FBI-1 Is Overexpressed in Gestational Trophoblastic Disease and Promotes Tumor Growth and Cell Aggressiveness of Choriocarcinoma via PI3K/Akt Signaling.

Human placental trophoblasts can be considered pseudomalignant, with tightly controlled proliferation, apoptosis, and invasiveness. Gestational trophoblastic disease (GTD) represents a family of heterogeneous trophoblastic lesions with aberrant apoptotic and proliferative activities and dysregulation of cell signaling pathways. We characterize the oncogenic effects of factor that binds to the inducer of short transcripts of HIV-1 [FBI-1, alias POZ and Krüppel erythroid myeloid ontogenic factor (POKEMON)/ZBTB7A] in GTD and its role in promoting cell aggressiveness in vitro and tumor growth in vivo. IHC studies showed increased nuclear expression of FBI-1, including hydatidiform moles, choriocarcinoma (CCA), and placental site trophoblastic tumor, in GTD. In JAR and JEG-3 CCA cells, ectopic FBI-1 expression opposed apoptosis through repression of proapoptotic genes (eg, BAK1, FAS, and CASP8). FBI-1 overexpression also promoted Akt activation, as indicated by Akt-pS473 phosphorylation. FBI-1 overexpression promoted mobility and invasiveness of JEG-3 and JAR, but not in the presence of the phosphoinositide 3-kinase inhibitor LY294002. These findings suggest that FBI-1 could promote cell migration and invasion via phosphoinositide 3-kinase/Akt signaling. In vivo, nude mice injected with CCA cells with stable FBI-1 knockdown demonstrated reduced tumor growth compared with that in control groups. These findings suggest that FBI-1 is clinically associated with the progression of, and may be a therapeutic target in, GTD, owing to its diverse oncogenic effects on dysregulated trophoblasts.

Functional selection in SH3-mediated activation of the PI3 kinase.

The phosphoinositide-3 kinase (PI3K), a heterodimeric enzyme, plays a pivotal role in cellular metabolism and survival. Its deregulation is associated with major human diseases, particularly cancer. The p85 regulatory subunit of PI3K binds to the catalytic p110 subunit via its C-terminal domains, stabilising it in an inhibited state. Certain Src homology 3 (SH3) domains can activate p110 by binding to the proline-rich (PR) 1 motif located at the N-terminus of p85. However, the mechanism by which this N-terminal interaction activates the C-terminally bound p110 remains elusive. Moreover, the intrinsically poor ligand selectivity of SH3 domains raises the question of how they can control PI3K. Combining structural, biophysical, and functional methods, we demonstrate that the answers to both these unknown issues are linked: PI3K-activating SH3 domains engage in additional "tertiary" interactions with the C-terminal domains of p85, thereby relieving their inhibition of p110. SH3 domains lacking these tertiary interactions may still bind to p85 but cannot activate PI3K. Thus, p85 uses a functional selection mechanism that precludes nonspecific activation rather than nonspecific binding. This separation of binding and activation may provide a general mechanism for how biological activities can be controlled by promiscuous protein-protein interaction domains.

Downregulation of ASPP2 in choriocarcinoma contributes to increased migratory potential through Src signaling pathway activation.

Gestational choriocarcinoma is a malignant tumor derived from placental trophoblast and the most aggressive member of gestational trophoblastic disease (GTD). Apoptosis-stimulating protein of p53-2 (ASPP2) is a member of ASPP family that transactivates p53 and thereby functions as a tumor suppressor. In this study, the expression profile of ASPP2 in choriocarcinoma was examined in comparison with normal placentas and hydatidiform moles, the latter being a type of GTD that carries malignant potential. Downregulation of ASPP2 messenger RNA and protein was demonstrated in choriocarcinoma by quantitative PCR and immunohistochemistry. ASPP2-transfected choriocarcinoma cells (JEG-3 and JAR) showed an increase in apoptosis and a decrease in cell migration as detected by TdT-mediated dUTP nick end labeling and wound healing assays, respectively, illustrating the complex action of ASPP2 on cell functions other than programmed cell death. Activated Src is known to be important in tumor progression. Transfection of ASPP2 but not ASPP1, another tumor-suppressive ASPP, was found to be related to subsequent decreased Src-pY416 phosphorylation, suggesting an inactivating effect of ASPP2 on Src. Moreover, this ASPP2-mediated inactivation of Src could be abolished by RNA interference with C-terminal Src kinase (Csk), a kinase that can inhibit Src activation. Our findings suggested that the ability of ASPP2 to attenuate Src activation was specific to ASPP2 in a Csk-dependent manner. Taken together, we demonstrated a loss of tumor-suppressive ASPP2 in choriocarcinoma with effects on cell migration and apoptosis. We also unveiled a possible mechanistic link between ASPP2 and Csk/Src signaling pathway, implicating the multiple cellular functions of ASPP2.

AKTIP loss is enriched in ERα-positive breast cancer for tumorigenesis and confers endocrine resistance.

Recurrent deletion of 16q12.2 is observed in luminal breast cancer, yet the causal genomic alterations in this region are largely unknown. In this study, we identify that loss of AKTIP, which is located on 16q12.2, drives tumorigenesis of estrogen receptor alpha (ERα)-positive, but not ERα-negative, breast cancer cells and is associated with poor prognosis of patients with ERα-positive breast cancer. Intriguingly, AKTIP-depleted tumors have increased ERα protein level and activity. Cullin-associated and neddylation-dissociated protein 1 (CAND1), which regulates the cullin-RING E3 ubiquitin ligases, protects ERα from cullin 2-dependent proteasomal degradation. Apart from ERα signaling, AKTIP loss triggers JAK2-STAT3 activation, which provides an alternative survival signal when ERα is inhibited. AKTIP-depleted MCF7 cells and ERα-positive patient-derived organoids are more resistant to ERα antagonists. Importantly, the resistance can be overcome by co-inhibition of JAK2/STAT3. Together, our results highlight the subtype-specific functional consequences of AKTIP loss and provide a mechanistic explanation for the enriched AKTIP copy-number loss in ERα-positive breast cancer.

Overexpressed PAK4 promotes proliferation, migration and invasion of choriocarcinoma.

Gestational trophoblastic disease (GTD) includes frankly malignant choriocarcinoma (CCA) and placental site trophoblastic tumor and potentially malignant hydatidiform mole. p21-Activated kinase (PAK) 4 promotes cell motility. This study investigated the role of PAK4 in the pathogenesis of GTD. PAK4 messenger RNA and protein expressions in clinical samples and cell lines of normal placentas and GTD were determined by quantitative real-time polymerase chain reaction and western blot, respectively. The effects of human chorionic gonadotropin (hCG) and phosphoinositide 3 kinase (PI3K) on the expression and activation of PAK4 were investigated by treating CCA JEG3 and JAR cells with anti-hCG antibody and PI3K inhibitor, respectively. The effects of PAK4 on CCA cell proliferation, migration and invasion were assessed by corresponding functional assays. We demonstrated overexpression of PAK4 in GTD and CCA cell lines at both RNA and protein level. hCG is one of the upstream regulators of PAK4 expression, whereas activation of PAK4 is PI3K/PKB dependent in JEG3 and JAR cells. Significant correlation was found between PAK4 expression and proliferation index minichromosome maintenance complex component 7 (P = 0.007). In JEG3 and JAR cells, stably transfected PAK4 increased proliferation, migration and invasion, whereas small interfering RNA knockdown of PAK4 decreased proliferation, migration and invasion along with downregulated CDK6 and membrane-type 1 matrix metalloproteinase (MT1-MMP) and upregulated p16. We further found PAK4-mediated transcription of MT1-MMP in CCA cells by luciferase reporter assay. Our results demonstrated for the first time that overexpressed PAK4 was involved in the pathogenesis of GTD, promoting proliferation and enhancing cell migration and invasion in CCA cells.

Downregulation of ASPP1 in gestational trophoblastic disease: correlation with hypermethylation, apoptotic activity and clinical outcome.

Gestational trophoblastic disease encompasses a spectrum of trophoblastic lesions including true neoplasms such as choriocarcinomas and the potentially malignant hydatidiform moles, which may develop persistent disease requiring chemotherapy. ASPP1, a member of apoptosis-stimulating proteins of p53 (ASPPs), is a proapoptotic protein that can stimulate apoptosis through its interaction with p53. We evaluated the promoter methylation and expression profiles of ASPP1 in different trophoblastic tissues and its in vitro functional effect on two choriocarcinoma cell lines, namely JEG-3 and JAR. Significant downregulation of ASPP1 mRNA and protein levels was demonstrated in hydatidiform moles and choriocarcinomas, when compared with normal placentas by quantitative-PCR and immunohistochemistry. The ASPP1 mRNA level was significantly correlated with its hypermethylation status, evaluated with methylation-specific PCR, in placenta and gestational trophoblastic disease samples (P=0.024). Moreover, lower ASPP1 immunoreactivity was shown in hydatidiform moles that progressed to persistent gestational trophoblastic neoplasms than in those that regressed (P=0.045). A significant correlation was also found between expression of ASPP1 and proliferative indices (assessed by Ki67 and MCM7), apoptotic activity (M30 CytoDeath antibody), p53 and caspase-8 immunoreactivities. An in vitro study showed that ectopic expression of ASPP1 could trigger apoptosis through intrinsic and extrinsic pathways as indicated by an increase in cleaved caspase-9 and Fas ligand protein expression. The latter suggests a hitherto unreported novel link between ASPP1 and the extrinsic pathway of apoptosis. Our findings suggest that downregulation of ASPP1 by hypermethylation may be involved in the pathogenesis and progress of gestational trophoblastic disease, probably through its effect on apoptosis.

Strategic Combination Therapies for Ovarian Cancer.

Ovarian cancer remains the leading cause of gynecologic cancer-related deaths among women worldwide. The dismal survival rate is partially due to recurrence after standardized debulking surgery and first-line chemotherapy. In recent years, targeted therapies, including antiangiogenic agents or poly (ADP-ribose) polymerase inhibitors, represent breakthroughs in the treatment of ovarian cancer. As more therapeutic agents become available supplemented by a deeper understanding of ovarian cancer biology, a range of combination treatment approaches are being actively investigated to further improve the clinical outcomes of the disease. These combinations, which involve DNA-damaging agents, targeted therapies of signaling pathways and immunotherapies, simultaneously target multiple cancer pathways or hallmarks to induce additive or synergistic antitumor activities. Here we review the preclinical data and ongoing clinical trials for developing effective combination therapies in treating ovarian cancer. These emerging therapeutic modalities may reshape the treatment landscape of the disease.

p85ß regulates autophagic degradation of AXL to activate oncogenic signaling.

PIK3R2 encodes the p85ß regulatory subunit of phosphatidylinositol 3-kinase and is frequently amplified in cancers. The signaling mechanism and therapeutic implication of p85ß are poorly understood. Here we report that p85ß upregulates the protein level of the receptor tyrosine kinase AXL to induce oncogenic signaling in ovarian cancer. p85ß activates p110 activity and AKT-independent PDK1/SGK3 signaling to promote tumorigenic phenotypes, which are all abolished upon inhibition of AXL. At the molecular level, p85ß alters the phosphorylation of TRIM2 (an E3 ligase) and optineurin (an autophagy receptor), which mediate the selective regulation of AXL by p85ß, thereby disrupting the autophagic degradation of the AXL protein. Therapeutically, p85ß expression renders ovarian cancer cells vulnerable to inhibitors of AXL, p110, or PDK1. Conversely, p85ß-depleted cells are less sensitive to these inhibitors. Together, our findings provide a rationale for pharmacological blockade of the AXL signaling axis in PIK3R2-amplified ovarian cancer.

Deregulated Gab2 phosphorylation mediates aberrant AKT and STAT3 signaling upon PIK3R1 loss in ovarian cancer.

Copy number loss of PIK3R1 (p85α) most commonly occurs in ovarian cancer among all cancer types. Here we report that ovarian cancer cells manifest a spectrum of tumorigenic phenotypes upon knockdown of PIK3R1. PIK3R1 loss activates AKT and p110-independent JAK2/STAT3 signaling through inducing changes in the phosphorylation of the docking protein Gab2, thereby relieving the negative inhibition on AKT and promoting the assembly of JAK2/STAT3 signalosome, respectively. Additional mechanisms leading to AKT activation include enhanced p110α kinase activity and a decrease in PTEN level. PIK3R1 loss renders ovarian cancer cells vulnerable to inhibition of AKT or JAK2/STAT3. The combination of AKT and STAT3 inhibitors significantly increases the anti-tumor effect compared to single-agent treatments. Together, our findings provide a rationale for mechanism-based therapeutic approach that targets tumors with loss of PIK3R1.

Dysregulated stemness-related genes in gynecological malignancies.

In recent years, much attention has been paid to the concept of cancer stem cells (CSC) and self-renewal related pathways in cancer biology. This review outlines the dysregulated stemness-related genes or transcription factors in gynecological cancers. Hedgehog (Hh) and Notch signaling are important pathways in tissue pattern programming and cell fate determination during embryonic development. Hyperactivation of these two pathways was frequently observed in gynecological malignancies such as ovarian, endometrial and cervical cancers. In contrast, the expression profiles of pluripotency-regulating core transcriptional circuitry: Nanog, Oct4 and Sox2 appear heterogeneous. Among these transcription factors, overexpression of Nanog was found to exert a prominent effect in gynecological tumorigenesis, while dysregulations of Oct4 and Sox2 may vary in a context dependent manner. On the other hand, the isolation of putative CSC illustrates a hierarchy model of tumor heterogeneity, in which only a subset of cells among biologically distinct populations can initiate tumor growth. Re-activation of these pluripotent transcription factors (Nanog, Oct4 and/or Sox2) in association with distinct tumorigenic properties could be found in clones isolated from gynecological tumors using various approaches. Recent understanding on the roles of Hh and Notch signaling in enhancing CSC survival may help to better understand the mechanism of carcinogenesis and identify new pharmaceutical targets for gynecological malignancies.