CCNE1 Is a Putative Therapeutic Target for ARID1A-Mutated Ovarian Clear Cell Carcinoma

Background: Ovarian clear cell carcinoma (OCCC) is resistant to platinum chemotherapy and is characterized by poor prognosis. Today, the use of poly (ADP-ribose) polymerase (PARP) inhibitor, which is based on synthetic lethality strategy and characterized by cancer selectivity, is widely used for new types of molecular-targeted treatment of relapsed platinum-sensitive ovarian cancer. However, it is less effective against OCCC. Methods: We conducted siRNA screening to identify synthetic lethal candidates for the ARID1A mutation; as a result, we identified Cyclin-E1 (CCNE1) as a potential target that affects cell viability. To further clarify the effects of CCNE1, human OCCC cell lines, namely TOV-21G and KOC7c (ARID1A mutant lines), and RMG-I and ES2 (ARID1A wild type lines) were transfected with siRNA targeting CCNE1 or a control vector. Results: Loss of CCNE1 reduced proliferation of the TOV-21G and KOC7c cells but not of the RMG-I and ES2 cells. Furthermore, in vivo interference of CCNE1 effectively inhibited tumor cell proliferation in a xenograft mouse model. Conclusion: This study showed for the first time that CCNE1 is a synthetic lethal target gene to ARID1A-mutated OCCC. Targeting this gene may represent a putative, novel, anticancer strategy in OCCC treatment.     

Targeting Mitochondrial Metabolism in Clear Cell Carcinoma of the Ovaries

Ovarian clear cell carcinoma (OCCC) is a rare but chemorefractory tumor. About 50% of all OCCC patients have inactivating mutations of ARID1A, a member of the SWI/SNF chromatin-remodeling complex. Members of the SWI/SNF remodeling have emerged as regulators of the energetic metabolism of mammalian cells; however, the role of ARID1A as a modulator of the mitochondrial metabolism in OCCCs is yet to be defined. Here, we show that ARID1A loss results in increased mitochondrial metabolism and renders ARID1A-mutated cells increasingly and selectively dependent on it. The increase in mitochondrial activity following ARID1A loss is associated with increase in c-Myc expression and increased mitochondrial number and reduction of their size consistent with a higher mitochondrial cristae/outer membrane ratio. Significantly, preclinical testing of the complex I mitochondrial inhibitor IACS-010759 showed it extends overall survival in a preclinical model of ARID1A-mutated OCCC. These findings provide for the targeting mitochondrial activity in ARID1A-mutated OCCCs.     

Clinicopathological Significance of Loss of ARID1A Immunoreactivity in Ovarian Clear Cell Carcinoma

Recent genome-wide analysis has demonstrated that somatic mutations in ARID1A (BAF250) are the most common molecular genetic changes in ovarian clear cell carcinoma (OCCC). ARID1A mutations, which occur in approximately half of OCCC cases, lead to deletion of the encoded protein and inactivation of the putative tumor suppressor. In this study, we determined the significance of loss of ARID1A immunoreactivity with respect to several clinicopathological features in a total of 149 OCCCs. First, we demonstrated that ARID1A immunohistochemistry showed concordance with the mutational status in 91% of cases with 100% sensitivity and 66% specificity. Specifically, among 12 OCCC cases for which ARIDA mutational status was known, ARIDIA immunoreactivity was undetectable in all 9 cases harboring ARID1A mutations and was undetectable in one of 3 cases with wild-type ARID1A. With respect to the entire cohort, ARID1A immunoreactivity was undetectable in 88 (59%) of 149 OCCCs. There was no significant difference between ARID1A negative and positive cases in terms of histopathologic features, age, clinical stage, or overall survival. In conclusion, this study provides further evidence that mutations in ARID1A resulted in loss of ARID1A protein expression in OCCC, although no significant differences between ARID1A positive and negative cases were observed with respect to any clinicopathological features examined.     

Expression and Significance of CD44, CD47 and c-met in Ovarian Clear Cell Carcinoma

Aims: The aim of the present study is to investigate the differential expression of CD44, CD47 and c-met in ovarian clear cell carcinoma (OCCC), the correlation in their expression and their relationship with the biological behavior of OCCC. Methods: We used immunohistochemistry to examine the expression of CD44, CD47 and c-met in OCCC (86 cases) and investigated the effects of the expression and interaction of these molecules on the development of OCCC. Results: CD44, CD47 and c-met expression was significantly high in OCCC. Expression of CD44 and CD47 correlated with patient surgical stage, chemotherapy resistance and prognosis (all p < 0.05), and expression of c-met correlated with chemotherapy resistance and prognosis (all p < 0.05), but did not correlate with lymph node metastasis (all p > 0.05). The surgical stage, CD44, CD47 and c-met expression were independent risk factors for OCCC prognosis (all p < 0.05). Patients with low levels of CD44, CD47 and c-met showed better survival than those with high levels (all p < 0.05). There was a positive correlation between CD44 (or CD47) and c-met, as well as between CD44 and CD47 (the Spearman correlation coefficient r_s was 0.783, 0.776 and 0.835, respectively, all p < 0.01). Additionally, pairwise correlation analysis of these three markers shows that the high expression of CD44/CD47, CD44/c-met and CD47/c-met were correlated with patient surgical stage, chemotherapy resistance and prognosis (all p < 0.05), but did not correlate with lymph node metastasis (all p > 0.05). Conclusions: Expression of CD44, CD47 and c-met was upregulated in OCCC and pairwise correlation. CD44, CD47 and c-met may have synergistic effects on the development of OCCC and are prognostic factors for ovarian cancer.     

PI3K/PTEN/AKT Signaling Pathways in Germ Cell Development and Their Involvement in Germ Cell Tumors and Ovarian Dysfunctions

Several studies indicate that the PI3K/PTEN/AKT signaling pathways are critical regulators of ovarian function including the formation of the germ cell precursors, termed primordial germ cells, and the follicular pool maintenance. This article reviews the current state of knowledge of the functional role of the PI3K/PTEN/AKT pathways during primordial germ cell development and the dynamics of the ovarian primordial follicle reserve and how dysregulation of these signaling pathways may contribute to the development of some types of germ cell tumors and ovarian dysfunctions.     

PTEN and Other PtdIns(3,4,5)P3 Lipid Phosphatases in Breast Cancer

The phosphoinositide 3-kinase (PI3K)/AKT signalling pathway is hyperactivated in ~70% of breast cancers. Class I PI3K generates PtdIns(3,4,5)P₃ at the plasma membrane in response to growth factor stimulation, leading to AKT activation to drive cell proliferation, survival and migration. PTEN negatively regulates PI3K/AKT signalling by dephosphorylating PtdIns(3,4,5)P₃ to form PtdIns(4,5)P₂. PtdIns(3,4,5)P₃ can also be hydrolysed by the inositol polyphosphate 5-phosphatases (5-phosphatases) to produce PtdIns(3,4)P₂. Interestingly, while PTEN is a bona fide tumour suppressor and is frequently mutated/lost in breast cancer, 5-phosphatases such as PIPP, SHIP2 and SYNJ2, have demonstrated more diverse roles in regulating mammary tumourigenesis. Reduced PIPP expression is associated with triple negative breast cancers and reduced relapse-free and overall survival. Although PIPP depletion enhances AKT phosphorylation and supports tumour growth, this also inhibits cell migration and metastasis in vivo, in a breast cancer oncogene-driven murine model. Paradoxically, SHIP2 and SYNJ2 are increased in primary breast tumours, which correlates with invasive disease and reduced survival. SHIP2 or SYNJ2 overexpression promotes breast tumourigenesis via AKT-dependent and independent mechanisms. This review will discuss how PTEN, PIPP, SHIP2 and SYNJ2 distinctly regulate multiple functional targets, and the mechanisms by which dysregulation of these distinct phosphoinositide phosphatases differentially affect breast cancer progression.     

The Involvement of RhoA and Wnt-5a in the Tumorigenesis and Progression of Ovarian Epithelial Carcinoma

Background

Ras homolog gene family member A (RhoA) is involved in Wnt-5a–induced migration of gastric and breast cancer cells. We investigated the roles of RhoA and Wnt-5a in ovarian carcinoma.

Methods

RhoA and Wnt-5a mRNA and protein expression in normal fallopian tube epithelium, benign tumors, primary ovarian carcinomas, and metastatic omentum were quantified. RhoA or Wnt-5a was knocked down in OVCAR3 ovarian carcinoma cells using siRNAs and cell phenotype and expression of relevant molecules were assayed.

Results

RhoA and Wnt-5a mRNA and protein expression were found to be significantly higher in metastatic omentum than in ovarian carcinomas, benign tumors, and normal fallopian tube epithelium (p < 0.05), and positively associated with differentiation and FIGO staging (stage I/II vs. stage III/IV) in ovarian carcinoma (p < 0.05). RhoA and Wnt-5a expression were positively correlated in ovarian carcinoma (p = 0.001, R² = 0.1669). RhoA or Wnt-5a knockdown downregulated RhoA and Wnt-5a expression; reduced cell proliferation; promoted G₁ arrest and apoptosis; suppressed lamellipodia formation, cell migration, and invasion; and reduced PI3K, Akt, p70S6k, Bcl-xL, survivin, and VEGF mRNA or protein expression.

Conclusions

This is the first demonstration that RhoA and Wnt-5a are associated with ovarian carcinogenesis and apoptosis inhibition; there might be positive correlation between RhoA and Wnt-5a expression. RhoA is a potential tumorigenesis, differentiation, and progression biomarker in ovarian carcinoma.     

The Correlation of Mutations and Expressions of Genes within the PI3K/Akt/mTOR Pathway in Breast Cancer—A Preliminary Study

There is an urgent need to seek new molecular biomarkers helpful in diagnosing and treating breast cancer. In this elaboration, we performed a molecular analysis of mutations and expression of genes within the PI3K/Akt/mTOR pathway in patients with ductal breast cancer of various malignancy levels. We recognized significant correlations between the expression levels of the studied genes. We also performed a bioinformatics analysis of the data available on the international database TCGA and compared them with our own research. Studies on mutations and expression of genes were conducted using High-Resolution Melt PCR (HRM-PCR), Allele-Specific-quantitative PCR (ASP-qPCR), Real-Time PCR molecular methods in a group of women with ductal breast cancer. Bioinformatics analysis was carried out using web source Ualcan and bc-GenExMiner. In the studied group of women, it was observed that the prevalence of mutations in the studied PIK3CA and AKT1 genes was 29.63%. It was stated that the average expression level of the PIK3CA, PIK3R1, PTEN genes in the group of breast cancer patients is lower in comparison to the control group, while the average expression level of the AKT1 and mTOR genes in the studied group was higher in comparison to the control group. It was also indicated that in the group of patients with mutations in the area of the PIK3CA and AKT1 genes, the PIK3CA gene expression level is statistically significantly lower than in the group without mutations. According to our knowledge, we demonstrate, for the first time, that there is a very strong positive correlation between the levels of AKT1 and mTOR gene expression in the case of patients with mutations and without mutations.     

The Inhibitory Response to PI3K/AKT Pathway Inhibitors MK-2206 and Buparlisib Is Related to Genetic Differences in Pancreatic Ductal Adenocarcinoma Cell Lines

The aberrant activation of the phosphoinositide 3-kinase (PI3K)/ protein kinase B (AKT) pathway is common in pancreatic ductal adenocarcinomas (PDAC). The application of inhibitors against PI3K and AKT has been considered as a therapeutic option. We investigated PDAC cell lines exposed to increasing concentrations of MK-2206 (an AKT1/2/3 inhibitor) and Buparlisib (a pan-PI3K inhibitor). Cell proliferation, metabolic activity, biomass, and apoptosis/necrosis were evaluated. Further, whole-exome sequencing (WES) and RNA sequencing (RNA-seq) were performed to analyze the recurrent aberrations and expression profiles of the inhibitor target genes and the genes frequently mutated in PDAC (Kirsten rat sarcoma virus (KRAS), Tumor protein p53 (TP53)). MK-2206 and Buparlisib demonstrated pronounced cytotoxic effects and limited cell-line-specific effects in cell death induction. WES revealed two sequence variants within the direct target genes (PIK3CA c.1143C > G in Colo357 and PIK3CD c.2480C > G in Capan-1), but a direct link to the Buparlisib response was not observed. RNA-seq demonstrated that the expression level of the inhibitor target genes did not affect the efficacy of the corresponding inhibitors. Moreover, increased resistance to MK-2206 was observed in the analyzed cell lines carrying a KRAS variant. Further, increased resistance to both inhibitors was observed in SU.86.86 carrying two TP53 missense variants. Additionally, the presence of the PIK3CA c.1143C > G in KRAS-variant-carrying cell lines was observed to correlate with increased sensitivity to Buparlisib. In conclusion, the present study reveals the distinct antitumor effects of PI3K/AKT pathway inhibitors against PDAC cell lines. Aberrations in specific target genes, as well as KRAS and TP53, individually or together, affect the efficacy of the two PI3K/AKT pathway inhibitors.     

G-Protein-Coupled Lysophosphatidic Acid Receptors and Their Regulation of AKT Signaling

A hallmark of G-protein-coupled receptors (GPCRs) is their ability to recognize and respond to chemically diverse ligands. Lysophospholipids constitute a relatively recent addition to these ligands and carry out their biological functions by activating G-proteins coupled to a large family of cell-surface receptors. This review aims to highlight salient features of cell signaling by one class of these receptors, known as lysophosphatidic acid (LPA) receptors, in the context of phosphatidylinositol 3-kinase (PI3K)–AKT pathway activation. LPA moieties efficiently activate AKT phosphorylation and activation in a multitude of cell types. The interplay between LPA, its receptors, the associated Gαi/o subunits, PI3K and AKT contributes to the regulation of cell survival, migration, proliferation and confers chemotherapy-resistance in certain cancers. However, detailed information on the regulation of PI3K–AKT signals induced by LPA receptors is missing from the literature. Here, some urgent issues for investigation are highlighted.     

Hyaluronan Synthase and Hyaluronidase Expression in Serous Ovarian Carcinoma is Related to Anatomic Site and Chemotherapy Exposure

The present study investigated the expression and clinical role of hyaluronan synthases (HAS1-3) and hyaluronidases (Hyal1-3) in serous ovarian carcinoma. HAS and HYAL mRNA expression was analyzed in 97 tumors (61 effusions, 27 primary carcinomas, 9 solid metastases) using PCR and further studied for association with clinicopathologic parameters, including survival. HAS1 mRNA was overexpressed in effusions compared to primary carcinomas and solid metastases (p < 0.001), and an alternatively spliced HAS1 was expressed only in effusions. HAS2 mRNA was overexpressed in solid metastases and primary carcinomas compared to effusions (p = 0.043), and HAS3 mRNA was overexpressed in primary carcinomas and effusions compared to solid metastases (p = 0.008). HYAL1 mRNA was absent in all specimens, whereas HYAL2 was expressed as two splice variants, of which HYAL2-var2 was overexpressed in solid metastases compared to effusions and primary carcinomas (p < 0.001). HYAL3 mRNA was expressed as wild-type and variant 1–3 form, the latter more highly in primary carcinomas and effusions compared to solid metastases (p = 0.006). HAS1 mRNA was overexpressed in pre- compared to post-chemotherapy effusions (p < 0.001), with opposite finding for HYAL2-var1 and HYAL3-WT (p = 0.016 and p = 0.024, respectively). Higher HYAL2-var1 and HAS1 splice variant mRNA expression in effusions was associated with longer (p = 0.033) and shorter (p = 0.047) overall survival, respectively. These data are the first to document a role for HAS and Hyal members in tumor progression in ovarian carcinoma, as evidenced by their differential expression as function of anatomic site and chemotherapy exposure, with a possible prognostic role for patients with malignant effusions.     

Endometrial Epithelial ARID1A Is Required for Uterine Immune Homeostasis during Early Pregnancy

A growing body of work suggests epigenetic dysregulation contributes to endometriosis pathophysiology and female infertility. The chromatin remodeling complex subunit AT-rich interaction domain 1A (ARID1A) must be properly expressed to maintain normal uterine function. Endometrial epithelial ARID1A is indispensable for pregnancy establishment in mice through regulation of endometrial gland function; however, ARID1A expression is decreased in infertile women with endometriosis. We hypothesized that ARID1A performs critical operations in the endometrial epithelium necessary for fertility besides maintaining gland function. To identify alterations in uterine gene expression resulting from loss of epithelial ARID1A, we performed RNA-sequencing analysis on pre-implantation uteri from Ltf^iCre/+Arid1a^f/f and control mice. Differential expression analysis identified 4181 differentially expressed genes enriched for immune-related ingenuity canonical pathways including agranulocyte adhesion and diapedesis and natural killer cell signaling. RT-qPCR confirmed an increase in pro-inflammatory cytokine and macrophage-related gene expression but a decrease in natural killer cell signaling. Immunostaining confirmed a uterus-specific increase in macrophage infiltration. Flow cytometry delineated an increase in inflammatory macrophages and a decrease in uterine dendritic cells in Ltf^iCre/+Arid1a^f/f uteri. These findings demonstrate a role for endometrial epithelial ARID1A in suppressing inflammation and maintaining uterine immune homeostasis, which are required for successful pregnancy and gynecological health.     

Resistin Promotes the Expression of Vascular Endothelial Growth Factor in Ovary Carcinoma Cells

Resistin is a novel hormone that is secreted by human adipocytes and mononuclear cells and is associated with obesity, insulin resistance and inflammation. Recently, resistin has been postulated to play a role in angiogenesis. Here, we investigated the hypothesis that resistin regulates ovary carcinoma production of vascular endothelial growth factor (VEGF) and the angiogenic processes. We found that in human ovarian epithelial carcinoma cells (HO-8910), resistin (10–150 ng/mL) enhanced both VEGF protein and mRNA expression in a time- and concentration-dependent manner, as well as promoter activity. Furthermore, resistin enhanced DNA-binding activity of Sp1 with VEGF promoter in a PI3K/Akt-dependent manner. PI3K/Akt activated by resistin led to increasing interaction with Sp1, triggering a progressive phosphorylation of Sp1 on Thr453 and Thr739, resulting in the upregulation of VEGF expression. In an in vitro angiogenesis system for endothelial cells (EA.hy926) co-cultured with HO-8910 cells, we observed that the addition of resistin stimulated endothelial cell tube formation, which could be abolished by VEGF neutralizing antibody. Our findings suggest that the PI3K/Akt-Sp1 pathway is involved in resistin-induced VEGF expression in HO-8910 cells and indicates that antiangiogenesis therapy may be beneficial treatment against ovarian epithelial carcinoma, especially in obese patients.     

BTG1 Expression Correlates with the Pathogenesis and Progression of Ovarian Carcinomas

BTG (B-cell translocation gene) can inhibit cell proliferation, metastasis, and angiogenesis and regulate cell cycle progression and differentiation in a variety of cell types. We aimed to clarify the role of BTG1 in ovarian carcinogenesis and progression. A BTG1-expressing plasmid was transfected into ovarian carcinoma cells and their phenotypes and related proteins were examined. BTG1 mRNA expression was detected in ovarian normal tissue (n = 17), ovarian benign tumors (n = 12), and ovarian carcinoma (n = 64) using real-time RT-PCR. Ectopic BTG1 expression resulted in lower growth rate, high cisplatin sensitivity, G₁ arrest, apoptosis, and decreased migration and invasion. Phosphoinositide 3-kinase, protein kinase B, Bcl-xL, survivin, vascular endothelial growth factor, and matrix metalloproteinase-2 mRNA and protein expression was reduced in transfectants as compared to control cells. There was higher expression of BTG1 mRNA in normal tissue than in carcinoma tissue (p = 0.001) and in benign tumors than in carcinoma tissue (p = 0.027). BTG1 mRNA expression in International Federation of Gynecology and Obstetrics (FIGO) stage I/II ovarian carcinomas was higher than that in FIGO stage III/IV ovarian carcinomas (p = 0.038). Altered BTG1 expression might play a role in the pathogenesis and progression of ovarian carcinoma by modulating proliferation, migration, invasion, the cell cycle, and apoptosis.     

PI3K/AKT/mTOR Signaling Pathway in Breast Cancer: From Molecular Landscape to Clinical Aspects

Breast cancer is a serious health problem worldwide, representing the second cause of death through malignancies among women in developed countries. Population, endogenous and exogenous hormones, and physiological, genetic and breast-related factors are involved in breast cancer pathogenesis. The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) is a signaling pathway involved in cell proliferation, survival, invasion, migration, apoptosis, glucose metabolism and DNA repair. In breast tumors, PIK3CA somatic mutations have been reported, located in exon 9 and exon 20. Up to 40% of PIK3CA mutations are estrogen receptor (ER) positive and human epidermal growth factor receptor 2 (HER2) -negative in primary and metastatic breast cancer. HER2 is overexpressed in 20–30% of breast cancers. HER1, HER2, HER3 and HER4 are membrane receptor tyrosine kinases involved in HER signaling to which various ligands can be attached, leading to PI3K/AKT activation. Currently, clinical studies evaluate inhibitors of the PI3K/AKT/mTOR axis. The main purpose of this review is to present general aspects of breast cancer, the components of the AKT signaling pathway, the factors that activate this protein kinase B, PI3K/AKT-breast cancer mutations, PI3K/AKT/mTOR-inhibitors, and the relationship between everolimus, temsirolimus and endocrine therapy.     

Comprehensive Mutational and Phenotypic Characterization of New Metastatic Cutaneous Squamous Cell Carcinoma Cell Lines Reveal Novel Drug Susceptibilities

Cutaneous squamous cell carcinoma (cSCC) is a common skin cancer. Most patients who develop metastases (2–5%) present with advanced disease that requires a combination of radical surgery and adjuvant radiation therapy. There are few effective therapies for refractory disease. In this study, we describe novel patient-derived cell lines from cSCC metastases of the head and neck (designated UW-CSCC1 and UW-CSCC2). The cell lines genotypically and phenotypically resembled the original patient tumor and were tumorogenic in mice. Differences in cancer-related gene expression between the tumor and cell lines after various culturing conditions could be largely reversed by xenografting and reculturing. The novel drug susceptibilities of UW-CSCC1 and an irradiated subclone UW-CSCC1-R to drugs targeting cell cycle, PI3K/AKT/mTOR, and DNA damage pathways were observed using high-throughput anti-cancer and kinase-inhibitor compound libraries, which correlate with either copy number variations, targetable mutations and/or the upregulation of gene expression. A secondary screen of top hits in all three cell lines including PIK3CA-targeting drugs supports the utility of targeting the PI3K/AKT/mTOR pathway in this disease. UW-CSCC cell lines are thus useful preclinical models for determining targetable pathways and candidate therapeutics.     

KRAS and MAPK1 Gene Amplification in Type II Ovarian Carcinomas

In this study, we examined the clinical significance of KRAS and MAPK1 amplification and assessed whether these amplified genes were potential therapeutic targets in type II ovarian carcinoma. Using fluorescence in situ hybridization, immunohistochemistry, and retrospectively collected clinical data, KRAS and MAPK1 amplifications were identified in 9 (13.2%) and 5 (7.4%) of 68 type II ovarian carcinoma tissue samples, respectively. Interestingly, co-amplification of KRAS and MAPK1 seemed to be absent in the type II ovarian carcinomas tested, except one case. Active phospho-ERK1/2 was identified in 26 (38.2%) out of 68 type II ovarian carcinomas and did not correlate with KRAS or MAPK1 amplification. There was no significant relationship between KRAS amplification and overall or progression-free survival in patients with type II ovarian carcinoma. However, patients with MAPK1 amplification had significantly poorer progression-free survival than patients without MAPK1 amplification. Moreover, type II ovarian carcinoma cells with concomitant KRAS amplification and mutation exhibited dramatic growth reduction following treatment with the MEK inhibitor PD0325901. These findings indicate that KRAS/MAPK1 amplification is critical for the growth of a subset of type II ovarian carcinomas. Additionally, RAS/RAF/MEK/ERK pathway-targeted therapy may benefit selected patients with type II ovarian carcinoma harboring KRAS/MAPK1 amplifications.