Makoto Saegusa

A Transcriptional Variant of Anaplastic Lymphoma Kinase Promotes Apoptosis in Ovarian High‐Grade Serous Carcinoma

ABSTRACTThe current study aims to delineate the role of a novel anaplastic lymphoma kinase (ALK) transcript, ALKATI, in ovarian high‐grade serous carcinoma (HGSC). Overexpressed ALKATI exhibited both cytoplasmic and nuclear localization in HGSC cells, whereas full‐length ALK was predominantly cytoplasmic. ALKATI interacts with the DNA repair protein, poly (ADP ribose) polymer 1 (PARP1), and cells stably overexpressing ALKATI (OE‐ ALKATI) were more sensitive to cisplatin‐induced apoptosis. Consistent with this, cleaved PARP1 levels were higher in HGSC tissue samples in areas with nuclear ALK immunoreactivity. The ratio of antiapoptotic BCL2 relative to proapoptotic BAX was significantly increased in OE‐ALKATI cells, despite the increase in apoptosis, suggesting that ALKATI‐mediated apoptosis is independent of mitochondrion‐driven cell death. OE‐ALKATI decreased epithelial‐mesenchymal transition/cancer stem cell properties but did not alter proliferation rates, and nuclear ALK immunopositivity was not associated with clinicopathological factors or prognosis in HGSC. Together, our observations suggest that ALKATI sensitizes HGSC cells to apoptosis (probably though an association with PARP1) but this may have a relatively minor impact on tumor progression.

EBP50 regulates senescence and focal adhesion in endometrial carcinoma

Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a multifunctional scaffold protein that is highly expressed in polarized epithelial cells. Here, we focused on the functional roles of EBP50 in endometrial carcinoma (Em Ca). We analyzed immunohistochemical sections from 121 Em Ca and 30 normal samples. We also characterized EBP50 overexpression or knockout (KO) Em Ca cell lines. High levels of membranous (Me) EBP50 expression were observed in endometrial tissues from normal menstrual cycles, in contrast to the transient upregulation of cytoplasmic (Cyt) EBP50 in tissues in the proliferative phase; this was probably in response to estrogenic effects. There was a significant stepwise reduction of Me-EBP50 expression from grade (G) 1 to G3 Em Cas, which was consistent with the loss of glandular structures. Conversely, Cyt-EBP50 levels increased with in the higher tumor grades. Low Me-EBP50 expression was significantly associated with tumor lymphovascular invasion and short overall survival. Whereas EBP50 KO led to senescence and reduced proliferation and motility, overexpression elicited the opposite phenotypes. Moreover, the number of focal adhesions (FAs), which mediate cell migration, was significantly increased in EBP50 overexpressing cells but decreased in the KO cells. In conclusion, Me- and/or Cyt-EBP50 expression contributes to acceleration of cell motility through enhancement of FA formation, and inhibits senescence to promote cytokinesis. Together, these effects contribute to Em Ca aggressiveness.

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.

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

Abstract 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.