Estrogen Receptor ß Modulates Apoptosis Complexes and the Inflammasome to Drive the Pathogenesis of Endometriosis.

Alterations in estrogen-mediated cellular signaling play an essential role in the pathogenesis of endometriosis. In addition to higher estrogen receptor (ER) ß levels, enhanced ERß activity was detected in endometriotic tissues, and the inhibition of enhanced ERß activity by an ERß-selective antagonist suppressed mouse ectopic lesion growth. Notably, gain of ERß function stimulated the progression of endometriosis. As a mechanism to evade endogenous immune surveillance for cell survival, ERß interacts with cellular apoptotic machinery in the cytoplasm to inhibit TNF-α-induced apoptosis. ERß also interacts with components of the cytoplasmic inflammasome to increase interleukin-1ß and thus enhance its cellular adhesion and proliferation properties. Furthermore, this gain of ERß function enhances epithelial-mesenchymal transition signaling, thereby increasing the invasion activity of endometriotic tissues for establishment of ectopic lesions. Collectively, we reveal how endometrial tissue generated by retrograde menstruation can escape immune surveillance and develop into sustained ectopic lesions via gain of ERß function.

Targeting progesterone signaling prevents metastatic ovarian cancer.

Effective cancer prevention requires the discovery and intervention of a factor critical to cancer development. Here we show that ovarian progesterone is a crucial endogenous factor inducing the development of primary tumors progressing to metastatic ovarian cancer in a mouse model of high-grade serous carcinoma (HGSC), the most common and deadliest ovarian cancer type. Blocking progesterone signaling by the pharmacologic inhibitor mifepristone or by genetic deletion of the progesterone receptor (PR) effectively suppressed HGSC development and its peritoneal metastases. Strikingly, mifepristone treatment profoundly improved mouse survival (∼18 human years). Hence, targeting progesterone/PR signaling could offer an effective chemopreventive strategy, particularly in high-risk populations of women carrying a deleterious mutation in the BRCA gene.

Vaginal Squamous Cell Carcinoma Develops in Mice with Conditional Arid1a Loss and Gain of Oncogenic Kras Driven by Progesterone Receptor Cre.

Oncogenic KRAS mutations are a common finding in endometrial cancers. Recent sequencing studies indicate that loss-of-function mutations in the ARID1A gene are enriched in gynecologic malignant tumors. However, neither of these genetic insults alone are sufficient to develop gynecologic cancer. To determine the role of the combined effects of deletion of Arid1a and oncogenic Kras, Arid1aflox/flox mice were crossed with KrasLox-Stop-Lox-G12D/+ mice using progesterone receptor Cre (PgrCre/+). Histologic analysis and immunohistochemistry of survival studies were used to characterize the mutant mouse phenotype. Hormone dependence was evaluated by ovarian hormone depletion and estradiol replacement. Arid1aflox/flox; KrasLox-Stop-Lox-G12D/+; PgrCre/+ mice were euthanized early because of invasive vaginal squamous cell carcinoma. Younger mice had precancerous intraepithelial lesions. Immunohistochemistry supported the pathological diagnosis with abnormal expression and localization of cytokeratin 5, tumor protein P63, cyclin-dependent kinase inhibitor 2A, and Ki-67, the marker of proliferation. Ovarian hormone deletion in Arid1aflox/flox; KrasLox-Stop-Lox-G12D/+; PgrCre/+ mice resulted in atrophic vaginal epithelium without evidence of vaginal tumors. Estradiol replacement in ovarian hormone-depleted Arid1aflox/flox; KrasLox-Stop-Lox-G12D/+; PgrCre/+ mice resulted in lesions that resembled the squamous cell carcinoma in intact mice. Therefore, this mouse can be used to study the transition from benign precursor lesions into invasive vaginal human papillomavirus-independent squamous cell carcinoma, offering insights into progression and pathogenesis of this rare disease.

Building a virtual summer research experience in cancer for high school and early undergraduate students: lessons from the COVID-19 pandemic.

BACKGROUND: The COVID-19 pandemic posed a unique challenge for summer research programs in 2020, particularly for programs aimed at hands-on experience for younger trainees. The Indiana University Melvin and Bren Simon Comprehensive Cancer Center supports two pipeline programs, which traditionally immerse high school juniors, seniors, and early undergraduate students from underrepresented populations in science in hands-on projects in cancer biology labs. However, due to social distancing policies during the pandemic and reduction of research operations, these students were not physically allowed on campus. Thus, the authors set out to strategically pivot to a wholly virtual curriculum and evaluate the Virtual Summer Research Experience in Cancer outcomes. METHODS: The virtual program included four components: 1. a core science and professional development curriculum led by high school teachers and senior undergraduates; 2. faculty-delivered didactic sessions on cancer science; 3. mentored, virtual research projects with research faculty; and 4. online networking events to encourage vertical mentoring. Outcomes data were measured using a locally created 11-item Research Preparation Scale, daily electronic feedback, and weekly structured evaluation and feedback via Zoom. RESULTS: Outcome data suggested high self-reported satisfaction with the virtual program. Outcome data also revealed the importance of coordination between multiple entities for seamless program implementation. This includes the active recruitment and participation of high school teachers and further investment in information technology capabilities of institutions. CONCLUSIONS: Findings reveal a path to educate and train high school and early undergraduate students in cancer research when hands-on, in-person training is not feasible. Virtual research experiences are not only useful to engage students during public health crises but can provide an avenue for cancer centers to expand their cancer education footprints to remotely located schools and universities with limited resources to provide such experiences to their students.

Translational Applications of Linear and Circular Long Noncoding RNAs in Endometriosis.

Endometriosis is a chronic gynecologic disease that negatively affects the quality of life of many women. Unfortunately, endometriosis does not have a cure. The current medical treatments involve hormonal manipulation with unwanted side effects and high recurrence rates after stopping the medication. Sadly, a definitive diagnosis for endometriosis requires invasive surgical procedures, with the risk of complications, additional surgeries in the future, and a high rate of recurrence. Both improved therapies and noninvasive diagnostic tests are needed. The unique molecular features of endometriosis have been studied at the coding gene level. While the molecular components of endometriosis at the small RNA level have been studied extensively, other noncoding RNAs, such as long intergenic noncoding RNAs and the more recently discovered subset of long noncoding RNAs called circular RNAs, have been studied more limitedly. This review describes the molecular formation of long noncoding and the unique circumstances of the formation of circular long noncoding RNAs, their expression and function in endometriosis, and promising preclinical studies. Continued translational research on long noncoding RNAs, including the more stable circular long noncoding RNAs, may lead to improved therapeutic and diagnostic opportunities.

Uterine Epithelial Development and Enhancer of Zeste Homolog 2: It Is Important for More than Just Cancer.

This commentary highlights the article by Fang et al that describes the role of enhancer of zeste homolog 2 in endometrial development.

Distinct molecular pathways in ovarian endometrioid adenocarcinoma with concurrent endometriosis.

Women with endometriosis, a benign growth of endometrial tissue outside the uterine cavity, are at increased risk of specific histotypes of epithelial ovarian cancer, such as ovarian endometrioid adenocarcinoma (OEA). Women with OEA who have endometriosis at time of surgical staging demonstrate improved clinical prognosis compared to women with OEA without evidence of endometriosis. However, the molecular contributions of the endometriotic tumor microenvironment to these ovarian cancers remain poorly understood. As a starting point, we used a platform for genome-wide transcriptomic profiling to compare specimens of OEA from women with and without concurrent endometriosis and benign reproductive tract tissues, including proliferative endometrium and typical and atypical endometrioma samples (n = 20). Principle component analysis revealed distinct clustering between benign and malignant samples as well as malignant samples with and without concurrent endometriosis. Examination of gene signatures revealed that OEA with concurrent endometriosis contained a unique molecular signature compared to OEA without concurrent endometriosis, distinguished by 682 unique genes differentially expressed (fold change < or >1.5, p < 0.01). Bioinformatic analysis of these differentially expressed gene products using ingenuity pathway analysis revealed activation of NFkB signaling, an inflammatory signaling pathway constitutively active in endometriosis. DAVID functional annotation clustering further revealed enrichment in RAS signaling as both cytoskeleton organization and GTPase regulator activity relied heavily on RAS protein signal transduction. Gene set enrichment analysis highlighted immune and inflammatory nodes involved in OEA with concurrent endometriosis. These observations provide novel resources for understanding molecular subtleties potentially involved in OEA within the context of the endometriotic tumor microenvironment.

Recurrent BCAM-AKT2 fusion gene leads to a constitutively activated AKT2 fusion kinase in high-grade serous ovarian carcinoma.

High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. Excessive genomic rearrangements, which are expected to create fusion oncogenes, are the hallmark of this cancer. Here we report a cancer-specific gene fusion between BCAM, a membrane adhesion molecule, and AKT2, a key kinase in the PI3K signaling pathway. This fusion is present in 7% of the 60 patient cancers tested, a significant frequency considering the highly heterogeneous nature of this malignancy. Further, we provide direct evidence that BCAM-AKT2 is translated into an in-frame fusion protein in the patient's tumor. The resulting AKT2 fusion kinase is membrane-associated, constitutively phosphorylated, and activated as a functional kinase in cells. Unlike endogenous AKT2, whose activity is tightly regulated by external stimuli, BCAM-AKT2 escapes the regulation from external stimuli. Moreover, a BCAM-AKT2 fusion gene generated via chromosomal translocation using the CRISPR/Cas9 system leads to focus formation in both OVCAR8 and HEK-293T cell lines, suggesting that BCAM-AKT2 is oncogenic. Together, the results indicate that BCAM-AKT2 expression is a new mechanism of AKT2 kinase activation in HGSC. BCAM-AKT2 is the only fusion gene in HGSC that is proven to translate an aberrant yet functional kinase fusion protein with oncogenic properties. This recurrent genomic alteration is a potential therapeutic target and marker of a clinically relevant subtype for tailored therapy of HGSC.

ARID1A Is Essential for Endometrial Function during Early Pregnancy.

AT-rich interactive domain 1A gene (ARID1A) loss is a frequent event in endometriosis-associated ovarian carcinomas. Endometriosis is a disease in which tissue that normally grows inside the uterus grows outside the uterus, and 50% of women with endometriosis are infertile. ARID1A protein levels were significantly lower in the eutopic endometrium of women with endometriosis compared to women without endometriosis. However, an understanding of the physiological effects of ARID1A loss remains quite poor, and the function of Arid1a in the female reproductive tract has remained elusive. In order to understand the role of Arid1a in the uterus, we have generated mice with conditional ablation of Arid1a in the PGR positive cells (Pgrcre/+Arid1af/f; Arid1ad/d). Ovarian function and uterine development of Arid1ad/d mice were normal. However, Arid1ad/d mice were sterile due to defective embryo implantation and decidualization. The epithelial proliferation was significantly increased in Arid1ad/d mice compared to control mice. Enhanced epithelial estrogen activity and reduced epithelial PGR expression, which impedes maturation of the receptive uterus, was observed in Arid1ad/d mice at the peri-implantation period. The microarray analysis revealed that ARID1A represses the genes related to cell cycle and DNA replication. We showed that ARID1A positively regulates Klf15 expression with PGR to inhibit epithelial proliferation at peri-implantation. Our results suggest that Arid1a has a critical role in modulating epithelial proliferation which is a critical requisite for fertility. This finding provides a new signaling pathway for steroid hormone regulation in female reproductive biology and furthers our understanding of the molecular mechanisms that underlie dysregulation of hormonal signaling in human reproductive disorders such as endometriosis.

The role of MicroRNA molecules and MicroRNA-regulating machinery in the pathogenesis and progression of epithelial ovarian cancer.

MicroRNA molecules are small, single-stranded RNA molecules that function to regulate networks of genes. They play important roles in normal female reproductive tract biology, as well as in the pathogenesis and progression of epithelial ovarian cancer. DROSHA, DICER, and Argonaute proteins are components of the microRNA-regulatory machinery and mediate microRNA production and function. This review discusses aberrant expression of microRNA molecules and microRNA-regulating machinery associated with clinical features of epithelial ovarian cancer. Understanding the regulation of microRNA molecule production and function may facilitate the development of novel diagnostic and therapeutic strategies to improve the prognosis of women with epithelial ovarian cancer. Additionally, understanding microRNA molecules and microRNA-regulatory machinery associations with clinical features may influence prevention and early detection efforts.

CDKN2D-WDFY2 is a cancer-specific fusion gene recurrent in high-grade serous ovarian carcinoma.

Ovarian cancer is the fifth leading cause of cancer death in women. Almost 70% of ovarian cancer deaths are due to the high-grade serous subtype, which is typically detected only after it has metastasized. Characterization of high-grade serous cancer is further complicated by the significant heterogeneity and genome instability displayed by this cancer. Other than mutations in TP53, which is common to many cancers, highly recurrent recombinant events specific to this cancer have yet to be identified. Using high-throughput transcriptome sequencing of seven patient samples combined with experimental validation at DNA, RNA and protein levels, we identified a cancer-specific and inter-chromosomal fusion gene CDKN2D-WDFY2 that occurs at a frequency of 20% among sixty high-grade serous cancer samples but is absent in non-cancerous ovary and fallopian tube samples. This is the most frequent recombinant event identified so far in high-grade serous cancer implying a major cellular lineage in this highly heterogeneous cancer. In addition, the same fusion transcript was also detected in OV-90, an established high-grade serous type cell line. The genomic breakpoint was identified in intron 1 of CDKN2D and intron 2 of WDFY2 in patient tumor, providing direct evidence that this is a fusion gene. The parental gene, CDKN2D, is a cell-cycle modulator that is also involved in DNA repair, while WDFY2 is known to modulate AKT interactions with its substrates. Transfection of cloned fusion construct led to loss of wildtype CDKN2D and wildtype WDFY2 protein expression, and a gain of a short WDFY2 protein isoform that is presumably under the control of the CDKN2D promoter. The expression of short WDFY2 protein in transfected cells appears to alter the PI3K/AKT pathway that is known to play a role in oncogenesis. CDKN2D-WDFY2 fusion could be an important molecular signature for understanding and classifying sub-lineages among heterogeneous high-grade serous ovarian carcinomas.

PAPD5-mediated 3' adenylation and subsequent degradation of miR-21 is disrupted in proliferative disease.

Next-generation sequencing experiments have shown that microRNAs (miRNAs) are expressed in many different isoforms (isomiRs), whose biological relevance is often unclear. We found that mature miR-21, the most widely researched miRNA because of its importance in human disease, is produced in two prevalent isomiR forms that differ by 1 nt at their 3' end, and moreover that the 3' end of miR-21 is posttranscriptionally adenylated by the noncanonical poly(A) polymerase PAPD5. PAPD5 knockdown caused an increase in the miR-21 expression level, suggesting that PAPD5-mediated adenylation of miR-21 leads to its degradation. Exoribonuclease knockdown experiments followed by small-RNA sequencing suggested that PARN degrades miR-21 in the 3'-to-5' direction. In accordance with this model, microarray expression profiling demonstrated that PAPD5 knockdown results in a down-regulation of miR-21 target mRNAs. We found that disruption of the miR-21 adenylation and degradation pathway is a general feature in tumors across a wide range of tissues, as evidenced by data from The Cancer Genome Atlas, as well as in the noncancerous proliferative disease psoriasis. We conclude that PAPD5 and PARN mediate degradation of oncogenic miRNA miR-21 through a tailing and trimming process, and that this pathway is disrupted in cancer and other proliferative diseases.

Deletion of Arid1a in Reproductive Tract Mesenchymal Cells Reduces Fertility in Female Mice.

Women with endometriosis can suffer from decreased fecundity or complete infertility via abnormal oocyte function or impaired placental-uterine interactions required for normal pregnancy establishment and maintenance. Although AT-rich interactive domain 1A (SWI-like) (ARID1A) is a putative tumor suppressor in human endometrial cancers and endometriosis-associated ovarian cancers, little is known about its role in normal uterine function. To study the potential function of ARID1A in the female reproductive tract, we generated mice with a conditional knockout of Arid1a using anti-Müllerian hormone receptor 2-Cre Female Arid1a conditional knockout mice exhibited a progressive decrease in number of pups per litter, with a precipitous decline after the second litter. We observed no tumors in virgin mice, although one knockout mouse developed a uterine tumor after pregnancy. Unstimulated virgin female knockout mice showed normal oviductal, ovarian, and uterine histology. Uteri of Arid1a knockout mice showed a normal decidualization response and appropriate responses to estradiol and progesterone stimulation. In vitro studies using primary cultures of human endometrial stromal fibroblasts revealed that small interfering RNA knockdown of ARID1A did not affect decidualization in vitro. Timed pregnancy studies revealed the significant resorption of embryos at Embryonic Day 16.5 in knockout mice in the third pregnancy. In addition to evidence of implantation site hemorrhage, pregnant Arid1a knockout mice showed abnormal placental morphology. These results suggest that Arid1a supports successful pregnancy through its role in placental function.

Activin-like kinase 2 functions in peri-implantation uterine signaling in mice and humans.

Implantation of a blastocyst in the uterus is a multistep process tightly controlled by an intricate regulatory network of interconnected ovarian, uterine, and embryonic factors. Bone morphogenetic protein (BMP) ligands and receptors are expressed in the uterus of pregnant mice, and BMP2 has been shown to be a key regulator of implantation. In this study, we investigated the roles of the BMP type 1 receptor, activin-like kinase 2 (ALK2), during mouse pregnancy by producing mice carrying a conditional ablation of Alk2 in the uterus (Alk2 cKO mice). In the absence of ALK2, embryos demonstrate delayed invasion into the uterine epithelium and stroma, and upon implantation, stromal cells fail to undergo uterine decidualization, resulting in sterility. Mechanistically, microarray analysis revealed that CCAAT/enhancer-binding protein ß (Cebpb) expression is suppressed during decidualization in Alk2 cKO females. These findings and the similar phenotypes of Cebpb cKO and Alk2 cKO mice lead to the hypothesis that BMPs act upstream of CEBPB in the stroma to regulate decidualization. To test this hypothesis, we knocked down ALK2 in human uterine stromal cells (hESC) and discovered that ablation of ALK2 alters hESC decidualization and suppresses CEBPB mRNA and protein levels. Chromatin immunoprecipitation (ChIP) analysis of decidualizing hESC confirmed that BMP signaling proteins, SMAD1/5, directly regulate expression of CEBPB by binding a distinct regulatory sequence in the 3' UTR of this gene; CEBPB, in turn, regulates the expression of progesterone receptor (PGR). Our work clarifies the conserved mechanisms through which BMPs regulate peri-implantation in rodents and primates and, for the first time, uncovers a linear pathway of BMP signaling through ALK2 to regulate CEBPB and, subsequently, PGR during decidualization.

Cytokine stimulation of MUC4 expression in human female reproductive tissue carcinoma cell lines and endometrial cancer.

MUC4, a transmembrane glycoprotein, interferes with cell adhesion, and promotes EGFR signaling in cancer. Studies in rat models have demonstrated steroid hormonal regulation of endometrial MUC4 expression. In this study, qRT-PCR screening of mouse tissues determined that Muc4 mRNA also was robustly expressed in mouse uteri. Previous studies from our labs have demonstrated MUC4 mRNA was expressed at levels <1% of MUC1 mRNA in human endometrium and endometriotic tissue. Multiple human endometrial adenocarcinoma cell lines were assayed for MUC4 mRNA expression revealing extremely low basal expression in the Ishikawa, RL-95-2, AN3CA, and KLE lines. Moderate to high expression was observed in HEC50 and HEC-1A cells. MUC4 mRNA expression was not affected by progesterone and/or estrogen treatment, but was greatly stimulated at both mRNA and protein levels by proinflammatory cytokines (IFN-γ and TNF-α), particularly when used in combination. In endometrial tissue, MUC4 mRNA levels did not change significantly between normal or cancerous samples; although, a subset of patients with grade 1 and 2 tumors displayed substantially higher expression. Likewise, immunostaining of human endometrial adenocarcinoma tissues revealed little to no staining in many patients (low MUC4), but strong staining in some patients (high MUC4) independent of cancer grade. In cases where staining was observed, it was heterogeneous with some cells displaying robust MUC4 expression and others displaying little or no staining. Collectively, these observations demonstrate that while MUC4 is highly expressed in the mouse uterus, it is not a major mucin in normal human endometrium. Rather, MUC4 is a potential marker of endometrial adenocarcinoma in a subset of patients.

Role of microRNAs in cancers of the female reproductive tract: insights from recent clinical and experimental discovery studies.

microRNAs (miRNAs) are small RNA molecules that represent the top of the pyramid of many tumorigenesis cascade pathways as they have the ability to affect multiple, intricate, and still undiscovered downstream targets. Understanding how miRNA molecules serve as master regulators in these important networks involved in cancer initiation and progression open up significant innovative areas for therapy and diagnosis that have been sadly lacking for deadly female reproductive tract cancers. This review will highlight the recent advances in the field of miRNAs in epithelial ovarian cancer, endometrioid endometrial cancer and squamous-cell cervical carcinoma focusing on studies associated with actual clinical information in humans. Importantly, recent miRNA profiling studies have included well-characterized clinical specimens of female reproductive tract cancers, allowing for studies correlating miRNA expression with clinical outcomes. This review will summarize the current thoughts on the role of miRNA processing in unique miRNA species present in these cancers. In addition, this review will focus on current data regarding miRNA molecules as unique biomarkers associated with clinically significant outcomes such as overall survival and chemotherapy resistance. We will also discuss why specific miRNA molecules are not recapitulated across multiple studies of the same cancer type. Although the mechanistic contributions of miRNA molecules to these clinical phenomena have been confirmed using in vitro and pre-clinical mouse model systems, these studies are truly only the beginning of our understanding of the roles miRNAs play in cancers of the female reproductive tract. This review will also highlight useful areas for future research regarding miRNAs as therapeutic targets in cancers of the female reproductive tract.

High-grade serous ovarian cancer arises from fallopian tube in a mouse model.

Although ovarian cancer is the most lethal gynecologic malignancy in women, little is known about how the cancer initiates and metastasizes. In the last decade, new evidence has challenged the dogma that the ovary is the main source of this cancer. The fallopian tube has been proposed instead as the primary origin of high-grade serous ovarian cancer, the subtype causing 70% of ovarian cancer deaths. By conditionally deleting Dicer, an essential gene for microRNA synthesis, and Pten, a key negative regulator of the PI3K pathway, we show that high-grade serous carcinomas arise from the fallopian tube in mice. In these Dicer-Pten double-knockout mice, primary fallopian tube tumors spread to engulf the ovary and then aggressively metastasize throughout the abdominal cavity, causing ascites and killing 100% of the mice by 13 mo. Besides the clinical resemblance to human serous cancers, these fallopian tube cancers highly express genes that are known to be up-regulated in human serous ovarian cancers, also demonstrating molecular similarities. Although ovariectomized mice continue to develop high-grade serous cancers, removal of the fallopian tube at an early age prevents cancer formation--confirming the fallopian tube origin of the cancer. Intriguingly, the primary carcinomas are first observed in the stroma of the fallopian tube, suggesting that these epithelial cancers have a mesenchymal origin. Thus, this mouse model demonstrates a paradigm for the origin and initiation of high-grade serous ovarian carcinomas, the most common and deadliest ovarian cancer.

Functional analysis of miR-34c as a putative tumor suppressor in high-grade serous ovarian cancer.

Altered microRNA expression patterns are implicated in the formation of many human diseases, including ovarian cancer. Our laboratory previously created Dicer(fl/fl)/Pten(fl/fl)/Amhr2(cre/+) mice, which developed high-grade serous carcinomas originating from mouse fallopian tubes, while neither Dicer(fl/fl)/Amhr2(cre/+) nor Pten(fl/fl)/Amhr2(cre/+) mice developed tumors. To explore miRNAs involved in the tumorigenesis in the double-knockout (DKO) mice, tumor cell lines were established from mouse primary tumors, and the most abundant miRNAs present in mouse normal fallopian tubes, let-7b and miR-34c, were expressed in these cell lines. We found that miR-34c had a more dramatic effect on inhibiting tumor cell viability than let-7b. The action of miR-34c induced tumor cell cycle arrest in G1 phase and apoptosis, and was accompanied with the regulation of key genes involved in cell proliferation and cell cycle G1/S transition. miR-34c suppressed the expression of Ezh2 and Mybl2, which may transcriptionally and functionally activate Cdkn1c. Furthermore, miR-34c levels are extremely low in human serous adenocarcinomas compared with human normal fallopian tubes. Expression of miR-34c in human ovarian cancer cells phenocopied its effects in DKO mouse tumor cells. However, miR-34b/c(-/-)/Pten(fl/fl)/Amhr2(cre/+) mice failed to develop high-grade serous carcinomas, implicating a combination of miRNAs in the tumorigenesis process. Thus, while miR-34c is a putative tumor suppressor in high-grade serous ovarian carcinoma with potential therapeutic advantages, screening of additional miRNAs for their effects alone and in combination with miR-34c is highly warranted to uncover miRNAs that synergize with miR-34c against cancer.

Transcriptomic analyses of ovarian clear-cell carcinoma with concurrent endometriosis.

Introduction: Endometriosis, a benign inflammatory disease whereby endometrial-like tissue grows outside the uterus, is a risk factor for endometriosis-associated ovarian cancers. In particular, ovarian endometriomas, cystic lesions of deeply invasive endometriosis, are considered the precursor lesion for ovarian clear-cell carcinoma (OCCC). Methods: To explore this transcriptomic landscape, OCCC from women with pathology-proven concurrent endometriosis (n = 4) were compared to benign endometriomas (n = 4) by bulk RNA and small-RNA sequencing. Results: Analysis of protein-coding genes identified 2449 upregulated and 3131 downregulated protein-coding genes (DESeq2, P< 0.05, log2 fold-change > |1|) in OCCC with concurrent endometriosis compared to endometriomas. Gene set enrichment analysis showed upregulation of pathways involved in cell cycle regulation and DNA replication and downregulation of pathways involved in cytokine receptor signaling and matrisome. Comparison of pathway activation scores between the clinical samples and publicly-available datasets for OCCC cell lines revealed significant molecular similarities between OCCC with concurrent endometriosis and OVTOKO, OVISE, RMG1, OVMANA, TOV21G, IGROV1, and JHOC5 cell lines. Analysis of miRNAs revealed 64 upregulated and 61 downregulated mature miRNA molecules (DESeq2, P< 0.05, log2 fold-change > |1|). MiR-10a-5p represented over 21% of the miRNA molecules in OCCC with endometriosis and was significantly upregulated (NGS: log2fold change = 4.37, P = 2.43e-18; QPCR: 8.1-fold change, P< 0.05). Correlation between miR-10a expression level in OCCC cell lines and IC50 (50% inhibitory concentration) of carboplatin in vitro revealed a positive correlation (R2 = 0.93). MiR-10a overexpression in vitro resulted in a significant decrease in proliferation (n = 6; P< 0.05) compared to transfection with a non-targeting control miRNA. Similarly, the cell-cycle analysis revealed a significant shift in cells from S and G2 to G1 (n = 6; P< 0.0001). Bioinformatic analysis predicted that miR-10a-5p target genes that were downregulated in OCCC with endometriosis were involved in receptor signaling pathways, proliferation, and cell cycle progression. MiR-10a overexpression in vitro was correlated with decreased expression of predicted miR-10a target genes critical for proliferation, cell-cycle regulation, and cell survival including [SERPINE1 (3-fold downregulated; P< 0.05), CDK6 (2.4-fold downregulated; P< 0.05), and RAP2A (2-3-fold downregulated; P< 0.05)]. Discussion: These studies in OCCC suggest that miR-10a-5p is an impactful, potentially oncogenic molecule, which warrants further studies.

Unintended Consequences of Antibiotic Therapy on the Microbiome Delivers a Gut Punch in Ovarian Cancer.

While the early use of antibiotics during chemotherapy may be lifesaving, antibiotic therapy is associated with worse outcomes in patients with ovarian cancer during platinum chemotherapy. The study by Chambers and colleagues in this issue of Cancer Research provides mechanistic insights into how disrupting the gut microbiome with broad-spectrum antibiotics negatively influences the survival of patients with ovarian cancer and highlights the impact of the gut microbiome on tumor progression and response to therapy. Treatment of ovarian cancer models with a broad-spectrum antibiotic cocktail (ABX, vancomycin, neomycin sulfate, metronidazole, ampicillin) changed the gut microbiome and increased tumor growth and development of cisplatin resistance. Stem cells, reported to drive resistance to chemotherapy and disease recurrence in ovarian cancer, were enriched as a surprising consequence of ABX-induced microbiome disruption. Immune-competent and immune-deficient mice revealed that ABX treatment enhanced the cisplatin-induced stemness and provided evidence for immune surveillance of ovarian cancer stem cells through the gut microbiome. Two gut-derived metabolites, indole-3-propionic acid and indoxyl sulfate, suppressed by ABX treatment and reestablished with cecal microbial transplantation colonization of ABX-treated mice, were identified as potential effectors connecting the gut microbiome to ovarian cancer growth. This clinically relevant study opens new therapeutic opportunities for patients-one aimed at interventions to increase platinum sensitivity and another aimed at preventing the potential adverse effects of broad-spectrum antibiotic treatment. Both represent paradigm changes to standard care. See related article by Chambers et al., p. 4654.