraepithelial neoplasias (CINs), and 429 matched normal tissues. RESULTS: API5 overexpression promoted cell proliferation and colony formation in CaSki cells, whereas API5 knockdown inhibited the both properties in HeLa cells. Immunohistochemical staining showed that API5 expression increased during the normal to tumor transition of cervical carcinoma (P < 0.001), and this increased expression was significantly associated with tumor stage (P = 0.004), tumor grade (P < 0.001), and chemo-radiation response (P = 0.004). API5 expression levels were positively associated with pERK1/2 in cervical cancer (P < 0.001) and high grade CIN (P = 0.031). In multivariate analysis, API5+ (P = 0.039) and combined API5+/pERK1/2+ (P = 0.032) were independent prognostic factors for overall survival. CONCLUSIONS: API5 expression is associated with pERK1/2 in a subset of cervical cancer patients and its expression predicts poor overall survival, supporting that API5 may be a promising novel target for therapeutic interventions.