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.     

ARID1A Mutations and PI3K/AKT Pathway Alterations in Endometriosis and Endometriosis-Associated Ovarian Carcinomas

Endometriosis is a common gynecological disease affecting 6%–10% of women of reproductive age and is characterized by the presence of endometrial-like tissue in localizations outside of the uterine cavity as, e.g., endometriotic ovarian cysts. Mainly, two epithelial ovarian carcinoma subtypes, the ovarian clear cell carcinomas (OCCC) and the endometrioid ovarian carcinomas (EnOC), have been molecularly and epidemiologically linked to endometriosis. Mutations in the gene encoding the AT-rich interacting domain containing protein 1A (ARID1A) have been found to occur in high frequency in OCCC and EnOC. The majority of these mutations lead to a loss of expression of the ARID1A protein, which is a subunit of the SWI/SNF chromatin remodeling complex and considered as a bona fide tumor suppressor. ARID1A mutations frequently co-occur with mutations, leading to an activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway, such as mutations in PIK3CA encoding the catalytic subunit, p110α, of PI3K. In combination with recent functional observations, these findings strongly suggest cooperating mechanisms between the two pathways. The occurrence of ARID1A mutations and alterations in the PI3K/AKT pathway in endometriosis and endometriosis-associated ovarian carcinomas, as well as the possible functional and clinical implications are discussed in this review.     

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.     

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.     

Oncogenic MicroRNAs Characterization in Clear Cell Renal Cell Carcinoma

A key challenge for the improvement of clear cell renal cell carcinoma (ccRCC) management could derive from a deeper characterization of the biology of these neoplasms that could greatly improve the diagnosis, prognosis and treatment choice. The aim of this study was to identify specific miRNAs that are deregulated in tumor vs. normal kidney tissues and that could impact on the biology of ccRCC. To this end we selected four miRNAs (miR-21-5p, miR-210-3p, miR-185-5p and miR-221-3p) and their expression has been evaluated in a retrospective cohort of formalin-fixed paraffin-embedded (FFPE) tissues from 20 ccRCC patients who underwent surgical nephrectomy resection. miR-21-5p and miR-210-3p resulted the most significantly up-regulated miRNAs in this patient cohort, highlighting these onco-miRNAs as possible relevant players involved in ccRCC tumorigenesis. Thus, this study reports the identification of specific oncogenic miRNAs that are altered in ccRCC tissues and suggests that they might be useful biomarkers in ccRCC management.     

The Prognostic Significance of FKBP1A and Its Related Immune Infiltration in Liver Hepatocellular Carcinoma

Liver hepatocellular carcinoma (LIHC) remains a global health challenge with poor prognosis and high mortality. FKBP1A was first discovered as a receptor for the immunosuppressant drug FK506 in immune cells and is critical for various tumors and cancers. However, the relationships between FKBP1A expression, cellular distribution, tumor immunity, and prognosis in LIHC remain unclear. Here, we investigated the expression level of FKBP1A and its prognostic value in LIHC via multiple datasets including ONCOMINE, TIMER, GEPIA, UALCAN, HCCDB, Kaplan–Meier plotter, LinkedOmics, and STRING. Human liver tissue microarray was employed to analyze the characteristics of FKBP1A protein including the expression level and pathological alteration in cellular distribution. FKBP1A expression was significantly higher in LIHC and correlated with tumor stage, grade and metastasis. The expression level of the FKBP1A protein was also increased in LIHC patients along with its accumulation in endoplasmic reticulum (ER). High FKBP1A expression was correlated with a poor survival rate in LIHC patients. The analysis of gene co-expression and the regulatory pathway network suggested that FKBP1A is mainly involved in protein synthesis, metabolism and the immune-related pathway. FKBP1A expression had a significantly positive association with the infiltration of hematopoietic immune cells including B cells, CD8⁺ T cells, CD4⁺ T cells, macrophages, neutrophils, and dendritic cells. Moreover, M2 macrophage infiltration was especially associated with a poor survival prognosis in LIHC. Furthermore, FKBP1A expression was significantly positively correlated with the expression of markers of M2 macrophages and immune checkpoint proteins such as PD-L1, CTLA-4, LAG3 and HAVCR2. Our study demonstrated that FKBP1A could be a potential prognostic target involved in tumor immune cell infiltration in LIHC.     

Beclin 1 Expression in Ovarian Tissues and Its Effects on Ovarian Cancer Prognosis

Beclin 1 is an autophagy-associated protein involved in apoptosis and drug resistance, as well as various malignancies. We investigated the expression of Beclin 1 protein in ovarian epithelial tissues and correlated it with the prognosis of ovarian cancer. Beclin 1 protein expression was determined using immunohistochemistry in 148 patients with ovarian epithelial cancer, 26 with ovarian borderline tumor, 25 with benign ovarian tumor, and 30 with normal ovarian tissue. The relationships between Beclin 1 protein expression and ovarian cancer pathological characteristics were analyzed. The risk factors for ovarian cancer prognosis were analyzed using Cox’s regression model. A survival curve was plotted from the follow-up data of 93 patients with ovarian cancer to analyze the effects of Beclin 1 expression on the prognosis of ovarian cancer. The positive rates of Beclin 1 were significantly higher in ovarian epithelial cancer (148) and borderline tumor (26) than in benign ovarian tumor (25) or normal ovarian tissue (30) (all p < 0.001). The surgical stage and Beclin 1 expression were both independent risk factors for ovarian cancer prognosis (both p < 0.05). Patients with high Beclin 1 levels showed better survival than those with low Beclin 1 levels (p = 0.009). Beclin 1 protein is upregulated in ovarian epithelial cancer and is a prognostic factor of ovarian cancer.     

Zafirlukast Induces VHL- and HIF-2α-Dependent Oxidative Cell Death in 786-O Clear Cell Renal Carcinoma Cells

Mutations in the Von Hippel–Lindau (VHL) gene are the driving force in many forms of clear cell renal cell carcinoma (ccRCC) and promote hypoxia-inducible factor (HIF)-dependent tumor proliferation, metastasis and angiogenesis. Despite the progress that has already been made, ccRCC generally remain resistant to conventional therapies and ccRCC patients suffer from metastasis and acquired resistance, highlighting the need for novel therapeutic options. Cysteinyl leukotriene receptor 1 (CysLTR1) antagonists, like zafirlukast, are administered in bronchial asthma to control eicosanoid signaling. Intriguingly, long-term use of zafirlukast decreases cancer risk and leukotriene receptor antagonists inhibit tumor growth, but the mechanisms still remain unexplored. Therefore, we aim to understand the mechanisms of zafirlukast-mediated cell death in ccRCC cells. We show that zafirlukast induces VHL-dependent and TNFα-independent non-apoptotic and non-necroptotic cell death in ccRCC cells. Cell death triggered by zafirlukast could be rescued with antioxidants and the PARP-1 inhibitor Olaparib, and additionally relies on HIF-2α. Finally, MG-132-mediated proteasome inhibition sensitized VHL wild-type cells to zafirlukast-induced cell death and inhibition of HIF-2α rescued zafirlukast- and MG-132-triggered cell death. Together, these results highlight the importance of VHL, HIF and proteasomal degradation in zafirlukast-induced oxidative cell death with potentially novel therapeutic implications for ccRCC.     

Prognostic Impact of Membranous/Nuclear Epidermal Growth Factor Receptor Localization in Clear Cell Renal Cell Carcinoma

EGFR is overexpressed in the majority of clear cell renal cell carcinomas (CCRCCs). Although EGFR deregulation was found to be of great significance in CCRCC biology, the EGFR overexpression is not associated with EGFR-targeted therapy responsiveness. Moreover, the prognostic role of EGFR expression remains controversial. In the present study, we evaluated the role played by EGFR overexpression in CCRCC and its prognostic significance associated with different immunohistochemical localization patterns. In our study, the Total Score (TS) related to membranous-cytoplasmic EGFR expression showed a significant correlation with grade, pathologic stage (pT), and Stage, Size, Grade, and Necrosis (SSIGN) score, and a negative correlation with nuclear EGFR expression. No significant correlations were shown between nuclear EGFR and clinic-pathological features. Additionally, a correlation between SGLT1 expression levels and pT was described. Multivariate analysis identifies pT and SSIGN score as independent prognostic factors for CCRCC. A significantly increased survival rate was found in the case of positive expression of nuclear EGFR and SGLT1. Based on our findings, SGLT1 and nuclear EGFR overexpression defines a subgroup of CCRCC patients with good prognosis. Membranous-cytoplasmic EGFR expression was shown to be a poor prognostic factor and could define a CCRCC subgroup with poor prognosis that should be responsive to anti-EGFR therapies.     

Characteristics of Clear Cell Papillary Renal Cell Carcinoma (ccpRCC)

Renal cell carcinomas (RCCs) is a group of various malignant tumours of the renal cortex displaying distinct clinical, morphologic, and genetic features. Clear cell papillary renal cell carcinoma (ccpRCC), belonging to this group, shares morphologic features with both clear cell renal cell carcinoma (ccRCC) and papillary renal cell carcinoma (pRCC) and therefore, more strict diagnostic criteria should be developed to avoid misdiagnosis. Despite overlapping features, ccpRCC has also distinct clinical behaviour, histologic characteristics (morphologic and immunohistochemical), and genomic features. The concepts concerning this tumour are constantly developing since its biological potential and molecular basis remains to be fully unravelled. First reports indicated the presence of ccpRCC in end-stage renal disease, and they underlined the enriched development in this group of patients; however, currently, it is known that such tumours can also occur spontaneously in the normal kidney. Numerous studies have demonstrated that clinical outcomes and prognosis of ccpRCC patients is highly favourable. Till now, no convincing evidence of metastatic ccpRCC or death caused by the disease has been found. Therefore, it is of high importance to correctly differentiate ccpRCC from other subtypes of RCC with a much worse prognosis and to introduce appropriate management.     

Vitamin D Analogs Regulate the Vitamin D System and Cell Viability in Ovarian Cancer Cells

Background: Ovarian cancer (OC) is one of the most lethal cancers in women. The active form of vitamin D₃, 1,25-dihydroxyvitamin D₃ (1,25D₃, calcitriol) has anticancer activity in several cancers, including ovarian cancer, but the required pharmacological doses may cause hypercalcemia. We hypothesized that newly developed, low calcemic, vitamin D analogs (an1,25Ds) may be used as anticancer agents instead of calcitriol in ovarian cancer cells. Methods: We used two patient-derived high-grade serous ovarian cancer (HGSOC) cell lines with low (13781) and high (14433) mRNA expression levels of the gene encoding 1,25-dihydroxyvitamin D₃ 24-hydroxylase CYP24A1, one of the main target genes of calcitriol. We tested the effect of calcitriol and four structurally related series of an1,25Ds (PRI-1906, PRI-1907, PRI-5201, PRI-5202) on cell number, viability, the expression of CYP24A1, and the vitamin D receptor (VDR). Results: CYP24A1 mRNA expression increased in a concentration-dependent manner after treatment with all compounds. In both cell lines, after 4 h, PRI-5202 was the most potent analog (in 13781 cells: EC₅₀ = 2.98 ± 1.10 nmol/L, in 14433 cells: EC₅₀ = 0.92 ± 0.20 nmol/L), while PRI-1907 was the least active one (in 13781 cells: EC₅₀ = n/d, in 14433 cells: EC₅₀ = n/d). This difference among the analogs disappeared after 5 days of treatment. The 13781 cells were more sensitive to the an1,25Ds compared with 14433 cells. The an1,25Ds increased nuclear VDR levels and reduced cell viability, but only in the 13781 cell line. Conclusions: The an1,25Ds had different potencies in the HGSOC cell lines and their efficacy in increasing CYP24A1 expression was cell line- and chemical structure-dependent. Therefore, choosing sensitive cancer cell lines and further optimization of the analogs’ structure might lead to new treatment options against ovarian cancer.     

Focus on PD-1/PD-L1 as a Therapeutic Target in Ovarian Cancer

Ovarian cancer is considered one of the most aggressive and deadliest gynecological malignancies worldwide. Unfortunately, the therapeutic methods that are considered the gold standard at this moment are associated with frequent recurrences. Survival in ovarian cancer is associated with the presence of a high number of intra tumor infiltrating lymphocytes (TILs). Therefore, immunomodulation is considered to have an important role in cancer treatment, and immune checkpoint inhibitors may be useful for restoring T cell-mediated antitumor immunity. However, the data presented in the literature until now are not sufficient to allow for the identification and selection of patients who really respond to immunotherapy among those with ovarian cancer. Although there are some studies with favorable results, more prospective trials are needed in this sense. This review focuses on the current and future perspectives of PD-1/L1 blockade in ovarian cancer and analyzes the most important immune checkpoint inhibitors used, with the aim of achieving optimal clinical outcomes. Future studies and trials are needed to maximize the efficacy of immune checkpoint blockade therapy in ovarian cancer, as well as in all cancers, in general.     

High Levels of KAP1 Expression Are Associated with Aggressive Clinical Features in Ovarian Cancer

KAP1 is an universal corepressor for Kruppel-associated box zinc finger proteins in both normal and tumor cells. In this study, the biological function and clinical significance of KAP1 expression in ovarian cancer were investigated. Immunohistological staining of KAP1 was evaluated in 111 patients with ovarian epithelial cancer, 15 with ovarian borderline tumor, and 20 normal ovarian tissue. The correlations of KAP1 expression with clinicopathological features were studied. Kaplan-Meier analysis and Cox proportional hazard modeling were used to assess overall survival to analyze the effect of KAP1 expression on the prognosis of ovarian cancer. The positive rates of KAP1 were significantly higher in ovarian epithelial cancer (55.7%) and borderline tumor (20.0%) than in normal ovarian tissue (5.0%) (all p < 0.01). KAP1 expression correlated significantly with clinical stage (χ² = 14.57, p < 0.0001), pathological grade (χ² = 6.06, p = 0.048) and metastases (χ² =10.38, p = 0.001). Patients with high KAP 1 levels showed poor survival (p < 0.0001). Multivariate analysis showed that KAP1 high expression was an independent predictor for ovarian cancer patients (hazard ratio = 0.463; 95% confidence interval = 0.230–0.9318, p = 0.031). Functionally, depletion of KAP1 by siRNA inhibited ovarian cancer cell proliferation, cell migration. KAP1 expression correlated with aggressive clinical features in ovarian cancer. High KAP1 expression was a prognostic factor of ovarian cancer.     

The Impact of m1A Methylation Modification Patterns on Tumor Immune Microenvironment and Prognosis in Oral Squamous Cell Carcinoma

N1-methyladenosine (m1A) modification widely participates in the occurrence and progression of numerous diseases. Nevertheless, the potential roles of m1A in the tumor immune microenvironment (TIME) are still not fully understood. Based on 10 m1A methylation regulators, we comprehensively explored the m1A modification patterns in 502 patients with oral squamous cell carcinoma (OSCC). The m1A modification patterns were correlated with TIME characteristics and the m1A score was established to evaluate the effect of the m1A modification patterns on individual OSCC patients. The TIME characteristics and survival outcomes under the three m1A modification patterns were significantly distinct. OSCC patients in the high m1A score group were characterized by poorer prognosis, lower immune infiltration, lower ssGSEA score, lower expression levels of immune checkpoint molecules, and higher tumor mutation loads. The present study revealed that m1A modification might be associated with the TIME in OSCC, and has potential predictive ability for the prognosis of OSCC.