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.

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.