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Ubiquitin C-terminal hydrolase L1 (UCHL1) is a de-ubiquitinating enzyme enriched in neuronal and gonadal tissues known to regulate the cellular stores of mono-ubiquitin and protein turnover. While its function in maintaining proper motor neuron function is well established, investigation into its role in the health and function of reproductive processes is only just beginning to be studied. Single-cell-sequencing analysis of all ovarian cells from the murine perinatal period revealed that Uchl1 is very highly expressed in the developing oocyte population, an observation which was corroborated by high levels of oocyte-enriched UCHL1 protein expression in oocytes of all stages throughout the mouse reproductive lifespan. To better understand the role UCHL1 may be playing in oocytes, we utilized a UCHL1-deficient mouse line, finding reduced number of litters, reduced litter sizes, altered folliculogenesis, morphologically abnormal oocytes, disrupted estrous cyclicity and apparent endocrine dysfunction in these animals compared to their wild-type and heterozygous littermates. These data reveal a novel role of UCHL1 in female fertility as well as overall ovarian function, and suggest a potentially essential role for the ubiquitin proteasome pathway in mediating reproductive health. Summary Sentence Ubiquitin C-terminal hydrolase L1 (UCHL1) is required for proper ovarian folliculogenesis, estrous cyclicity, and fertility in the female mouse.

High grade serous ovarian cancer (HGSOC) is a lethal gynecologic malignancy in which chemoresistant recurrence rates remain high. Furthermore, HGSOC patients have demonstrated overall low response rates to clinically available immunotherapies. Amphiregulin (AREG), a low affinity epidermal growth factor receptor ligand is known to be significantly upregulated in HGSOC patient tumors following neoadjuvant chemotherapy exposure. While much is known about AREG’s role in oncogenesis and classical immunity, it is function in tumor immunology has been comparatively understudied. Therefore, the objective of this present study was to elucidate how increased AREG exposure impacts the ovarian tumor immune microenvironment (OTIME). Using NanoString IO 360 and protein analysis, it was revealed that treatment with recombinant AREG led to prominent upregulation of genes associated with ovarian pathogenesis and immune evasion ( CXCL8 , CXCL1 , CXCL2 ) along with increased STAT3 activation in HGSOC cells. In vitro co-culture assays consisting of HGSOC cells and peripheral blood mononuclear cells (PBMCs) stimulated with recombinant AREG (rAREG) led to significantly enhanced tumor cell viability. Moreover, PBMCs stimulated with rAREG exhibited significantly lower levels of IFNy and IL-2 . In vivo rAREG treatment promoted significant reductions in circulating levels of IL-2 and IL-5. Intratumoral analysis of rAREG treated mice revealed a significant reduction in CD8 + T cells coupled with an upregulation of PD-L1. Finally, combinatorial treatment with an AREG neutralizing antibody and carboplatin led to a synergistic reduction of cell viability in HGSOC cell lines OVCAR8 and PEA2. Overall, this study demonstrates AREG’s ability to modulate cytotoxic responses within the OTIME and highlights its role as a novel HGSOC immune target.

The high rate of ovarian cancer recurrence and chemoresistance necessitates further research into how chemotherapy affects the tumor immune microenvironment (TIME). While studies have shown that immune infiltrate increases following neoadjuvant (NACT) chemotherapy, there lacks a comprehensive understanding of chemotherapy-induced effects on immunotranscriptomics and cancer-related pathways and their relationship with immune infiltrate and patient responses. In this study, we performed NanoString nCounter ® PanCancer IO360 analysis of 31 high grade serous ovarian cancer (HGSOC) patients with matched pre-treatment biopsy and post-NACT tumor. We observed increases in pro-tumorigenic and immunoregulatory pathways and immune infiltrate following NACT, with striking increases in a cohort of genes centered on the transcription factors ATF3 and EGR1 . Using quantitative PCR, we analyzed several of the top upregulated genes in HGSOC cell lines, noting that two of them, ATF3 and AREG , were consistently upregulated with chemotherapy exposure and significantly increased in platinum resistant cells compared to their sensitive counterparts. Furthermore, we observed that pre-NACT immune infiltrate and pathway scores were not strikingly related to platinum free interval (PFI), but post-NACT immune infiltrate, pathway scores, and gene expression were. Finally, we found that higher levels of a cohort of proliferative and DNA damage-related genes was related to shorter PFI. This study underscores the complex alterations in the ovarian TIME following chemotherapy exposure and begins to untangle how immunologic factors are involved in mediating chemotherapy response, which will allow for the future development of novel immunologic therapies to combat chemoresistance.

Current safety data on chemotherapy during pregnancy are based on studies which focus on the mother and do not explore reproductive health and fecundity potential within the exposed offspring. We designed this randomized ex vivo animal study to evaluate the effect of chemotherapy on the developing ovarian reserve in the exposed offspring. Specimens (100 postnatal day zero C57BL/6 mouse ovaries) were randomized to control or chemotherapy drug exposure and maintained in a hanging well organ culture. Murine ovarian reserve establishment mirrors activity seen in the human fetus but with a significant time shift of the transition to meiotic arrest to the postnatal period. Exposures included: doxorubicin, cyclophosphamide, paclitaxel, docetaxel, and cisplatin. Doxorubicin resulted in a significant loss of 95.2% (p < 0.0001) of oocyte density compared to controls. Cyclophosphamide also caused depletion of 50.5% (p < 0.0001) of oocyte density. Cisplatin, docetaxel, and paclitaxel all demonstrated unique phenotypical changes on the ovaries and their oocytes, without a significant decrease in oocyte density over a five-day exposure. Exposure to chemotherapy may result in profound loss of oogonia during the transition to mature oocytes.

BackgroundOutcomes for high grade serous ovarian cancer (HGSOC) patients have remained dismal due to the inevitable development of chemotherapy resistance with recurrent disease.1 In order to better tailor treatment approaches and uncover opportunities for novel treatments, we need to better understand factors contributing to chemotherapy resistance. Recent studies have shown that immune-related gene expression profiles may serve as prognostic indicators of response to chemotherapy and clinical outcomes in solid tumors, including ovarian cancer.2–7 Moreover, immunologic factors have been shown to mediate chemotherapy resistance8 Reports in the literature show that common ovarian cancer therapeutics, including chemotherapy, PARP inhibitors, and bevacizumab, modulate tumor cell expressed PD-L1 levels through immunologic signaling pathways.9–12 However, very little research has addressed the effect of these treatments on other immune ligands or the differences in immunologic responses between platinum-sensitive and platinum-resistant HGSOC cell lines.MethodsThe HGSOC cell lines OVCAR4 (naturally platinum-resistant), PEO1 and PEO4 (matched platinum-sensitive and -resistant lines from the same patient), were treated with common ovarian cancer therapeutics (carboplatin/paclitaxel, olaparib, and bevacizumab), in the presence or absence of peripheral blood mononuclear cells. Western blot was employed to identify levels of immune ligands of interest and a proteome profiler was used to detect broad immunologic changes in response to standard of care therapeutics.ResultsOlaparib and bevacizumab treatment strikingly upregulated levels of tumor cell expressed immune ligands ICOSL and PVRL2. Platinum status or presence of an immune component had no bearing on the effect. Moreover, blockade of PVRL2 using siRNA or monoclonal antibodies suppressed STAT3 signaling. When examining the effect of these therapeutics on cytokine levels in HGSOC cell lines treated in immune cell co-culture, OVCAR4 cells displayed marked changes in cytokine levels, particularly CXCL10, CXCL12, SERPINE1, IL1A, and IL1RA. While PEO1 and PEO4 cells displayed more subtle cytokine changes compared to OVCAR4 cells, differences in basal levels and treatment responses were observed between the platinum-sensitive and -resistant lines, most strikingly higher basal levels of SERPINE1 and CCL5/RANTES in PEO4 cells, and a robust increase in IL8 levels in response to chemotherapy in only PEO1 cells and not PEO4.ConclusionsIn conclusion, common ovarian cancer chemotherapeutics and targeted agents induce tumor cell intrinsic immunologic effects that could potentially be exploited as combinatorial therapeutic targets. Differences in immunologic responses may help define platinum-sensitive and -resistant disease. These results will require further exploration in immune-competent mouse models and human HGSOC tissue.ReferencesCortez AJ, Tudrej P, Kujawa KA, Lisowska KM. Advances in ovarian cancer therapy. Cancer Chemother Pharmacol 2018;81(1):17–38.James NE, Miller K, LaFranzo N, Lips E, Woodman M, Ou J, Ribeiro JR. Immune modeling analysis reveals immunologic signatures associated with improved outcomes in high grade serous ovarian cancer. Front Oncol 2021;11:622182.Liu R, Hu R, Zeng Y, Zhang W, Zhou H-H. Tumour immune cell infiltration and survival after platinum-based chemotherapy in high-grade serous ovarian cancer subtypes: a gene expression-based computational study. EBioMedicine 2020;51:102602.Liu J, Meng H, Nie S, Sun Y, Jiang P, Li S, et al. Identification of a prognostic signature of epithelial ovarian cancer based on tumor immune microenvironment exploration. Genomics. 2020.Ding J, Zhang Q, Chen S, Huang H, He L. Construction of a new tumor immunity-related signature to assess and classify the prognostic risk of ovarian cancer. Aging (Albany, NY). 2020;12.Wu Y, Xia L, Zhao P, Deng Y, Guo Q, Zhu J, et al. Immune profiling reveals prognostic genes in high-grade serous ovarian cancer. Aging (Albany, NY). 2020;12(12):11398–11415.Montfort A, Owen S, Piskorz AM, Supernat A, Moore L, Al-Khalidi S, et al. Combining measures of immune infiltration shows additive effect on survival prediction in high-grade serous ovarian carcinoma. Br J Cancer 2020;122(12):1803–1810.Liu W, Wang Y, Xie Y, Dai T, Fan M, Lu C, Zou Y. Cisplatin remodels the tumor immune microenvironment via the transcription factor EB in ovarian cancer. Cell Death Discov. 2021;7(1):136.Peng J, Hamanishi J, Matsumura N, Abiko K, Murat K, Baba T, Yamaguchi K, Horikawa N, Hosoe Y, Murphy SK, Konishi I, Mandai M. Chemotherapy induces programmed cell death-Ligand 1 overexpression via the nuclear factor-κB to foster an immunosuppressive tumor microenvironment in Ovarian cancer. Cancer Res 2015;75(23):5034–45.Jiao S, Xia W, Yamaguchi H, Wei Y, Chen M-K, Hsu J-M, et al. PARP inhibitor upregulates PD-L1 expression and enhances cancer-associated immunosuppression. Clin Cancer Res 2017;23(14):3711–3720.Xue C, Xu Y, Ye W, Xie Q, Gao H, Xu B, et al. Expression of PD-L1 in ovarian cancer and its synergistic antitumor effect with PARP inhibitor. Gynecol Oncol 2020;157(1):222–233.Zhang L, Chen Y, Li F, Bao L, Liu W. Atezolizumab and bevacizumab attenuate cisplatin resistant Ovarian cancer cells progression synergistically via suppressing epithelial-Mesenchymal transition. Front Immunol 2019;10:867.

BackgroundHigh grade serous ovarian cancer (HGSOC) is a lethal gynecologic malignancy with a five-year survival rate of only 39 percent.1 Despite the fact that ovarian tumors are considered immunologic, HGSOC patients respond poorly to PD-1 based immunotherapy. Hence, the need to identify novel prognostic and therapeutic immunologic factors is crucial. Our previous investigation uncovered intratumoral levels of immune co-inhibitory receptor LAG-3 as a marker of improved HGSOC patient survival. For this current study we sought to evaluate the prognostic utility of serum-based LAG-3, as well as determine how these circulating levels change in response to HGSOC standard of care therapy.MethodsThis study was approved by the Women and Infants Institutional Review Board; approval number 1057626. HGSOC serum samples were obtained from the Department of Special Testing and the Program in Women’s Oncology Gynecologic Tissue Bank at Women and Infants Hospital. A total of 43 HGSOC treatment naïve serum samples were tested for serum LAG-3 and in 20 of these patients, samples from on- and post- frontline platinum-based chemotherapy were also analyzed. A commercially available ELISA kit was employed to detect serum LAG-3.ResultsThere was no statistically significant change in pre-, on-, and post- serum LAG-3 levels following frontline chemotherapy, however median levels of LAG-3 decreased once patients initiated therapy and remained stable post-treatment. Spearman Rank Correlation analysis revealed a significant relationship between progression-free survival (PFS) and pre-treatment serum LAG-3 levels (r=0.36, p=0.017). Furthermore, it was found that patients with a PFS of 6 months or less exhibited a significantly (p=0.0031) lower mean rank of pre-treatment serum LAG-3 levels, compared to patients with a PFS of 18 months or longer. Finally, Kaplan Meier curve analysis revealed a statistically significant association between longer patient PFS and higher pre-treatment LAG-3 concentrations, when stratified by both median(HR=0.4916, log-rank p=0.022) and quartile LAG-3 serum levels (HR=0.2679, log-rank p=0.0031).ConclusionsThis study demonstrates for the first time that circulating immune checkpoint receptors have prognostic capabilities in ovarian cancer. Our findings suggest that LAG-3 is a marker for improved patient PFS. Future directions include an expansion of this original cohort in order to validate and further assess the clinical prognostic utility of serum LAG-3 in HGSOC.ReferenceSurvival Rates for Ovarian Cancer, American Cancer Society, [https://www.cancer.org/cancer/ovarian-cancer/detection-diagnosis-staging/survival-rates.html]

Epithelial Ovarian Cancer (EOC) is a deadly gynecologic malignancy in which patients frequently develop recurrent disease following initial platinum-taxane chemotherapy. Analogous to many other cancer subtypes, EOC clinical trials have centered upon immunotherapeutic approaches, most notably programmed cell death 1 (PD-1) inhibitors. While response rates to these immunotherapies in EOC patients have been low, evidence suggests that ovarian tumors are immunogenic and that immune-related genomic profiles can serve as prognostic markers. This review will discuss recent advances in the development of immune-based prognostic signatures in EOC that predict patient clinical outcomes, as well as emphasize specific research areas that need to be addressed to drive this field forward.

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, with an overall 5-year survival of only 47%. As the development of novel targeted therapies is drastically necessary in order to improve patient survival, current EOC clinical trials have heavily focused on immunotherapeutic approaches, centered upon programmed cell death 1 (PD-1) inhibitors. While PD-1 monotherapies have only exhibited modest responses for patients, it has been theorized that in order to enhance EOC patient response to immunotherapy, combinatorial regimens must be investigated. In this review, unique challenges to EOC PD-1 response will be discussed, along with a comprehensive description of both preclinical and clinical studies evaluating PD-1-based combinatorial therapies. Promising aspects of PD-1-based combinatorial approaches are highlighted, while also discussing specific preclinical and clinical areas of research that need to be addressed, in order to optimize EOC patient immunotherapy response.

Human epididymis protein-4 (HE4/ WFDC2 ) has been well-studied as an ovarian cancer clinical biomarker. To improve our understanding of its functional role in high grade serous ovarian cancer, we determined transcriptomic differences between ovarian tumors with high- versus low- WFDC2 mRNA levels in The Cancer Genome Atlas dataset. High- WFDC2 transcript levels were significantly associated with reduced survival in stage III/IV serous ovarian cancer patients. Differential expression and correlation analyses revealed secretory leukocyte peptidase inhibitor ( SLPI / WFDC4 ) as the gene most positively correlated with WFDC2 , while A kinase anchor protein-12 was most negatively correlated. WFDC2 and SLPI were strongly correlated across many cancers. Gene ontology analysis revealed enrichment of oxidative phosphorylation in differentially expressed genes associated with high- WFDC2 levels, while extracellular matrix organization was enriched among genes associated with low- WFDC2 levels. Immune cell subsets found to be positively correlated with WFDC2 levels were B cells and plasmacytoid dendritic cells, while neutrophils and endothelial cells were negatively correlated with WFDC2 . Results were compared with DepMap cell culture gene expression data. Gene ontology analysis of k-means clustering revealed that genes associated with low- WFDC2 were also enriched in extracellular matrix and adhesion categories, while high- WFDC2 genes were enriched in epithelial cell proliferation and peptidase activity. These results support previous findings regarding the effect of HE4/ WFDC2 on ovarian cancer pathogenesis in cell lines and mouse models, while adding another layer of complexity to its potential functions in ovarian tumor tissue. Further experimental explorations of these findings in the context of the tumor microenvironment are merited.

Patients with ovarian cancer exhibit low response rates to anti-programmed cell death protein-1 (PD-1) based therapies, despite ovarian tumors demonstrating measurable immune responses. Therefore, the aim of the present study was to comparatively examine expression of notable immune co-stimulatory and co-inhibitory receptors in order identify the most abundant receptors that could potentially serve as therapeutic targets to enhance immunotherapy response in high grade serous ovarian cancer (HGSOC). The Cancer Genome Atlas (TCGA) was employed to compare levels of various HGSOC and pan-cancer cohorts. To confirm these findings at the protein level, immunofluorescence of select receptors was performed in 29 HGSOC patient tissue samples. TCGA and Kaplan Meier analysis was employed to determine the association of highly expressed immune receptors with clinical outcomes. TIM-3 and OX40 exhibited the highest expression in HGSOC at both the gene and protein level, with TIM-3 demonstrating highest levels on both CD8+ and CD4+ T cell subsets. Pan-cancer analysis determined that TIM-3 and OX40 levels were similar to those in immunotherapy-responsive cancers, while PD-1 exhibited much lower expression in HGSOC. Finally, OX40 was most strongly associated with improved patient survival. Overall, the current study suggested that TIM-3 and OX40 are frequently expressed intratumoral immune receptors in HGSOC and thus represent promising immune targets. Furthermore, the present analysis strongly suggested that OX40 was significantly associated with a longer survival and could potentially be utilized as a prognostic factor for improved patient outcomes in HGSOC.