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The number of deaths due to oral squamous cell carcinoma (OSCC), a malignant tumor of the oral cavity, is on the increase. We examined fibrinogen (FIB) expression in patients with OSCC and developed novel immunoprofile classification methods that include FIB. The plasma FIB level in patients with OSCC was elevated compared with that in patients with non-tumor oral disease (non-T); using a cut-off point of 342 mg/dL, we found the area under the curve-receiver operating characteristic level for OSCC was 0.745. Similarly, FIB expression in OSCC tissues was significantly higher compared with that in non-T tissues. Hierarchical clustering based on the immunoprofile of several markers including FIB, p53, and p16 revealed four groups that could be used to categorize OSCC cases (referred to as immunoprofile subtypes [IPS], I-IV). Tumors in IPS-II, which were FIB+/p53+, were associated with a significantly worse overall survival (OS) when compared with the other subtypes. We conclude that our IPS classification system can facilitate prognostic evaluation in OSCC, and that quantification of FIB is an important component of the classification strategy for this disease.

Background Although anaplastic lymphoma kinase (ALK) overexpression is overexpressed in several primary solid tumor types, its role in endometrial carcinoma (Em Ca) remains unclear. Methods We evaluated expression of ALK and its related molecules in clinical samples consisting of 168 Em Ca tissues. We also used Em Ca cell lines to evaluate the functional role of ALK. Results Cytoplasmic ALK immunoreactivity in the absence of chromosomal rearrangement was positively correlated with ALK mRNA expression, and was significantly higher in Grade (G) 3 Em Ca than in G1 or G2 tumors. ALK immunoreactivity was also significantly associated with expression of cancer stem cell (CSC)-related molecules (cytoplasmic CD133, ALDH1, Sox2) and neuroendocrine markers (CD56 and synaptophysin). Although the proliferative index was significantly higher in ALK-positive Em Ca when compared to ALK- negative malignancies, there was no association between ALK expression and other clinicopathological factors in this disease. In Em Ca cell lines, full-length ALK overexpression increased proliferation, decreased susceptibility to apoptosis, enhanced cancer stem cell features, and accelerated cell mobility, whereas these phenotypes were abrogated in ALK-knockdown cells. Finally, tumors with either wild-type ALK or high ALK mRNA expression were associated with a poorer prognosis when compared to Em Ca with either mutant ALK or low ALK mRNA expression. Conclusion Full-length ALK overexpression occurs in a subset of Em Ca, particularly in G3 tumors, and contributes to establishment and maintenance of aggressive phenotypic characteristics through modulation of several biological processes.

Background: Although a lack of functional PTEN contributes to tumorigenesis in a wide spectrum of human malignancies, little is known about the functional role of its overexpression in the tumors. The current study focused on PTEN overexpression in endometrial carcinoma (Em Ca). Methods: The functional impact of PTEN overexpression was assessed by Em Ca cell lines. Immunohistochemical analyses were also conducted using 38 Em Ca with morular lesions. Results: Em Ca cell lines stably overexpressing PTEN (H6-PTEN) exhibited epithelial-mesenchymal transition (EMT)-like features, probably through b-catenin/Slug-meditated suppression of E-cadherin. PTEN overexpression also inhibited cell proliferation, accelerated cellular senescence, increased apoptotic features, and enhanced migration capability. Moreover, H6-PTEN cells exhibited cancer stem cell (CSC)-like properties, along with high expression of aldehyde dehydrogenase 1 (ALDH1) and CD44s, a large ALDH 1high population, enriched spheroid formation, and b-catenin-mediated upregulation of cyclin D2, which is required for persistent CSC growth. In clinical samples, immunoreactivities for PTEN, as well as CSC-related molecules, were significantly higher in morular lesions as compared to the surrounding carcinomas. PTEN score was positively correlated with expression of nuclear b-catenin, cytoplasmic CD133, and CD44v6, and negatively with cell proliferation. Finally, estrogen receptor-a(ERa)-dependent expression of Ezrin-radixin-moesin-binding phophoprotein-50 (EBP50), a multifunctional scaffolding protein, acts as a negative regulator of morular formation by Em Ca cells through interacting with PTEN and b-catenin. Conclusion: In the abscess of ERa/EBP50 expression, PTEN overexpression and nuclear b-catenin stabilization promote the establishment and maintenance of morular phenotype associated with EMT/CSC-like features in Em Ca cells.

Patients with ovarian clear cell carcinoma (OCCC) experience frequent recurrence, which is most likely due to chemoresistance. We used shotgun proteomics analysis and identified upregulation of ezrin-binding phosphoprotein 50 (EBP50) in recurrent OCCC samples. Cytoplasmic and/or nuclear (Cyt/N), but not membranous, EBP50 immunoreactivity was significantly higher in recurrent OCCC as compared with that of primary tumors. OCCC cells expressing cytoplasmic EBP50 were significantly less susceptible to cisplatin (CDDP)-induced apoptosis compared with cells expressing membranous EBP50. Abrogation of resistance following knockdown of cytoplasmic EBP50 was accompanied by decreased XIAP and BCL2, increased BAX and increased caspase-3 cleavage. We found that poly (ADP-ribose) polymerase1 (PARP1), which is involved in DNA damage detection and repair, binds to EBP50 through its PDZ1 domain. CDDP treatment of cells expressing cytoplasmic (but not membranous) EBP50 increased nuclear PARP1 expression, whereas knockdown of EBP50 cells decreased PARP1 expression and activity following CDDP treatment. Finally, OCCC patients with a combination of Cyt/N EBP50 and high PARP1 score had worst the prognosis for overall and progression-free survival. Together, our data suggest that cytoplasmic EBP50 inhibits apoptosis and promotes OCCC survival through stabilization of PARP1 activity and modulation of the XIAP/BCL2/BAX axis. This may increase the likelihood of tumor recurrence, and we therefore suggest a combined analysis for EBP50 and PARP1 may have great utility in OCCC prediction and prognosis.

Glioblastomas (GBM) contain numerous hypoxic foci associated with a rare fraction of glioma stem cells (GSCs). Left-right determination factor (LEFTY) and Nodal, members of the transforming growth factor β (TGF-β) superfamily, have glycogen synthase kinase 3β (GSK-3β) phosphorylation motifs and are linked with stemness in human malignancies. Herein, we investigated the roles of LEFTY and Nodal in GBM hypoxic foci. In clinical samples, significantly higher expression of LEFTY, Nodal, phospho (p) GSK-3β, pSmad2, and Nestin, as well as higher apoptotic and lower proliferation rates, were observed in nonpseudopalisading (non-Ps) perinecrotic lesions as compared to Ps and non-necrotic tumor lesions, with a positive correlation between LEFTY, Nodal, pGSK-3β, or pSmad2 scores. In KS-1, a GBM cell line that lacks endogenous Nodal expression, treatment with the hypoxic mimetic CoCl2 increased LEFTY, pGSK-3β, and pSmad2 levels, but decreased pAkt levels. Moreover, the promoter for LEFTY, but not Nodal, was activated by Smad2 or TGF-β1, suggesting that overexpression of LEFTY and Nodal may be due to Akt-independent GSK-3β inactivation, with or without cooperation of the TGF-β1/Smad2 axis. LEFTY and Nodal overexpression increased proliferation rates and reduced susceptibility to CoCl2 -induced apoptosis, and increased the expression of epithelial-mesenchymal transition (EMT)/GSC-related markers. An increased ALDH1high population and more efficient spheroid formation was also observed in LEFTY-overexpressing cells. These findings suggest that LEFTY and Nodal may contribute to cell survival in non-Ps GBM perinecrotic lesions, leading to alterations in apoptosis, proliferation, or EMT/GCS features.

Background We previously demonstrated that ovarian high grade serous carcinomas (OHGSeCa) and ovarian clear cell carcinomas (OCCCa) with an HNF-1β+/p53+/ARID1A+ immunophenotype were associated with the worst unfavorable prognosis. To clarify the molecular mechanisms underlying this finding, we focused on alterations in the p53 signaling pathway in these tumors. Methods Changes in cell phenotype and function following knockdown of wild-type p53 (p53-KD) were assessed using OCCCa cells expressing endogenous HNF-1β and ARID1A. The prognostic significance of molecules that were deregulated following p53-KD was also examined using 129 OCCCa/OHGSeCa cases. Results p53-KD cells had increased expression of Snail, phospho-Akt (pAkt), and pGSK3β, and decreased E-cadherin expression, leading to epithelial-mesenchymal transition (EMT)/cancer stem cell (CSC) features. The cells also exhibited acceleration of cell motility and inhibition of cell proliferation and apoptosis. Next generation sequencing assay revealed that fibronectin (FN) expression was significantly increased in the p53 KD-cells, in line with our observation that wild-type p53 (but not mutant p53) repressed FN1 promoter activity. In addition, treatment of OCCCa cells with FN significantly increased cell migration capacity and decreased cell proliferation rate, independent of induction of EMT features. In clinical samples, FN/p53 scores were significantly higher in OCCCa/OHGSeCa with the HNF-1β+/p53+/ARID1A+ immunophenotype when compared to others. Moreover, high FN/high p53 expression was associated with the worst overall survival and progression-free survival in OCCCa/OHGSeCa patients. Conclusion These findings suggest that upregulation of FN following loss of p53 function may impact the biological behavior of OCCCa/OHGSeCa, particularly in tumors with HNF-1β+/p53+/ARID1A+ immunophenotype, through alterations in cell mobility and cell proliferation. The accompanying induction of EMT/CSC properties and inhibition of apoptosis due to p53 abnormalities also contribute to the establishment and maintenance of tumor phenotypic characteristics.

Background Glioblastoma (GBM) is the most aggressive form of brain tumor and has vascular-rich features. The S100A4/non-muscle myosin IIA (NMIIA) axis contributes to aggressive phenotypes in a variety of human malignancies, but little is known about its involvement in GBM tumorigenesis. Herein, we examined the role of the S100A4/NMIIA axis during tumor progression and vasculogenesis in GBM. Methods We performed immunohistochemistry for S100A4, NMIIA, and two hypoxic markers, hypoxia-inducible factor-1α (HIF-1α) and carbonic anhydrase 9 (CA9), in samples from 94 GBM cases. The functional impact of S100A4 knockdown and hypoxia were also assessed using a GBM cell line. Results In clinical GBM samples, overexpression of S100A4 and NMIIA was observed in both non-pseudopalisading (Ps) and Ps (-associated) perinecrotic lesions, consistent with stabilization of HIF-1α and CA9. CD34(+) microvascular densities (MVDs) and the interaction of S100A4 and NMIIA were significantly higher in non-Ps perinecrotic lesions compared to those in Ps perinecrotic areas. In non-Ps perinecrotic lesions, S100A4(+)/HIF-1α(−) GBM cells were recruited to the surface of preexisting host vessels in the vascular-rich areas. Elevated vascular endothelial growth factor A (VEGFA) mRNA expression was found in S100A4(+)/HIF-1α(+) GBM cells adjacent to the vascular-rich areas. In addition, GBM patients with high S100A4 protein expression had significantly worse OS and PFS than did patients with low S100A4 expression. Knockdown of S100A4 in the GBM cell line KS-1 decreased migration capability, concomitant with decreased Slug expression; the opposite effects were elicited by blebbistatin-dependent inhibition of NMIIA. Conclusion S100A4(+)/HIF-1α(−) GBM cells are recruited to (and migrate along) preexisting vessels through inhibition of NMIIA activity. This is likely stimulated by extracellular VEGF that is released by S100A4(+)/HIF-1α(+) tumor cells in non-Ps perinecrotic lesions. In turn, these events engender tumor progression via acceleration of pro-tumorigenic vascular functions.

Background S100A1 expression is deregulated in a variety of human malignancies, but its role in normal and malignant endometrial cells is unclear. Methods We used endometrial carcinoma (Em Ca) cell lines to evaluate the physical and functional interaction of S100A1 with p53 and its negative regulator, mouse double minute 2 (MDM2). We also evaluated the expression of S100A1, p53, and MDM2 in clinical samples consisting of 89 normal endometrial and 189 Em Ca tissues. Results S100A1 interacted with MDM2 but not p53 in Em Ca cell lines. Treatment of cells stably overexpressing S100A1 with Nutlin-3A, an inhibitor of the p53/MDM2 interaction, increased expression of p53-target genes including p21waf1 and BAX. S100A1 overexpression enhanced cellular migration, but also sensitized cells to the antiproliferative and proapoptotic effects of Adriamycin, a genotoxic agent; these phenotypes were abrogated when S100A1 was knocked down using shRNA. In clinical samples from normal endometrium, S100A1 expression was significantly higher in endometrial glandular cells of the middle/late secretory and menstrual stages when compared to cells in the proliferative phases; high S100A1 was also positively correlated with expression of MDM2 and p21waf1 and apoptotic status, and inversely correlated with Ki-67 scores. However, such correlations were absent in Em Ca tissues. Conclusion The interaction between S100A1 and MDM2 may modulate proliferation, susceptibility to apoptosis, and migration through alterations in p53 signaling in normal- but not malignant-endometrial cells.

Although proteomic profiles for ovarian epithelial carcinoma (OECa) have been widely investigated, no single marker or set of predictors has been clinically implemented mainly because their reliability and validity have not yet been well established. To establish immunohistochemical (IHC) panels for prognosis prediction of OECa for use in daily pathology practice, the expression patterns of 12 IHC markers, p53, HNF-1β, ARID1A, estrogen receptor-α, progesterone receptor, vimentin, PTEN, PIK3CA, WT1, left-right determination factor, β-catenin, and Ki-67 were investigated using 282 OECas. Hierarchical clustering analysis revealed 7 major immunoprofile groups (IPGs I-VII) that could be used to categorize OECa tumors independent of histotypes. Based on the results of the cluster analysis and protein expression statuses, we further demonstrated the effective classification of OECa tumors into simplified immunoprofile panels using only 4 IHC markers including HNF-1β, p53, ARID1A, and WT1. The tumors in IPG VII with HNF1β+/p53+/ARID1A+ immunophenotype demonstrated a significantly worse overall survival and progression-free survival as compared with the other IPGs. Multivariate Cox regression analysis also revealed that the immunophenotype (HNF1β+/p53+/ARID1A+) and clinical stage were significant and independent prognostic factors for overall survival and progression-free survival in advanced OECa. In conclusion, we identified immunoprofiles in OECa using a panel of 4 IHC markers, which could identify tumors by the immunophenotype that is associated with the most unfavorable prognosis and thus facilitate prognosis prediction of advanced OECa.

Deregulated full-length anaplastic lymphoma kinase (ALK) overexpression has been found in some primary solid tumors, but little is known about its role in ovarian high-grade serous carcinoma (HGSC). The current study focused on the functional roles of ALK in HGSC. Cytoplasmic ALK immunoreactivity without chromosomal rearrangement and gene mutations was significantly higher in HGSC compared with non-HGSC-type ovarian carcinomas, and was significantly associated with several unfavorable clinicopathologic factors and poor prognosis. HGSC cell lines stably overexpressing ALK exhibited increased cell proliferation, enhanced cancer stem cell features, and accelerated cell mobility, whereas these phenotypes were abrogated in ALK-knockdown cells. Expression of the nervous system-associated gene, ELAVL3, and the corresponding protein (commonly known as HuC) was significantly increased in cells overexpressing ALK. Expression of SRY-box transcription factor (Sox)2 and Sox3 (genes associated with the neural progenitor population) increased in ALK-overexpressing but not ALK-knockdown cells. Furthermore, overexpression of Sox2 or Sox3 enhanced both ALK and ELAVL3 promoter activities, suggesting the existence of ALK/Sox/HuC signaling loops. Finally, ALK overexpression was attributed to increased expression of neuroendocrine markers, including synaptophysin, CD56, and B-cell lymphoma 2, in HGSC tissues. These findings suggest that overexpression of full-length ALK may influence the biological behavior of HGSC through cooperation with ELAVL3 and Sox factors, leading to the establishment and maintenance of the aggressive phenotypic characteristics of HGSC.

S100A4 is a small calcium-binding protein that exerts its biological functions by interacting with nonmuscle myosin IIA (NMIIA) and p53. Although S100A4 promotes metastasis in several tumors, little is known about its involvement in the progression of ovarian high-grade serous carcinomas (HGSCs). Herein, we focused on functional roles of the S100A4/NMIIA/p53 axis in these tumors. In HGSC cell lines harboring mutant p53, knockdown (KD) of S100A4 reduced the expression of several epithelial-mesenchymal transition/cancer stem cell markers and the ALDH1high population, consistent with an inhibition of stemness features. S100A4-KD also increased apoptosis, decreased cell proliferation, and accelerated cell mobility. This was accompanied by increased Snail expression, which, in turn, was likely due to loss of p53 function. In contrast, specific inhibition of NMIIA by blebbistatin induced phenotypes that-with the exception of cell proliferation and mobility-were opposite to those observed in S100A4-KD cells. In clinical samples, cytoplasmic and/or nuclear interactions between S100A4, NMIIA, and mutant p53 were observed. In addition, high expression of S100A4, but not NMIIA or p53, was a significant and independent unfavorable prognostic factor in HGSC patients. These findings suggest that, via its interaction with NMIIA and p53, overexpressed S100A4 may induce epithelial-mesenchymal transition/cancer stem cell properties in HGSC and elicit several other tumor-associated phenotypes.

We established the KU-Lu-8 monoclonal antibody (MoAb) using a lung cancer cell line as an antigen and a random immunization method. The KU-Lu-8 MoAb recognizes basigin (BSG), which is a transmembrane-type glycoprotein that is strongly expressed on the cell membranes of lung cancer cells. This study aimed to clarify the relationships between BSG expression and clinicopathological parameters and determine the prognostic significance of BSG expression in pulmonary adenocarcinoma (AC) patients. To evaluate the significance of BSG expression in lung cancer, we immunohistochemically analyzed 113 surgically resected pulmonary adenocarcinomas, and the associations between BSG expression and various clinicopathological parameters were evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to investigate the effects of BSG expression on survival. Clinicopathologically, BSG expression was significantly associated with tumor differentiation, vascular invasion, lymphatic invasion, and a poor prognosis. In particular, BSG expression was significantly correlated with poorer survival in patients with stage I AC. The high BSG expression group (compared with the low BSG expression group) exhibited adjusted hazard ratios for mortality of 4.694. BSG expression is indicative of a poor prognosis in AC patients, particularly in those with stage I disease.

// Masashi Akiya 1 , Masaaki Yamazaki 1 , Toshihide Matsumoto 1 , Yusuke Kawashima 2 , Yasuko Oguri 1 , Sabine Kajita 1 , Daiki Kijima 1 , Risako Chiba 1 , Ako Yokoi 1 , Hiroyuki Takahashi 1 , Yoshio Kodera 2 and Makoto Saegusa 1 1 Department of Pathology, Kitasato University School of Medicine, Sagamihara, Kanagawa 252-0374, Japan 2 Center for Disease Proteomics, School of Science, Kitasato University, Sagamihara, Kanagawa 252-0374, Japan Correspondence to: Makoto Saegusa, email: msaegusa@med.kitasato-u.ac.jp Keywords: LEFTY, ovarian clear cell carcinoma, cell proliferation, apoptosis, TGF-beta Received: November 22, 2016 Accepted: June 11, 2017 Published: June 29, 2017 ABSTRACT To identify proteins involved in ovarian clear cell carcinoma (OCCCa), shotgun proteomics analysis was applied using formalin-fixed and paraffin-embedded samples of ovarian carcinoma. Analysis of 1521 proteins revealed that 52 were differentially expressed between four OCCCa and 12 non-OCCCa samples. Of the highly expressed proteins in OCCCa, we focused on left-right determination factor (LEFTY), a novel member of the transforming growth factor-β superfamily. In 143 cases of ovarian epithelial carcinoma including 99 OCCCas and 44 non-OCCCas, LEFTY expression at both mRNA and protein levels was significantly higher in OCCCas compared with non-OCCCas, with the mRNA expression of LEFTY1 being predominant compared to that of LEFTY2 . OCCCa cells stably overexpressing LEFTY1 showed reduced cell proliferation, along with decreased pSmad2 expression, and also either displayed an activated p53/p21 waf1 pathway or increased p27 kip1 expression, directly or indirectly. Moreover, the treatment of stable cell lines with cisplatin led to increased apoptotic cells, together with the inhibition of protein expression of a pSmad2-mediated X-linked inhibitor of apoptosis and a decreased bcl2/bax ratio. Blocking LEFTY1 expression with a specific short hairpin RNA inhibited cisplatin-induced apoptosis, probably through the increased expression of both XIAP and bcl2, but not bax. In clinical samples, a significantly higher number of apoptotic cells and lower Ki-67 labeling indices were observed in OCCCas with a high LEFTY score relative to those with a low score. These findings suggest that LEFTY may be an excellent OCCCa-specific molecular marker, which has anti-tumor effects in altering cell proliferation and cellular susceptibility to apoptosis.

Uterine carcinosarcoma (UCS) represents a true example of cancer associated with epithelial-mesenchymal transition (EMT), which exhibits cancer stem cell (CSC)-like traits. Although S100A4 is an inducer of EMT, little is known about its involvement in UCS tumorigenesis. Herein, we focused on the functional role of S100A4 during development of UCS. Expression of S100A4 and molecules associated with its function were also examined in 35 UCS cases. In endometrial carcinoma cell lines, S100A4 promoter activity and mRNA levels were significantly increased by the transfection of NF-κB/p65, independent of a putative κB-binding site in the promoter. Cells stably overexpressing S100A4 showed enhancement of CSC properties, along with decreased cell proliferation and acceleration of cell migration. These phenotypes were abrogated in S100A4-knockdown cells. A combination of S100A4 antibody-mediated co-immunoprecipitation and shotgun proteomics analysis revealed that S100A4 strongly interacted with non-muscle myosin II (NMII) heavy chains, including myosin 9 and myosin 14. Specific inhibition of NMII by blebbistatin phenocopied S100A4 overexpression and induced a fibroblast-like morphology. In clinical samples, S100A4 score was significantly higher in sarcomatous as compared with carcinomatous components of UCS, and was positively correlated with ALDH1, Slug, and vimentin scores, and inversely with Ki-67 labeling indices. These findings suggest that an S100A4/NMII-related signaling cascade may contribute to the establishment and maintenance of EMT/CSC properties, along with changes in cell proliferation and migration capability. These events may be initiated in carcinomatous components in UCS and lead to divergent sarcomatous differentiation. The S100A4/non-muscle myosin II-related signal cascade may contribute to the establishment and maintenance of epithelial mesenchymal transition/cancer stem cell properties, along with changes in cell proliferation and migration capability. These events may be initiated in carcinomatous components in uterine carcinosarcoma and lead to divergent sarcomatous differentiation.

We focused on specific molecular events during the development of endometriosis-associated ovarian endometrioid carcinoma (OEmCa) and ovarian clear cell carcinoma (OCCCa). Alterations in β-catenin (encoded by CTNNB1) and microsatellite instability (MSI), as well as changes in the expression levels of HNF-1β and DNA mismatch repair (MMR) proteins were investigated in 50 OEmCas and 21 OCCCas with endometriotic lesions. Mutations of CTNNB1 were identified in 28 (56%) of the 50 OEmCa cases and 26 (41.9%) of the 62 coexisting endometriosis lesions. MSI-high (H) was observed in 7 (14.6%) of the 48 OEmCa and 14 (23.3%) of the 60 coexisting endometriosis, and was significantly associated with loss of MMR protein expression, but not CTNNB1 mutations. Nonidentical CTNNB1 status between 2 different epithelial lesions within endometriosis was observed in 8 of 10 informative endometriosis cases that had adjacent OEmCa. Similar findings for MSI features were also found in 2 of 3 informative cases, suggesting that endometriotic lesions may predominantly consist of polyclonal cells. In contrast, high HNF-1β expression was significantly associated with SLC3A1 expression, which plays a major role in HNF-1β-triggered induction of reactive oxygen species in OCCCas, independent of abnormalities in both β-catenin and MSI/MMR status. Finally, 4 inflammatory parameters associated with repeated hemorrhaging in endometriosis were significantly higher in endometriosis with MSI-high when compared with that with MSS, independent of both β-catenin and HNF-1β status. In conclusion, different molecular pathways including alterations in β-catenin, MSI, and HNF-1β levels may contribute to tumorigenesis in endometriosis-associated carcinoma.

BackgroundRotator cuff tears (RCTs) are often associated with severe shoulder pain. Non-steroidal anti-inflammatory drugs, not recommended for long-term use, do not effectively manage RCT-induced pain, resulting in reduced quality of life. To improve management, a better understanding of the fundamental properties of RCT pain is needed. Here, we aimed to compare the expression levels of nerve growth factor (NGF) and cyclooxygenase-2 (COX-2) mRNA in the synovial tissues of patients with RCT-induced pain and patients with non-painful recurrent shoulder dislocation (RSD).MethodsThe study included 32 patients with RCT who underwent arthroscopic rotator cuff repair and 28 patients with non-painful RSD who underwent arthroscopic Bankart repair. Synovial tissue samples were harvested from subacromial bursa and rotator interval of RCT patients and from the rotator interval of RSD patients. Samples were analyzed quantitatively expression levels for NGF and COX2 mRNA and NGF protein.ResultsNGF mRNA and protein levels were significantly higher in the rotator interval of RCT patients than in the rotator interval of RSD patients ( p = 0.0017, p = 0.012, respectively), while COX2 mRNA levels did not differ significantly between the two patient groups. In RCT patients, COX2 mRNA was more highly expressed in the rotator interval than in the subacromial bursa ( p = 0.038), whereas the mRNA and protein levels of NGF did not differ between the two tissues. The expression of NGF mRNA in the synovium of the rotator interval was significantly correlated with the numeric rating scale of pain (ρ = 0.38, p = 0.004).ConclusionNGF mRNA and protein levels were elevated in patients with painful RCT compared with those in patients with non-painful RSD, whereas COX-2 levels were comparable in the two patient groups. These findings provide insights into novel potential strategies for clinical management of RCT.

Background Research suggests that vascular endothelial growth factor (VEGF) levels in the synovial fluid of knee osteoarthritis (KOA) patients are positively correlated with KOA severity. The relationship between synovial VEGF levels and pain in human KOA patients is not fully understood, and the role of VEGF in the pain pathway remains unclear. Methods We harvested synovial membrane (SM) from 102 patients with radiographic evidence of KOA (unilateral Kellgren/Lawrence [K/L] grade 2–4) during total knee arthroplasty. Patients scored their pain on a 0 to 10 cm visual analog scale (VAS). VEGF levels in the SM of KOA patients with strong/severe (VAS ≥ 6) and mild/moderate pain (VAS

Background The left-right determination factor (LEFTY) is a novel member of the TGF-β/Smad2 pathway and belongs to the premenstrual/menstrual repertoire in human endometrium, but little is known about its functional role in endometrial carcinomas (Em Cas). Herein, we focused on LEFTY expression and its association with progesterone therapy in Em Cas. Methods Regulation and function of LEFTY, as well as its associated molecules including Smad2, ovarian hormone receptors, GSK-3β, and cell cycle-related factors, were assessed using clinical samples and cell lines of Em Cas. Results In clinical samples, LEFTY expression was positively correlated with estrogen receptor-α, but not progesterone receptor (PR), status, and was inversely related to phosphorylated (p) Smad2, cyclin A2, and Ki-67 levels. During progesterone therapy, expression of LEFTY, pSmad2, and pGSK-3β showed stepwise increases, with significant correlations to morphological changes toward secretory features and decreased Ki-67 values. In Ishikawa cells, an Em Ca cell line that expresses PR, progesterone treatment reduced proliferation and induced increased expression of LEFTY and pGSK-3β, although LEFTY promoter regions were inhibited by transfection of PR. Moreover, inhibition of GSK-3β resulted in increased LEFTY expression through a decrease in its ubiquitinated form, suggesting posttranslational regulation of LEFTY protein via GSK-3β suppression in response to progesterone. In addition, overexpression or knockdown of LEFTY led to suppression or enhancement of Smad2-dependent cyclin A2 expression, respectively. Conclusion Upregulation of LEFTY may serve as a useful clinical marker for the therapeutic effects of progesterone for Em Cas, leading to inhibition of tumor cell proliferation through alteration in Smad2-dependent transcription of cyclin A2. Electronic supplementary material The online version of this article (10.1186/s12964-017-0211-0) contains supplementary material, which is available to authorized users.

Pseudopalisades (Ps) around necrotic foci are severely hypoxic and overexpress hypoxia-inducible factor (HIF) in glioblastoma (GBM). Hypoxic regions have been proposed as one of several distinct niches for cancer stem cells (CSCs) in GBM, but little is known about the association between Ps features and CSC properties. Herein, we focused on the biological role of Ps lesions. In clinical cases of GBM, expression of hypoxia-related molecules including HIF-1α, Glut-1, p27(Kip1), and pAkt was significantly increased in perinecrotic Ps lesions compared with nonnecrotic areas and perinecrotic lesions lacking Ps features. Significantly higher expression levels of several CSC-related markers, including CD133, Sox2, CD44s, and aldehyde dehydrogenase (ALDH) 1, were also observed in Ps lesions, which were positively correlated with expression of hypoxia-related molecules and pAkt. Ps lesions also showed increased number of apoptotic cells and decreased bcl-2 and survivin expression compared with the surrounding tissue. Short-term exposure of astrocytoma cell lines to cobalt chloride, which is known to mimic the effect of hypoxia, caused an increase in expression of both hypoxia- and CSC-related markers, in line with increases in the ALDH(high) cell population and number of spheroids. Inhibition of endogenous Akt by LY294002 resulted in decreased expression of Sox2, ALDH1, and CD133, leading to enhancement of cobalt chloride-mediated apoptotic events due to altered ratio of bcl-2 to bax expression. These findings suggest that Ps lesions within GBM may serve as a specialized hypoxic niche, in which the HIF-1α/pAkt axis is activated, in response to severe hypoxia.

Hepatocyte nuclear factor-1β (HNF-1β) is a transcriptional factor that has an important role in endometriosis-ovarian clear cell carcinoma (OCCC) sequence by modulating cell kinetics and glucose metabolism. However, little is known about the detailed molecular mechanisms that govern its regulation and function. Herein, we focus on upstream and downstream regulatory factors of HNF-1β in OCCCs. In clinical samples, HNF-1β expression was positively correlated with the active form of NF-κB/p65 in OCCCs, and closely linked with a low nuclear grade and non-solid architecture. In cell lines, transfection of p65 resulted in increased HNF-1β mRNA and protein expression in TOV-21G cells (OCCC cell line with endogenous HNF-1β expression), in line with activation of the promoter, probably through interacting with the basic transcriptional machinery. Suppression of endogenous HNF-1β expression by siRNA increased apoptosis in TOV-21G cells, while treatment of Hec251 cells (endometrial carcinoma cell line with extremely low endogenous HNF-1β expression) stably overexpressing exogenous HNF-1β with doxorubicin abrogated apoptosis of the cells, along with increased ratio of bcl-2 relative to bax. Moreover, overexpression of HNF-1β led to upregulation of bcl-2 expression at the transcriptional level in TOV-21G cells, which provided evidence for a positive correlation between HNF-1β and bcl-2 expression in OCCCs. These data, therefore, suggest that association between HNF-1β and NF-κB signaling may participate in cell survival by alteration of apoptotic events, particularly in mitochondria-mediated pathways, through upregulation of bcl-2 expression in OCCCs.

The factors conferring the increased malignancy on lung adenocarcinoma with micropapillary component (AC-MPC) remain to be elucidated. On proteomics based on 2-dimensional gel electrophoresis, 19 proteins differentially expressed by more than 1.5-fold between AC-MPC and conventional adenocarcinoma (CAC); in particular, vimentin, one of the proteins, was 3.5-fold up-regulated in AC-MPC. Subsequent semi-quantitative investigation by immunohistochemistry with large cohorts comprised 101 AC-MPC and 119 CAC, respectively, of different stages revealed that vimentin was expressed in MPC of 95 (94.1%) AC-MPC and the expression scores were higher than those of well- and moderately differentiated CAC, as well as the background non-MPC of the AC-MPC (P < 0.0001), but not significantly different from those of poorly differentiated CAC (P = 0.561). Even within the AC-MPC entity, higher vimentin expression was correlated with more frequent vascular invasion and more advanced node metastasis (P < 0.02), and multivariate analysis showed that high vimentin expression and worse node statuses were independent indicators of adverse prognosis (P < 0.048). In conclusion, vimentin expression is prevalent and markedly up-regulated in MPC, which might reflect the biological essence of poorer differentiation or dedifferentiation of MPC, and this might have a role in the acquisition and increase of invasiveness and consequent more malignant nature of MPC.

Summary Nerve growth factor (NGF) functions to modulate osteoarthritis (OA)-associated pain. Although recent studies suggest that tumour necrosis factor (TNF)-α and interleukin (IL)-1β mediate NGF activity in human synovial fibroblasts, the regulation of NGF expression in human synovial macrophages remains unclear. Here, we examined the role of macrophages in the production and regulation of synovial (SYN) NGF in osteoarthritic knee joints by examining the mRNA expression of TNF-α and IL-1β in freshly isolated CD14-positive (macrophage-rich fraction) and CD14-negative cells (fibroblast-rich fraction) in synovial tissue from OA patients by quantitative polymerase chain reaction. We also examined the effects of IL-1β and TNF-α on NGF mRNA expression in cultured CD14-positive (macrophage-rich fraction) and CD14-negative cells (fibroblast-rich fraction). In addition, to examine the contribution of macrophages to NGF, TNF-α and IL-1β expression, we injected clodronate liposomes systemically into STR/Ort mice, an osteoarthritis animal model, to deplete macrophages. TNF-α and IL-1β mRNA levels in CD14-positive cells from the SYN of OA patients was significantly higher than that in CD14-negative cells, while NGF expression did not differ markedly between the two cell fractions. In addition, treatment of human cultured CD14-positive and -negative cells with IL-1β and TNF-α enhanced NGF mRNA and protein levels. Expression of NGF, IL-1β and TNF-α was also reduced significantly in STR/Ort mice upon macrophage depletion. These findings suggest that IL-1β and TNF-α regulate NGF expression and production in synovial macrophages and fibroblasts in osteoarthritic joints.

We established the KU-Lu-8 monoclonal antibody (MoAb) using a lung cancer cell line as an antigen and a random immunization method. The KU-Lu-8 MoAb recognizes basigin (BSG), which is a transmembrane-type glycoprotein that is strongly expressed on the cell membranes of lung cancer cells. This study aimed to clarify the relationships between BSG expression and clinicopathological parameters and determine the prognostic significance of BSG expression in pulmonary adenocarcinoma (AC) patients. To evaluate the significance of BSG expression in lung cancer, we immunohistochemically analyzed 113 surgically resected pulmonary adenocarcinomas, and the associations between BSG expression and various clinicopathological parameters were evaluated. Kaplan-Meier survival analysis and Cox proportional hazards models were used to investigate the effects of BSG expression on survival. Clinicopathologically, BSG expression was significantly associated with tumor differentiation, vascular invasion, lymphatic invasion, and a poor prognosis. In particular, BSG expression was significantly correlated with poorer survival in patients with stage I AC. The high BSG expression group (compared with the low BSG expression group) exhibited adjusted hazard ratios for mortality of 4.694. BSG expression is indicative of a poor prognosis in AC patients, particularly in those with stage I disease.

Background Recent studies have suggested that the tumor necrosis factor-α (TNF-α) pathway is a potential target for the management of osteoarthritis (OA). Transforming growth factor (TGF)-β-activated kinase 1 (TAK1) is essential in several cytokine-mediated cascades, including the TNF-α, interleukin-1 (IL-1), and TGF-β pathways. The role of TAK1 in synovial tissue in OA is not fully understood. Using synovial cells harvested from OA patients during surgery, we investigated whether TAK1 inhibition suppresses production of TNF-α-induced extracellular matrix degrading enzymes and expression of pain-related molecules. Methods Synovial tissues were harvested from ten subjects with radiographic evidence of osteoarthritis (OA) during total knee arthroplasty. Synovial cells were cultured and stimulated with control (culture media), 10 ng/mL human recombinant TNF-α, or 10 ng/mL TNF-α and 10 μM of the TAK1 inhibitor (5Z)-7-oxozeaenol for 24 h. Real-time polymerase chain reaction (PCR) analysis was used to monitor expression of mRNA of the extracellular matrix degrading enzymes matrix metalloproteinase-3 (MMP-3) and a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 4 (ADAMTS-4); and of the pain-related molecules cyclooxygenase-2 (COX-2), microsomal prostaglandin E synthase-1 (mPGES-1), and nerve growth factor (NGF). MMP-3 and NGF protein concentrations in cell supernatant were measured by enzyme-linked immunosorbent assay (ELISA). COX-2, mPGES-1 and ADAMTS-4 protein expression was also evaluated by western blotting. Results TNF-α stimulated increases in ADAMTS-4 and MMP3 mRNA (2.0-fold and 1.6-fold, respectively, p

Summary Atypical polypoid adenomyoma (APA) is an uncommon polypoid lesion of the uterus. To clarify the mechanism of its histogenesis, we examined the functional role of β -catenin, with reference to expression of p21 waf1 , cyclin D1, cyclin E, CD10, and α –smooth muscle actin (SMA), as well as cell proliferation, in 7 lesions. In the epithelial components, expression of nuclear β -catenin, p21 waf1 , and cyclin D1 was increased in a stepwise fashion from normal tissue through complex atypical hyperplasia and adenomyoma to APA lesions, particularly in squamous morular areas, whereas cell proliferation, as well as cyclin E expression, was significantly decreased in the latter. Similar findings were evident in the stromal lesions, with the exception of a case of nuclear β -catenin. In addition, coexpression of CD10 and α -SMA markers was observed in the stromal components in 3 APA cases, in line with the results of normal secretory endometrial and adenomyoma samples, suggesting that cells progress to myofibromatous cells in response to differentiation-promoting events. Finally, β-catenin gene ( CTNNB1 ) mutations were detected in all APA cases, the single nucleotide substitutions being in the epithelial but not the stromal components. These findings suggest that activation of β -catenin signaling, probably secondary to the gene abnormalities, plays an important role in the formation of the complex epithelial architecture in APAs, leading to inhibition of cell proliferation through overexpression of p21 waf1 . In contrast, changes in the stromal cell phenotype may occur through a shift from CD10 to α -SMA immunopositivity, independent of CTNNB1 status.

Uterine carcinosarcomas (U-CSs) are considered monoclonal in origin, but little is known about the mechanisms for establishment of heterologous sarcomatous components. Here, we examine the functional roles of nuclear factor κB (NF-κB)/p65 and Sox9 in the transcriptional regulation of alpha-1 type II collagen (COL2A1), a hallmark of chondrogenesis, during morphologic change in the direction of the chondrocytic phenotype. In 32 cases of U-CS, both phosphorylated p65 and Sox9 expression were colocalized in Col2A1-positive sarcomatous components, particularly in cartilaginous elements, with strongly positive correlation (ρ = 0.72, P = .005). A positive association of Col2A1 expression between protein (immunohistochemistry) and messenger RNA (in situ hybridization) assays was evident in sarcomatous components, whereas 9 cases also showed distinct positive signals for the messenger RNA without protein expression in carcinomatous elements, probably through a posttranscriptional and/or posttranslational modulation mechanism. In the Ishikawa endometrial cancer line, overexpression of p65 could activate transcription of COL2A1 promoter-intron reporters through binding to specific NF-κB sites in the first intron, along with up-regulation of Sox9. Exogenous induction of Sox9 also caused an increase in transcription of COL2A1, in contrast to a repression of the p65-mediated COL2A1 transcription, suggesting the existence of a negative feedback loop. These data, therefore, suggest that NF-κB/p65 signaling, as well as Sox9, may contribute to changes in the morphology of U-CS cells toward the chondrocytic phenotype through modulation of COL2A1 transcription.

Aims β-Catenin signalling participates in the regulation of epithelial-mesenchymal transition (EMT)/cancer stem cell (CSC) properties. The aim of this study was to investigate the role of β-catenin in resistance to neoadjuvant chemoradiotherapy in patients with rectal cancer, especially pertaining to its association with EMT/CSC features. Methods and results A total of 109 cases of locally advanced rectal cancer, along with a colon cancer cell line, were investigated. Nuclear β-catenin accumulation in pretreatment-biopsied samples was inversely associated with the therapeutic efficacy of chemoradiotherapy in resected rectal cancer. In resected tumours, nuclear β-catenin was predominantly observed in EMT-like lesions with decreased E-cadherin and increased Snail expression, along with expression of CSC-related markers. The EMT-like lesions also showed significant decreases in both apoptosis and cell proliferation as compared with non-EMT lesions. In-vitro culture of a colon cancer cell line in STK2 was sufficient to induce EMT/CSC properties together with nuclear β-catenin accumulation, and showed inhibition of cell proliferation and resistance to doxorubicin treatment. Conclusion Nuclear β-catenin accumulation may contribute to chemoradioresistance in locally advanced rectal cancer, probably through its regulation of EMT/CSC properties. In addition, nuclear β-catenin in pretreatment-biopsied samples is useful in predicting the efficacy of chemoradiotherapy in patients with rectal cancer.

To clarify the biological differences between small cell lung carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC), we investigated the expression of two bHLH type transcription factors, human achaete-scute homolog 1 (hASH1) and hairy/enhancer of split 1 (HES1), which positively and negatively regulate the neuroendocrine differentiation of respiratory epithelial cells, respectively. Eighty-eight formalin-fixed and paraffin-embedded pulmonary carcinomas (32 SCLC, 32 LCNEC, 14 adenocarcinomas, and 10 squamous cell carcinomas) and 14 SCLC and 1 LCNEC derived cell lines were used. hASH1 and HES1 mRNA were detected using a highly sensitive in situ hybridization method with digoxigenin-labeled cRNA probes and biotinylated tyramide. The staining results were scored from 0 to 12 by multiplying the staining intensity by the percentage of positive tumor cells. The mean staining score of hASH1 mRNA was significantly higher in SCLC than in LCNEC (p