Entinostat, a selective HDAC1/2 inhibitor, potentiates the effects of olaparib in homologous recombination proficient ovarian cancer.

OBJECTIVE: Poly ADP ribose polymerase inhibitors (PARPi) are most effective in BRCA1/2 mutated ovarian tumors. Better treatments are needed for homologous recombination HR-proficient cancer, including CCNE1 amplified subtypes. We have shown that histone deacetylase inhibitors (HDACi) sensitize HR-proficient ovarian cancer to PARPi. In this study, we provide complementary preclinical data for an investigator-initiated phase 1/2 clinical trial of the combination of olaparib and entinostat in recurrent, HR-proficient ovarian cancer. METHODS: We assessed the in vitro effects of the combination of olaparib and entinostat in SKOV-3, OVCAR-3 and primary cells derived from CCNE1 amplified high grade serous ovarian cancer (HGSOC) patients. We then tested the combination in a SKOV-3 xenograft model and in a patient-derived xenograft (PDX) model. RESULTS: Entinostat potentiates the effect of olaparib in reducing cell viability and clonogenicity of HR-proficient ovarian cancer cells. The combination reduces peritoneal metastases in a SKOV-3 xenograft model and prolongs survival in a CCNE1 amplified HR-proficient PDX model. Entinostat also enhances olaparib-induced DNA damage. Further, entinostat decreases BRCA1, a key HR repair protein, associated with decreased Ki-67, a proliferation marker, and increased cleaved PARP, a marker of apoptosis. Finally, entinostat perturbs replication fork progression, which increases genome instability. CONCLUSION: Entinostat inhibits HR repair by reducing BRCA1 expression and stalling replication fork progression, leading to irreparable DNA damage and ultimate cell death. This work provides preclinical support for the clinical trial of the combination of olaparib and entinostat in HR-proficient ovarian cancer and suggests potential benefit even for CCNE1 amplified subtypes.

CCNE1 and BRD4 co-amplification in high-grade serous ovarian cancer is associated with poor clinical outcomes.

OBJECTIVE: High-grade serous ovarian cancer (HGSOC) is the most common and lethal histological subtype of epithelial ovarian cancer. HGSOC with cyclin E1 gene (CCNE1) amplification and bromodomain and extraterminal 4 (BRD4) amplification have been associated with poor outcomes. Our objective was to evaluate clinical outcomes of HGSOC with co-amplification of CCNE1 and BRD4 and high protein expression of cyclin E and BRD4. METHODS: Copy number amplification data were extracted from The Cancer Genome Atlas (TCGA) for 579 HGSOC. Reverse phase protein array (RPPA) TCGA data were used to determine cyclin E and BRD4 protein expression in 482 HGSOC. Cyclin E and BRD4 protein expression by immunohistochemistry (IHC) was evaluated in a tissue microarray (TMA) of 110 HGSOC. Measured clinical outcomes were survival and platinum sensitivity. RESULTS: Of 30% of HGSOC with amplifications in CCNE1 or BRD4, 8% have both CCNE1 and BRD4 amplification. Protein expression of cyclin E and BRD4 are positively correlated, both by RPPA (r = 0.23; p < 0.001) and by IHC (r = 0.21; p = 0.025). Patients with CCNE1 and BRD4 co-amplified HGSOC have worse overall survival than patients without amplifications, 39.94 vs 48.06 months (p = 0.029). High protein expression of cyclin E, but not BRD4, was associated with poor overall survival (HR 1.62, 1.04-2.53, p = 0.033) and platinum resistance (p = 0.016). CONCLUSION: HGSOC with CCNE1 and BRD4 co-amplification are associated with poor overall survival. Further studies are warranted to determine the use of protein expression by IHC as a surrogate marker for CCNE1 and BRD4 co-amplified HGSOC.

Rethinking progesterone regulation of female reproductive cyclicity.

The progesterone receptor (PGR) is a ligand-activated transcription factor with key roles in the regulation of female fertility. Much has been learned of the actions of PGR signaling through the use of pharmacologic inhibitors and genetic manipulation, using mouse mutagenesis. Characterization of rats with a null mutation at the Pgr locus has forced a reexamination of the role of progesterone in the regulation of the female reproductive cycle. We generated two Pgr mutant rat models, using genome editing. In both cases, deletions yielded a null mutation resulting from a nonsense frame-shift and the emergence of a stop codon. Similar to Pgr null mice, Pgr null rats were infertile because of deficits in sexual behavior, ovulation, and uterine endometrial differentiation. However, in contrast to the reported phenotype of female mice with disruptions in Pgr signaling, Pgr null female rats exhibit robust estrous cycles. Cyclic changes in vaginal cytology, uterine histology, serum hormone levels, and wheel running activity were evident in Pgr null female rats, similar to wild-type controls. Furthermore, exogenous progesterone treatment inhibited estrous cycles in wild-type female rats but not in Pgr-null female rats. As previously reported, pharmacologic antagonism supports a role for PGR signaling in the regulation of the ovulatory gonadotropin surge, a result at variance with experimentation using genetic ablation of PGR signaling. To conclude, our findings in the Pgr null rat challenge current assumptions and prompt a reevaluation of the hormonal control of reproductive cyclicity.

Low total motile sperm in transgender women seeking hormone therapy.

PURPOSE: This study was undertaken to compare semen quality, hormonal status, and social factors in transgender women seeking fertility preservation with those of fertile cisgender men. Long-range goals are to establish standard practice measures ensuring optimum semen quality for cryopreservation and fertility preservation in transgender women. METHODS: This is a case-control study carried out at an academic medical center. Cases are transgender women seeking fertility preservation prior to initiation of hormone therapy. Controls are cisgender men recently fathering a child. All participants completed the Depression Anxiety Stress Scales 21 survey and additional survey questions related to personal behaviors. Complete semen analysis was carried out in a clinical andrology laboratory according to WHO guidelines, 5th edition. Serum follicle stimulating hormone, estradiol, and testosterone were measured at the time of semen analysis. RESULTS: Sperm concentration, total sperm per ejaculate, total motile sperm, volume, and normal sperm morphology were significantly lower in transgender females compared with fertile cisgender men. Other measures of semen parameters and hormone concentrations were not different between groups. Survey results indicated transgender women were more likely to have symptoms of depression, anxiety, and stress and utilize tucking and tight undergarments, compared with controls; however, both groups reported similar numbers of ejaculations per week. CONCLUSIONS: Although semen parameters were low, cryopreservation of sperm prior to hormone therapy is a viable fertility preservation option for most transgender women. The etiology of the differences in semen parameters is not known. Enhanced education related to personal behaviors or treatment to reduce effects of stressors prior to cryopreservation may improve future fertility potential.

Histone demethylase KDM4A and KDM4B expression in granulosa cells from women undergoing in vitro fertilization.

PURPOSE: To assess expression of the histone demethylases KDM4A and KDM4B in granulosa collected from women undergoing oocyte retrieval and to determine if expression was related to pregnancy outcome. METHODS: Cumulus and mural granulosa cells were obtained from women undergoing oocyte retrieval. KDM4A and KDM4B mRNA expression was determined by qRT-PCR. KDM4A and KDM4B proteins were immunohistochemically localized in ovarian tissue sections obtained from archival specimens. RESULTS: KDM4A and KDM4B protein was localized to oocytes, granulosa cells, and theca and luteal cells in ovaries from reproductive-aged women. KDM4A and KDM4B mRNA expression was overall higher in cumulus compared to mural granulosa. When comparing granulosa demethylase gene expression, KDM4A and KDM4B mRNA expression was higher in both cumulus and mural granulosa from not pregnant patients compared to patients in the pregnant-live birth group. CONCLUSIONS: Histone demethylases KDM4A and KDM4B mRNA are differentially expressed in cumulus and mural granulosa. Expression of both KDM4A and KDM4B mRNA was lower in cumulus granulosa and mural granulosa from pregnant compared to not pregnant patients. These findings suggest that altered expression of histone demethylases may impact epigenetic changes in granulosa cells associated with pregnancy.

Dysregulation of mitotic machinery genes precedes genome instability during spontaneous pre-malignant transformation of mouse ovarian surface epithelial cells.

BACKGROUND: Based in epidemiological evidence, repetitive ovulation has been proposed to play a role in the origin of ovarian cancer by inducing an aberrant wound rupture-repair process of the ovarian surface epithelium (OSE). Accordingly, long term cultures of isolated OSE cells undergo in vitro spontaneous transformation thus developing tumorigenic capacity upon extensive subcultivation. In this work, C57BL/6 mouse OSE (MOSE) cells were cultured up to passage 28 and their RNA and DNA copy number profiles obtained at passages 2, 5, 7, 10, 14, 18, 23, 25 and 28 by means of DNA microarrays. Gene ontology, pathway and network analyses were focused in passages earlier than 20, which is a hallmark of malignancy in this model. RESULTS: At passage 14, 101 genes were up-regulated in absence of significant DNA copy number changes. Among these, the top-3 enriched functions (>30 fold, adj p < 0.05) comprised 7 genes coding for centralspindlin, chromosome passenger and minichromosome maintenance protein complexes. The genes Ccnb1 (Cyclin B1), Birc5 (Survivin), Nusap1 and Kif23 were the most recurrent in over a dozen GO terms related to the mitotic process. On the other hand, Pten plus the large non-coding RNAs Malat1 and Neat1 were among the 80 down-regulated genes with mRNA processing, nuclear bodies, ER-stress response and tumor suppression as relevant terms. Interestingly, the earliest discrete segmental aneuploidies arose by passage 18 in chromosomes 7, 10, 11, 13, 15, 17 and 19. By passage 23, when MOSE cells express the malignant phenotype, the dysregulated gene expression repertoire expanded, DNA imbalances enlarged in size and covered additional loci. CONCLUSION: Prior to early aneuploidies, overexpression of genes coding for the mitotic apparatus in passage-14 pre-malignant MOSE cells indicate an increased proliferation rate suggestive of replicative stress. Concomitant down-regulation of nuclear bodies and RNA processing related genes suggests altered control of nuclear RNA maturation, features recently linked to impaired DNA damage response leading to genome instability. These results, combined with cytogenetic analysis by other authors in this model, suggest that transcriptional profile at passage 14 might induce cytokinesis failure by which tetraploid cells approach a near-tetraploid stage containing primary chromosome aberrations that initiate the tumorigenic drive.

Selective targeting of IRAK1 attenuates low molecular weight hyaluronic acid-induced stemness and non-canonical STAT3 activation in epithelial ovarian cancer.

Advanced epithelial ovarian cancer (EOC) survival rates are dishearteningly low, with ~25% surviving beyond 5 years. Evidence suggests that cancer stem cells contribute to acquired chemoresistance and tumor recurrence. Here, we show that IRAK1 is upregulated in EOC tissues, and enhanced expression correlates with poorer overall survival. Moreover, low molecular weight hyaluronic acid, which is abundant in malignant ascites from patients with advanced EOC, induced IRAK1 phosphorylation leading to STAT3 activation and enhanced spheroid formation. Knockdown of IRAK1 impaired tumor growth in peritoneal disease models, and impaired HA-induced spheroid growth and STAT3 phosphorylation. Finally, we determined that TCS2210, a known inducer of neuronal differentiation in mesenchymal stem cells, is a selective inhibitor of IRAK1. TCS2210 significantly inhibited EOC growth in vitro and in vivo both as monotherapy, and in combination with cisplatin. Collectively, these data demonstrate IRAK1 as a druggable target for EOC.

Preclinical antitumor activity of a nanoparticulate SN38.

The present studies were carried out to examine the efficacy of a nanoparticulate formulation of SN38, the potent topoisomerase I inhibitor and active metabolite of irinotecan. Metabolism of irinotecan to SN38 is inefficient and subject to considerable patient-to-patient variability. One approach to more controlled administration of the anticancer agent is direct administration of the active SN38. A nanoparticulate formulation of SN38 was prepared by a method of precipitation with compressed antisolvent. Nanoparticulate SN38 efficiently inhibited the proliferation of colorectal, ovarian, and mesothelial cancer cell lines in vitro. Concentrations resulting in 50 % inhibition of proliferation were approximately 1000 fold lower for nanoparticulate SN38 compared to irinotecan. In vivo effects were examined using colorectal and ovarian mouse model systems. In a mouse model of peritoneally disseminated ovarian cancer intraperitoneal administration of irinotecan was favorable to intravenous delivery however intraperitoneal delivery of nanoparticulate SN38 was significantly more effective than intraperitoneal irinotecan. In addition, in a mouse colorectal cancer model administration of nanoparticulate SN38 once weekly exhibited greater activity compared to daily or weekly administration of irinotecan. Additional studies demonstrated nanoparticulate SN38 administered as a combination therapy with mitomycin C was more effective than the combination of irinotecan and mitomycin C. Results from the present studies using preclinical colorectal and ovarian cancer model systems demonstrate the efficacy of nanoparticulate SN38 and substantiate continued development.

Inhibition of cell growth and induction of apoptosis in ovarian carcinoma cell lines CaOV3 and SKOV3 by natural withanolide Withaferin A.

OBJECTIVE: Withaferin A, a natural withanolide, has shown anti-cancer properties in various cancers including breast cancer, but its effects in ovarian cancer remain unexplored. Notch 1 and Notch3 are critically involved in ovarian cancer progression. We decided to examine the effects of Withaferin A in ovarian carcinoma cell lines and its molecular mechanism of action including its regulation of Notch. METHODS: The effects of Withaferin A were examined in CaOV3 and SKOV3 ovarian carcinoma cell lines using MTS assay, clonogenic assay, annexin V/propidium iodide flow cytometry, and cell cycle analysis. Western analysis was conducted to examine the molecular mechanisms of action. RESULTS: Withaferin A inhibited the growth and colony formation of CaOV3 and SKOV3 cells by inducing apoptosis and cell cycle arrest. These changes correlated with down-regulation of Notch1, Notch3, cdc25C, total and phosphorylated Akt, and bcl-2 proteins. CONCLUSIONS: Withaferin A inhibits CaOV3 and SKOV3 ovarian carcinoma cell growth, at least in part by targeting Notch1 and Notch3.

Evaluation of Placentation and the Role of the Aryl Hydrocarbon Receptor Pathway in a Rat Model of Dioxin Exposure.

BACKGROUND: Our environment is replete with chemicals that can affect embryonic and extraembryonic development. Dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), are compounds affecting development through the aryl hydrocarbon receptor (AHR). OBJECTIVES: The purpose of this investigation was to examine the effects of TCDD exposure on pregnancy and placentation and to evaluate roles for AHR and cytochrome P450 1A1 (CYP1A1) in TCDD action. METHODS: Actions of TCDD were examined in wild-type and genome-edited rat models. Placenta phenotyping was assessed using morphological, biochemical, and molecular analyses. RESULTS: TCDD exposures were shown to result in placental adaptations and at higher doses, pregnancy termination. Deep intrauterine endovascular trophoblast cell invasion was a prominent placentation site adaptation to TCDD. TCDD-mediated placental adaptations were dependent upon maternal AHR signaling but not upon placental or fetal AHR signaling nor the presence of a prominent AHR target, CYP1A1. At the placentation site, TCDD activated AHR signaling within endothelial cells but not trophoblast cells. Immune and trophoblast cell behaviors at the uterine-placental interface were guided by the actions of TCDD on endothelial cells. DISCUSSION: We identified an AHR regulatory pathway in rats activated by dioxin affecting uterine and trophoblast cell dynamics and the formation of the hemochorial placenta. https://doi.org/10.1289/EHP9256.

Granulosa cell genes that regulate ovarian follicle development beyond the antral stage: The role of estrogen receptor ß.

Follicle development beyond the preantral stage is dependent on gonadotropins. FSH signaling is crucial for the advancement of preantral follicles to the antral stage, and LH signaling is essential for further maturation of preovulatory follicles. Estrogen is intricately tied to gonadotropin signaling during the advanced stages of folliculogenesis. We observed that Erßnull ovarian follicles fail to develop beyond the antral stage, even after exogenous gonadotropin stimulation. As ERß is primarily expressed in the granulosa cells (GCs), we explored the gonadotropin-regulated GC genes that induce maturation of antral follicles. Synchronized follicle development was induced by administration of exogenous gonadotropins to wildtype 4-wk-old female rats. The GC transcriptome was analyzed via RNA-sequencing before and after gonadotropin stimulation. An Erßnull mutant model that fails to show follicle maturation was also included in order to identify the ERß-regulated genes involved at this step. We observed that specific groups of genes were differentially expressed in response to PMSG or hCG administration in wildtype rats. While some of the PMSG or hCG-induced genes showed a similar expression pattern in Erßnull GCs, a subset of PMSG- or hCG-induced genes showed a differential expression pattern in Erßnull GCs. These latter ERß-regulated genes included previously known FSH or LH target genes including Lhcgr, Cyp11a1, Cyp19a1, Pgr, Runx2, Egfr, Kiss1, and Ptgs2, which are involved in follicle development, oocyte maturation, and ovulation. We also identified novel ERß-regulated genes including Jaml, Galnt6, Znf750, Dusp9, Wnt16, and Mageb16 that failed to respond to gonadotropin stimulation in Erßnull GCs. Our findings indicate that the gonadotropin-induced spatiotemporal pattern of gene expression is essential for ovarian follicle maturation beyond the antral stage. However, expression of a subset of those gonadotropin-induced genes is dependent on transcriptional regulation by ERß.

ERß regulated ovarian kisspeptin plays an important role in oocyte maturation.

Kisspeptin (KISS1) signaling in the hypothalamic-pituitary (H-P) axis plays an essential role in regulating gonadotropin secretion. KISS1 and KISS1 receptor (KISS1R) are also expressed in the ovary; however, the role of intraovarian KISS1 signaling remains unclear. Granulosa cell (GC)-specific expression of KISS1, and oocyte-specific expression of KISS1R indicate that GC-derived KISS1 may act on oocytes. Expression of KISS1 in GCs is induced by gonadotropins but it is absent in estrogen receptor ß knockout (Erßnull) rat ovaries. We also observed that gonadotropin stimulation failed to induce maturation of Erßnull oocytes. Interestingly, KISS1 treatment of cumulus oocyte complexes (COCs) isolated from antral follicles promotes in vitro maturation of oocytes. Treatment of oocytes with KISS1 induced intracellular Ca2+ release, and increased activation of MAP kinase ERK1/2. KISS1 treatment also induced the expression of oocyte genes that are crucial for differentiation of GCs, and maturation of oocytes. Our findings suggest that ovarian KISS1-signaling plays an important role in gonadotropin induced follicle development and oocyte maturation.

The ability of the aryl hydrocarbon receptor to regulate ovarian follicle growth and estradiol biosynthesis in mice depends on stage of sexual maturity.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that mediates the toxicity of environmental chemicals and regulates many physiological functions, including processes in female reproduction. Previous studies demonstrated that Ahr deletion leads to slow ovarian follicle growth because of impaired estradiol production and reduced gonadotropin responsiveness in prepubertal mice. These studies, however, did not determine how Ahr deletion impairs estradiol production or whether the effects of Ahr deletion on follicle growth and estradiol production persist in adulthood. Thus, the present study evaluated the effect of Ahr deletion on steroid precursors in the estradiol biosynthesis pathway. Furthermore, this study evaluated follicle growth and estradiol biosynthesis in wild-type (WT) and Ahr knockout (AhrKO) antral follicles at different stages of sexual maturity. AhrKO antral follicles from prepubertal mice had slower growth, produced lower estradiol levels, and had reduced cyclin D2 (Ccnd2) expression compared to WT follicles. AhrKO follicles from adult mice, however, produced higher androgen levels and expressed higher levels of Ccnd2 compared to WT follicles. Furthermore, AhrKO follicles from adult mice had growth to that of WT follicles. These findings suggest that the AHR regulates follicle growth by altering factors involved in the estradiol biosynthesis pathway as well as key regulators of follicle growth and that this role of AHR depends on stage of sexual maturity.

Combination intraperitoneal chemotherapy is superior to mitomycin C or oxaliplatin for colorectal carcinomatosis in vivo.

BACKGROUND: Hyperthermic intraperitoneal (IP) chemotherapy after cytoreduction improves survival in patients with colorectal carcinomatosis of the peritoneal surface. Most protocols use single agents (mitomycin C or oxaliplatin) provided IP. The purpose of this study was to determine whether combination IP chemotherapy is superior to single-agent therapy in a mouse model. METHODS: Nu/Nu mice were injected IP with HT-29 colorectal cancer cells. Animals were treated with single agents or combinations. Primary end point was overall survival. Agents explored included oxaliplatin, mitomycin C, panitumumab, erlotinib, cetuximab, and irinotecan delivered IP as single agents; mitomycin C, panitumumab, and irinotecan in combination IP; and 5-fluorouracil-leucovorin-irinotecan (FOLFIRI) in combination delivered intravenously. RESULTS: Survival of mice receiving irinotecan or mitomycin C IP was greater than controls. Median survival of mice receiving intravenous FOLFIRI was also greater than control. However, survival of mice receiving IP irinotecan or mitomycin C was far greater than mice receiving intravenous FOLFIRI. For combination therapy, a positive interaction was observed with mitomycin C and irinotecan, whereas survival was greater than either agent individually. No interaction was observed between panitumumab and mitomycin C or irinotecan. However, an overall survival benefit was observed with the combination of irinotecan, mitomycin C, and panitumumab; at 120 days after cell injection, 100% of the triagent therapy group survived. CONCLUSIONS: IP therapy with mitomycin C or irinotecan provided a survival benefit compared with intravenous FOLFIRI. Combination IP therapy with mitomycin C, panitumumab, and irinotecan was superior to all other agents tested alone or in combination. This warrants further combination analysis and supports consideration for a phase I application.

Transcription factor Nfat1 deficiency causes osteoarthritis through dysfunction of adult articular chondrocytes.

Osteoarthritis (OA) is the most common form of joint disease in middle-aged and older individuals. Previous studies have shown that over-expression of matrix-degrading proteinases and proinflammatory cytokines is associated with osteoarthritic cartilage degradation. However, it remains unclear which transcription factors regulate the expression of these cartilage-degrading molecules in articular chondrocytes. This study demonstrated that mice lacking Nfat1, a member of the nuclear factor of activated T cells (NFAT) transcription factors, exhibited normal skeletal development but displayed loss of type II collagen (collagen-2) and aggrecan with over-expression of specific matrix-degrading proteinases and proinflammatory cytokines in young adult articular cartilage of load-bearing joints. These initial changes are followed by articular chondrocyte proliferation/clustering, progressive articular surface destruction, periarticular chondro-osteophyte formation and exposure of thickened subchondral bone, all of which resemble human OA. Forced expression of Nfat1 delivered with lentiviral vectors in cultured 3 month-old primary Nfat1 knockout (Nfat1(-/-)) articular chondrocytes partially or completely rescued the abnormal catabolic and anabolic activities of Nfat1(-/-) articular chondrocytes. These new findings revealed a previously unrecognized critical role of Nfat1 in maintaining the physiological function of differentiated adult articular chondrocytes through regulating the expression of specific matrix-degrading proteinases and proinflammatory cytokines. Nfat1 deficiency causes OA due to an imbalance between the catabolic and anabolic activities of adult articular chondrocytes, leading to articular cartilage degradation and failed repair activities in and around articular cartilage. These results may provide new insights into the aetiology, pathogenesis and potential therapeutic strategies for osteoarthritis.

A Gatekeeping Role of ESR2 to Maintain the Primordial Follicle Reserve.

Over the entire reproductive lifespan in mammals, a fixed number of primordial follicles serve as the source of mature oocytes. Uncontrolled and excessive activation of primordial follicles can lead to depletion of the ovarian reserve. We observed that disruption of estrogen receptor ß (ESR2) signaling results in increased activation of primordial follicles in Esr2-null (Esr2-/-) rats. However, follicle assembly was unaffected, and the total number of follicles remained comparable between neonatal wild-type and Esr2-/- ovaries. While the activated follicle counts were increased in Esr2-/- ovary, the number of primordial follicles were markedly decreased. Excessive recruitment of primordial follicles led to premature ovarian senescence in Esr2-/- rats and was associated with reduced levels of serum AMH and estradiol. Disruption of ESR2 signaling through administration of a selective antagonist (PHTPP) increased the number of activated follicles in wildtype rats, whereas a selective agonist (DPN) decreased follicle activation. In contrast, primordial follicle activation was not increased in the absence of ESR1, indicating that the regulation of primordial follicle activation is ESR2 specific. Follicle activation was also increased in Esr2 mutants lacking the DNA binding domain, suggesting a role for the canonical transcriptional activation function. Both primordial and activated follicles express ESR2, suggesting a direct regulatory role for ESR2 within these follicles. We also detected that loss of ESR2 augmented the activation of AKT, ERK, and mTOR pathways. Our results indicate that the lack of ESR2 upregulated both granulosa and oocyte factors, which can facilitate AKT and mTOR activation in Esr2-/- ovaries leading to increased activation of primordial follicles.

Chimeric NKG2D receptor-bearing T cells as immunotherapy for ovarian cancer.

Despite advancements in the treatment of ovarian cancer, this disease continues to be a leading cause of cancer death in women. Adoptive transfer of tumor-reactive T cells is a promising antitumor therapy for many cancers. We designed a chimeric receptor linking NKG2D, a natural killer (NK) cell-activating receptor, to the CD3zeta chain of the T-cell receptor to target ovarian tumor cells. Engagement of chimeric NKG2D receptors (chNKG2D) with ligands for NKG2D, which are commonly expressed on tumor cells, leads to T-cell secretion of proinflammatory cytokines and tumor cytotoxicity. In this study, we show that >80% of primary human ovarian cancer samples expressed ligands for NKG2D on the cell surface. The tumor samples expressed MHC class I-related protein A, MICB, and UL-16 binding proteins 1 and 3. ChNKG2D-expressing T cells lysed ovarian cancer cell lines. We show that T cells from ovarian cancer patients that express chNKG2D secreted proinflammatory cytokines when cultured with autologous tumor cells. In addition, we show that chNKG2D T cells can be used therapeutically in a murine model of ovarian cancer. These data indicate that treatment with chNKG2D-expressing T cells is a potential immunotherapy for ovarian cancer.

CD80 in immune suppression by mouse ovarian carcinoma-associated Gr-1+CD11b+ myeloid cells.

An elevated number of Gr-1+CD11b+ myeloid cells has been described in mice bearing transplantable tumors, and has been associated with immune suppression. We examined the role of such myeloid suppressor cells in mice bearing the spontaneously transformed syngeneic mouse ovarian surface epithelial cell line, 1D8. We observed high levels of CD80 expression by Gr-1+CD11b+ cells from spleen, ascites, and tumor tissue of mice bearing 1D8 ovarian carcinoma, whereas CD40 and CD86 were absent. CD80 expression was not detected on Gr-1+CD11b+ cells from naïve mice. However, the expression of CD80 by Gr-1+CD11b+ cells from naïve mice was promoted by coculture with 1D8 cells. Because irradiated 1D8 cells, but not 1D8-conditioned medium, up-regulate CD80 expression by Gr-1+CD11b+ cells, this phenomenon likely requires direct interaction. Gr-1+CD11b+ cells derived from 1D8 tumor-bearing mice provided significant suppression of antigen-specific immune responses, but Gr-1+CD11b+ cells from naïve mice did not. Both short interfering RNA-mediated knockdown and genetic knockout of CD80 expression by Gr-1+CD11b+ cells of 1D8 tumor-bearing mice alleviated the suppression of antigen-specific immune responses. Suppression via CD80 on Gr-1+CD11b+ myeloid cells was mediated by CD4+CD25+ T regulatory cells and required CD152. CD80 knockout or antibody blockade of either CD80 or CD152 retarded the growth of 1D8 tumor in mice, suggesting that expression of CD80 on Gr-1+CD11b+ myeloid cells triggered by 1D8 ovarian carcinoma suppresses antigen-specific immunity via CD152 signaling and CD4+CD25+ T regulatory cells. Thus, CD80-dependent responses to myeloid suppressor cells may contribute to tumor tolerance and the progression of ovarian carcinoma.

Integrative genomic analysis of protein kinase C (PKC) family identifies PKCiota as a biomarker and potential oncogene in ovarian carcinoma.

The protein kinase C (PKC) family plays a key regulatory role in a wide range of cellular functions as well as in various cancer-associated signal transduction pathways. Here, we investigated the genomic alteration and gene expression of most known PKC family members in human ovarian cancer. The DNA copy number of PKC family genes was screened by a high-resolution array-based comparative genomic hybridization in 89 human ovarian cancer specimens. Five PKC genes exhibited significant DNA copy number gains, including PKCiota (43.8%), PKCbeta1 (37.1%), PKCgamma (27.6%), PKCzeta (22.5%), and PKCtheta (21.3%). None of the PKC genes exhibited copy number loss. The mRNA expression level of PKC genes was analyzed by microarray retrieval approach. Two of the amplified PKC genes, PKCiota and PKCtheta, were significantly up-regulated in ovarian cancer compared with normal ovary. Increased PKCiota expression correlated with tumor stage or grade, and PKCiota overexpression was seen mostly in ovarian carcinoma but not in other solid tumors. The above results were further validated by real-time reverse transcription-PCR with 54 ovarian cancer specimens and 24 cell lines; overexpression of PKCiota protein was also confirmed by tissue array and Western blot. Interestingly, overexpressed PKCiota did not affect ovarian cancer cell proliferation or apoptosis in vitro. However, decreased PKCiota expression significantly reduced anchorage-independent growth of ovarian cancer cells, whereas overexpression of PKCiota contributed to murine ovarian surface epithelium transformation in cooperation with mutant Ras. We propose that PKCiota may serve as an oncogene and a biomarker of aggressive disease in human ovarian cancer.

An innovative immunotherapeutic strategy for ovarian cancer: CLEC10A and glycomimetic peptides.

BACKGROUND: Receptors specific for the sugar N-acetylgalactosamine (GalNAc) include the human type II, C-type lectin receptor macrophage galactose-type lectin/C-type lectin receptor family member 10A (MGL/CLEC10A/CD301) that is expressed prominently by human peripheral immature dendritic cells, dendritic cells in the skin, alternatively-activated (M2a) macrophages, and to lesser extents by several other types of tissues. CLEC10A is an endocytic receptor on antigen-presenting cells and has been proposed to play an important role in maturation of dendritic cells and initiation of an immune response. In this study, we asked whether a peptide that binds in the GalNAc-binding site of CLEC10A would serve as an effective tool to activate an immune response against ovarian cancer. METHODS: A 12-mer sequence emerged from a screen of a phage display library with a GalNAc-specific lectin. The peptide, designated svL4, and a shorter peptide consisting of the C-terminal 6 amino acids, designated sv6D, were synthesized as tetravalent structures based on a tri-lysine core. In silico and in vitro binding assays were developed to evaluate binding of the peptides to GalNAc-specific receptors. Endotoxin-negative peptide solutions were administered by subcutaneous injection and biological activity of the peptides was determined by secretion of cytokines and the response of peritoneal immune cells in mice. Anti-cancer activity was studied in a murine model of ovarian cancer. RESULTS: The peptides bound to recombinant human CLEC10A with high avidity, with half-maximal binding in the low nanomolar range. Binding to the receptor was Ca2+-dependent. Subcutaneous injection of low doses of peptides into mice on alternate days resulted in several-fold expansion of populations of mature immune cells within the peritoneal cavity. Peptide sv6D effectively suppressed development of ascites in a murine ovarian cancer model as a monotherapy and in combination with the chemotherapeutic drug paclitaxel or the immunotherapeutic antibody against the receptor PD-1. Toxicity, including antigenicity and release of cytotoxic levels of cytokines, was not observed. CONCLUSION: sv6D is a functional ligand for CLEC10A and induces maturation of immune cells in the peritoneal cavity. The peptide caused a highly significant extension of survival of mice with implanted ovarian cancer cells with a favorable toxicity and non-antigenic profile.