Nucleobindin 2 inhibits senescence in gastric carcinoma.

Here, we focused on the role of Nucleobindin 2 (NUCB2), a multifunctional protein, in gastric carcinoma (GC) progression. NUCB2 expression was investigated in 150 GC cases (20 non-invasive (pT1) and 130 invasive (pT2/pT3/pT4) tumors) by immunohistochemistry (IHC), and in situ hybridization for detection of the mRNA in 21 cases. Using GC cell lines, we determined whether NUCB2 expression was associated with specific cellular phenotypes. In GC clinical samples, NUCB2 was transcriptionally upregulated when compared to normal tissues. High NUCB2 expression was associated with clinicopathological factors including deep tumor invasion, lymphovascular invasion, lymph node metastasis, and advanced clinical stages, and was a significant independent predictor of unfavorable progression-free survival in 150 non-invasive and invasive GC patients. Similar findings were also evident in 72 invasive GC cases in which patients received post-operative chemotherapy, but not in 58 invasive tumors from patients who did not receive the chemotherapy. In cell lines, NUCB2 knockout inhibited proliferation, susceptibility to apoptosis, and migration capability by inducting cellular senescence; this was consistent with higher proliferation and apoptotic indices in the NUCB2 IHC-high compared to NUCB2 IHC-low GC cases. NUCB2-dependent inhibition of senescence in GC engenders aggressive tumor behavior by modulating proliferation, apoptosis, and migration.

Immunoglobulin G4-related thyroiditis associated with Graves' disease: A case report.

We report a case of immunoglobulin (ig)-g4-related thyroiditis associated with graves' disease. a 45-year-old man was diagnosed with graves' disease due to asymptomatic enlarged thyroid gland and high serum levels of thyrotropin receptor antibodies and thyroid hormones. surgical resection of the thyroid gland was performed because of further thyroid gland enlargement and severe fluctuations in the thyroid hormonal levels, despite medical therapy with a combination of an antithyroid drug and a thyroid hormone preparation. macroscopic examination of the resected thyroid gland revealed a grayish-white diffuse swelling, and histopathological findings revealed follicular destruction, chronic inflammatory cell infiltration with diffuse igg4-positive plasma cells (IgG4/IgG >40%), storiform fibrosis, and phlebitis obliterans throughout the thyroid tissue. Additionally, there were small foci of high columnar follicular components with scalloping, resembling Graves' disease. We propose that all patients with Graves' disease should be evaluated for coexisting IgG4-related thyroiditis to detect ophthalmopathies as soon as possible.

Anaplastic lymphoma kinase overexpression enhances aggressive phenotypic characteristics of endometrial carcinoma.

BACKGROUND: Although anaplastic lymphoma kinase (ALK) 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, patients with tumors harboring either wild-type ALK or high ALK mRNA expression had a poorer prognosis than those 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 the establishment and maintenance of aggressive phenotypic characteristics through modulation of several biological processes.

Interaction between membranous EBP50 and myosin 9 as a favorable prognostic factor in ovarian clear cell carcinoma.

Ezrin-radixin-moesin-binding phosphoprotein 50 (EBP50) is a scaffold protein that is required for epithelial polarity. Knockout (KO) of membranous EBP50 (Me-EBP50) in ovarian clear cell carcinoma (OCCC) cells induced an epithelial-mesenchymal transition (EMT)-like phenotype, along with decreased proliferation, accelerated migration capability, and induction of cancer stem cell (CSC)-like properties. Shotgun proteomics analysis of proteins that co-immunoprecipitated with EBP50 revealed that Me-EBP50 strongly interacts with myosin 9 (MYH9). Specific inhibition of MYH9 with blebbistatin phenocopied Me-EBP50 KO, and blebbistatin treatment potentiated the effects of Me-EBP50 KO. In OCCC cells from clinical samples, Me-EBP50 and MYH9 were co-localized at the apical plasma membrane. Patients with a combination of Me-EBP50-high and MYH9-high scores had the best prognosis for overall and progression-free survival. Our data suggest that Me-EBP50 has tumor-suppressive effects through the establishment and maintenance of epithelial polarization. By contrast, loss of Me-EBP50 expression induces EMT-like phenotypes, probably due to MYH9 dysfunction; this results in increased cell mobility and enhanced CSC-like properties, which in turn promote OCCC progression.

Nucleobindin-2 Mediates Transforming Growth Factor-ß1-Driven Phenotypes in Zinc Finger E-Box Binding Homeobox 1-High Uterine Carcinosarcoma.

Epithelial-mesenchymal transition is a hallmark of uterine carcinosarcoma (UCS). Here, shotgun proteomics analysis used to identify biomarkers associated with blebbistatin-mediated epithelial-mesenchymal transition in UCS indicated up-regulation of nucleobindin-2 (NUCB2) in endometrial carcinoma (Em Ca) cells. Expression of N-cadherin, Snail, Slug, and ZEB1 was reduced in NUCB2 knockout Em Ca cells, whereas ZEB1, Twist1, and vimentin were up-regulated in NUCB2-overexpressing Em Ca cells. NUCB2 knockout reduced cell proliferation and migration, whereas NUCB2 overexpression had the opposite effect. Treatment of Em Ca cells with transforming growth factor (TGF)-ß1 dramatically altered morphology toward a fibroblastic appearance; concomitantly, expression of NUCB2 and ZEB1 increased. The NUCB2 promoter was also activated by transfection of Smad2. In UCS tissues, NUCB2 expression was significantly higher in sarcomatous compared with carcinomatous components, which was consistent with increased TGF-ß1 mRNA expression in stromal and sarcomatous components compared with carcinomatous components. In addition, NUCB2 score correlated positively with ZEB1 and vimentin scores, whereas ZEB1 score correlated positively with Slug and vimentin scores and inversely with the E-cadherin score. Collectively, these data indicate that TGF-ß-dependent up-regulation of NUCB2 and ZEB1 contributes to the phenotypic characteristics of sarcomatous components in UCS.

TP53 positivity combined with high fibrinogen expression defines a subtype of oral squamous cell carcinoma with an unfavorable prognosis.

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.

PTEN overexpression and nuclear ß-catenin stabilization promote morular differentiation through induction of epithelial-mesenchymal transition and cancer stem cell-like properties in endometrial carcinoma.

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 ß-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 and CD44s, a large ALDH 1high population, enriched spheroid formation, and ß-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 ß-catenin, cytoplasmic CD133, and CD44v6, and negatively with cell proliferation. Finally, estrogen receptor-α (ERα)-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 ß-catenin. CONCLUSION: In the abscess of ERα/EBP50 expression, PTEN overexpression and nuclear ß-catenin stabilization promote the establishment and maintenance of morular phenotype associated with EMT/CSC-like features in Em Ca cells. Video Abstract.

A combination of stromal PD-L1 and tumoral nuclear ß-catenin expression as an indicator of colorectal carcinoma progression and resistance to chemoradiotherapy in locally advanced rectal carcinoma.

Programmed cell death-1 (PD-1) and its ligand (PD-L1) are significant mediators of immune suppression in the tumor microenvironment. We focused on the immunological impact of PD-1/PD-L1 signaling during tumor progression in colorectal carcinoma (CRC) and its association with resistance to neoadjuvant chemoradiotherapy (NCRT) in locally advanced rectal carcinoma (LAd-RC). Histopathological and immunohistochemical analyses of 100 CRC cases (including 34 RC) without NCRT and 109 NCRT-treated LAd-RC cases were performed. Membranous tumoral PD-L1 expression was identified in 9 of 100 (9%) CRC cases, including 1 of 34 (2.9%) RC cases, but PD-L1 immunopositivity was not associated with any clinicopathological factors, with the exception of deficient mismatch repair (dMMR) status. In contrast, stromal PD-L1+ immune cells, which frequently exhibited coexpression of PD-1 and CD8 markers, were significantly correlated with tumor vessel invasion, nuclear ß-catenin+ tumor budding cancer stem cell (CSC)-like features, and unfavorable prognosis. In the LAd-RC cases, stromal CD8+ (but not PD-L1+) immune cell infiltration in pretreatment-biopsied samples was significantly and positively associated with therapeutic efficacy. After NCRT, tumoral PD-L1 expression was observed in only 2 of 83 (2.4%) tumors, independent of dMMR status, whereas high stromal PD-L1+ and tumoral nuclear ß-catenin positivity were significantly linked to a poor response to NCRT and high tumor budding features. In addition, high stromal PD-L1 immunoreactivity was significantly associated with poorer overall survival. In conclusion, a combination of stromal PD-L1+ immune cells and nuclear ß-catenin+ tumor budding may contribute to tumor progression in CRC and resistance to NCRT in LAd-RC, through formation of niche-like lesions that exhibit immune resistance and CSC properties.

A functional role of S100A4/non-muscle myosin IIA axis for pro-tumorigenic vascular functions in glioblastoma.

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. Video abstract.

Functional interaction between S100A1 and MDM2 may modulate p53 signaling in normal and malignant endometrial cells.

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.

Regulation of Tumor Necrosis Factor-α by Peptide Lv in Bone Marrow Macrophages and Synovium.

Background: Bone marrow-derived monocytes/macrophages are recruited into synovial tissue, where they contribute to synovial inflammation in osteoarthritis through inflammatory cytokine production. Recent studies have suggested that V-Set and transmembrane domain-containing 4 (VSTM4) and its fragment, peptide Lv, exhibit immunosuppressive activity on T cells and vascular endothelial growth factor (VEGF)-like activity, respectively. Given that evidence suggests that VEGF may play a role in macrophage function, we investigated peptide Lv-mediated regulation of inflammatory cytokines in bone marrow macrophages (BMMs) and synovial inflammation. Method: To investigate the effects of peptide Lv, BMMs were stimulated with vehicle, LPS, or LPS + peptide Lv, and Tnfa, Il1b, Il6, and Ifng expression were evaluated using quantitative PCR (qPCR). TNF-α and IFN-γ production was measured using ELISA. To examine the effect of peptide Lv deficiency on macrophages and synovitis, peptide Lv-deficient mice were generated using genome editing. LPS-induced Tnfa and Ifng expression and TNF-α and IFN-γ production were evaluated in BMM isolated from wild-type and peptide Lv-deficient mice. Additionally, Tnfa and Ifng expression levels were compared between wild-type and peptide Lv-deficient mice before and after knee injury. Results: Peptide Lv suppressed the LPS-mediated elevation in TNF-α and IFN-γ. LPS stimulation significantly increased TNF-α and IFN-γ production in BMM derived from peptide Lv-deficient mice compared to wild-type mice. Synovial TNF-α expression in the injured knee was elevated in peptide Lv-deficient compared to wild-type mice. Conclusion: Peptide Lv suppressed TNF-α in macrophages and plays a role in synovial inflammation. Thus, peptide Lv may be a useful therapeutic target for synovitis.

Anaplastic Lymphoma Kinase Overexpression Is Associated with Aggressive Phenotypic Characteristics of Ovarian High-Grade Serous Carcinoma.

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.

Cytoplasmic EBP50 and elevated PARP1 are unfavorable prognostic factors in ovarian clear cell carcinoma.

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.

Increased nerve growth factor expression in the synovial tissues of patients with rotator cuff tears.

BACKGROUND: Rotator 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). METHODS: The 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. RESULTS: NGF 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). CONCLUSION: NGF 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.

Requirements of LEFTY and Nodal overexpression for tumor cell survival under hypoxia in glioblastoma.

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 ß (TGF-ß) superfamily, have glycogen synthase kinase 3ß (GSK-3ß) 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-3ß, 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-3ß, or pSmad2 scores. In KS-1, a GBM cell line that lacks endogenous Nodal expression, treatment with the hypoxic mimetic CoCl2 increased LEFTY, pGSK-3ß, and pSmad2 levels, but decreased pAkt levels. Moreover, the promoter for LEFTY, but not Nodal, was activated by Smad2 or TGF-ß1, suggesting that overexpression of LEFTY and Nodal may be due to Akt-independent GSK-3ß inactivation, with or without cooperation of the TGF-ß1/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.

S100A4/Nonmuscle Myosin IIA/p53 Axis Contributes to Aggressive Features in Ovarian High-Grade Serous Carcinoma.

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.

Upregulation of fibronectin following loss of p53 function is a poor prognostic factor in ovarian carcinoma with a unique immunophenotype.

BACKGROUND: We previously demonstrated that ovarian high grade serous carcinomas (OHGSeCa) and ovarian clear cell carcinomas (OCCCa) with an HNF-1ß+/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-1ß 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 pGSK3ß, 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 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-1ß+/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 an HNF-1ß+/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. Video Abstract.

S100A4/non-muscle myosin II signaling regulates epithelial-mesenchymal transition and stemness in uterine carcinosarcoma.

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.

Alterations in ß-catenin, microsatellite instability, and HNF-1ß levels are independently associated with ovarian endometriosis-associated tumorigenesis.

We focused on specific molecular events during the development of endometriosis-associated ovarian endometrioid carcinoma (OEmCa) and ovarian clear cell carcinoma (OCCCa). Alterations in ß-catenin (encoded by CTNNB1) and microsatellite instability (MSI), as well as changes in the expression levels of HNF-1ß 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-1ß expression was significantly associated with SLC3A1 expression, which plays a major role in HNF-1ß-triggered induction of reactive oxygen species in OCCCas, independent of abnormalities in both ß-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 ß-catenin and HNF-1ß status. In conclusion, different molecular pathways including alterations in ß-catenin, MSI, and HNF-1ß levels may contribute to tumorigenesis in endometriosis-associated carcinoma.