Placental syncytiotrophoblasts formed by the fusion of cytotrophoblasts constitute the interface between maternal and fetal circulations. The syncytium, composed of a continuous layer of syncytiotrophoblasts, assumes the fetal-maternal nutrient exchange, placental barrier, and endocrine functions important for the maintenance of normal pregnancy. Syncytin-1, an endogenous retroviral gene product, mediates the fusion of cytotrophoblasts. While the fusogenic function of syncytin-1 has been well established, little is known regarding its nonfusogenic activities. This study investigates the role of syncytin-1 in trophoblast proliferation. We found that syncytin-1 knockdown significantly inhibited BeWo cell growth and DNA synthesis. Moreover, time course studies on key cell cycle regulators demonstrated an upregulation of p15 and downregulation of CDK4, E2F1, PCNA, and c-Myc, which consequently led to a reduced level of CDK1. These results, together with those from flowcytometry analysis, indicated that syncytin-1 knockdown blocked the G1/S transition phase of the cell cycle. Moreover, syncytin-1 overexpression promoted CHO cell proliferation and led to changes opposite to those observed in syncytin-1 knockdown experiments, confirming the critical role of syncytin-1 for G1/S transition. Thus, syncytin-1, through both nonfusogenic and fusogenic, functions, may co-regulate the input (proliferation) and output (fusion) of the cytotrophoblast “pool”. Such co-regulation could be an efficient way to achieve the balance between these two opposing processes, which is required for syncytium homeostasis. Since decreased syncytin-1 expression has been shown to be associated with preeclamptic and hypoxic condition, insufficient replenishing of the cytotrophoblast “pool” may contribute to syncytium deficiency, a critical pathological change frequently found in preeclamptic placentas.

Epithelial ovarian cancer (EOC) is a highly lethal gynecologic malignancy arising from the fallopian tubes that has a high rate of chemoresistant recurrence and low five-year survival rate. The ovarian cancer biomarker HE4 is known to promote proliferation, metastasis, chemoresistance, and suppression of cytotoxic lymphocytes. In this study, we sought to examine the effects of HE4 on signaling within diverse cell types that compose the tumor microenvironment. HE4 was found to activate STAT3 signaling and promote upregulation of the pro-angiogenic STAT3 target genes IL8 and HIF1A in immune cells, ovarian cancer cells, and endothelial cells. Moreover, HE4 promoted increases in tube formation in an in vitro model of angiogenesis, which was also dependent upon STAT3 signaling. Clinically, HE4 and IL8 levels positively correlated in ovarian cancer patient tissue. Furthermore, HE4 serum levels correlated with microvascular density in EOC tissue and inversely correlated with cytotoxic T cell infiltration, suggesting that HE4 may cause deregulated blood vessel formation and suppress proper T cell trafficking in tumors. Collectively, this study shows for the first time that HE4 has the ability to affect signaling events and gene expression in multiple cell types of the tumor microenvironment, which could contribute to angiogenesis and altered immunogenic responses in ovarian cancer.

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Dual specificity phosphatase 6 (DUSP6) is a protein phosphatase that deactivates extracellular-signal-regulated kinase (ERK). Since the ovarian cancer biomarker human epididymis protein 4 (HE4) interacts with the ERK pathway, we sought to determine the relationship between DUSP6 and HE4 and elucidate DUSP6's role in epithelial ovarian cancer (EOC). Viability assays revealed a significant decrease in cell viability with pharmacological inhibition of DUSP6 using (E/Z)-BCI hydrochloride in ovarian cancer cells treated with carboplatin or paclitaxel, compared to treatment with either agent alone. Quantitative PCR was used to evaluate levels of ERK pathway response genes to BCI in combination with recombinant HE4 (rHE4), carboplatin, and paclitaxel. Expression of EGR1, a promoter of apoptosis, was higher in cells co-treated with BCI and paclitaxel or carboplatin than in cells treated with chemotherapeutic agents alone, while expression of the proto-oncogene c-JUN was decreased with co-treatment. The effect of BCI on the expression of these two genes opposed that of rHE4. Pathway focused quantitative PCR also revealed suppression of ERBB3 in cells co-treated with BCI plus carboplatin or paclitaxel. Finally, expression levels of DUSP6 in EOC tissue were evaluated by immunohistochemistry, revealing significantly increased levels of DUSP6 in serous EOC tissue compared to adjacent normal tissue. A positive correlation between HE4 and DUSP6 levels was determined by Spearman Rank correlation. In conclusion, DUSP6 inhibition sensitizes ovarian cancer cells to chemotherapeutic agents and alters gene expression of ERK response genes, suggesting that DUSP6 could plausibly function as a novel therapeutic target to reduce chemoresistance in EOC.

Human epididymis protein 4 (HE4) has received much attention recently due to its diagnostic and prognostic abilities for epithelial ovarian cancer. Since its inclusion in the Risk of Ovarian Malignancy Algorithm (ROMA), studies have focused on its functional effects in ovarian cancer. Here, we aimed to investigate the role of HE4 in invasion, haptotaxis, and adhesion of ovarian cancer cells. Furthermore, we sought to gain an understanding of relevant transcriptional profiles and protein kinase signaling pathways mediated by this multifunctional protein. Exposure of OVCAR8 ovarian cancer cells to recombinant HE4 (rHE4) promoted invasion, haptotaxis toward a fibronectin substrate, and adhesion onto fibronectin. Overexpression of HE4 or treatment with rHE4 led to upregulation of several transcripts coding for extracellular matrix proteins, including SERPINB2, GREM1, LAMC2, and LAMB3. Gene ontology indicated an enrichment of terms related to extracellular matrix, cell migration, adhesion, growth, and kinase phosphorylation. LAMC2 and LAMB3 protein levels were constitutively elevated in cells overexpressing HE4 and were upregulated in a time-dependent manner in cells exposed to rHE4 in the media. Deposition of laminin-332, the heterotrimer comprising LAMC2 and LAMB3 proteins, was increased in OVCAR8 cells treated with rHE4 or conditioned media from HE4-overexpressing cells. Enzymatic activity of matriptase, a serine protease that cleaves laminin-332 and contributes to its pro-migratory functional activity, was enhanced by rHE4 treatment in vitro. Proteomic analysis revealed activation of focal adhesion kinase signaling in OVCAR8 cells treated with conditioned media from HE4-overexpressing cells. Focal adhesions were increased in cells treated with rHE4 in the presence of fibronectin. These results indicate a direct role for HE4 in mediating malignant properties of ovarian cancer cells and validate the need for HE4-targeted therapies that will suppress activation of oncogenic transcriptional activation and signaling cascades.

Ovarian cancers are known to evade immunosurveillance and to orchestrate a suppressive immune microenvironment. Here we examine the role of human epididymis protein 4 (HE4), an ovarian cancer biomarker, in immune evasion. Through modified subtractive hybridization analyses we have characterized the gene targets of HE4 in human peripheral blood mononuclear cells (PBMCs), and established a preliminary mechanism for HE4-mediated immune failure in ovarian tumours. Upon exposure of purified PMBCs to HE4, osteopontin (OPN) and dual-specificity phosphatase 6 (DUSP6) emerged as the most suppressed and up-regulated genes, respectively. SKOV3 and OVCAR8, human ovarian carcinoma cell lines, exhibited enhanced proliferation in conditioned media from HE4-exposed PBMCs, an effect that was attenuated by the addition of recombinant OPN or OPN-inducible cytokines [interleukin (IL)-12 and interferon (IFN)-Ɣ]. Additionally, upon co-culture with PBMCs, HE4-silenced SKOV3 cells were found to be more susceptible to cytotoxic cell death. The relationship between HE4 and OPN was reinforced further through the analysis of serous ovarian cancer patient samples. In these biopsy specimens, the number of OPN T cells correlated positively with progression free survival (PFS) and inversely with serum HE4 level. Taken together, these findings show that HE4 enhances ovarian cancer tumorigenesis by compromising OPN-mediated T cell activation.

While selective overexpression of serum clinical biomarker Human epididymis secretory protein 4 (HE4) is indicative of ovarian cancer tumorigenesis, much is still known about the mechanistic role of the HE4 gene or gene product. Here, we examine the role of the secretory glycoprotein HE4 in ovarian cancer immune evasion. Through modified subtractive hybridization analyses of human peripheral blood mononuclear cells (PBMCs), we have characterized gene targets of HE4 and established a preliminary mechanism of HE4-mediated immune failure in ovarian tumors. Dual specificity phosphatase 6 (DUSP6) emerged as the most upregulated gene in PBMCs upon in vitro exposure to HE4. DUSP6 was found to be upregulated in CD8+ cells and CD56+ cells. HE4 exposure reduced Erk1/2 phosphorylation specifically in these cell populations and the effect was erased by co-incubation with a DUSP6 inhibitor, (E)-2-benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI). In co-culture with PBMCs, HE4-silenced SKOV3 human ovarian cancer cells exhibited enhanced proliferation upon exposure to external HE4, while this effect was partially attenuated by adding BCI to the culture. Additionally, the reversal effects of BCI were erased in the co-culture with CD8+ / CD56+ cell deprived PBMCs. Taken together, these findings show that HE4 enhances tumorigenesis of ovarian cancer by compromising cytotoxic CD8+ and CD56+ cells through upregulation of self-produced DUSP6.

Objective: The goal of this study is to comprehensively determine the most clinically relevant immune checkpoint receptor in epithelial ovarian cancer (EOC). Methods: Stage III, Grade III EOC formalin-fixed, paraffin-embedded (FFPE) tumors from ten patients were submitted to Cofactor Genomics to undergo RNA sequencing and machine learning analysis to determine immune cell content and levels of the ten most studied immune escape genes. Patient samples were stratified by high progression-free survival (PFS) of 65 months or greater (n=5) and low PFS of 7 months or less (n=5). All patient tumors submitted were from primary debulking surgery and were naïve to chemotherapy. Immunohistochemistry (IHC) was employed to validate findings. Results: The two immune escape genes ICOS and CTLA-4 were found to be the most predictive biomarkers differentiating short and long PFS. ICOS median transcripts per million (TPM) were 418 and 1,621 in patients with short and long PFS, respectively (p<0.03), while median TPM for CTLA-4 were 1,294 and 4,045 in patients with short and long PFS, respectively (p<0.03). In addition, PD-1 exhibited a median TPM of 817 in patients with short PFS compared to 2455 in patients with long PFS (p<0.05). Immune cell profiling revealed a statistically significant (p<0.05) lower percentage of M2 macrophages in patients with a long PFS (1.07%) compared to patients with a short PFS (2.13%). Higher percentages of CD4+ T cells, CD8+ T cells, CD19+ B cells, and Tregs were detected in patients with a longer PFS compared to short; however, these differences did not reach statistical significance. It was discovered that immune escape genes PD-1, PD-L1, CTLA-4, OX40, TIM3, and ICOS all significantly correlated (p<0.05) to the percentage of CD8+ T cells and total immune cell content. CD47 levels significantly inversely correlated to total immune cell percentages (p<0.02). OX40 was the only immune escape gene that significantly correlated to percentage of CD4+ T cells (p<0.03). PD-1, PD-L1, CTLA-4, OX40, and ICOS all correlated significantly to the percentage of CD19+ B cells (p<0.02). Finally, PD-L1, CTLA-4, OX40, TIM3, and ICOS all significantly correlated to Treg levels (p<0.03). Conclusions: Immune modeling analysis revealed ICOS and CTLA-4 as the most predictive immune biomarkers for PFS in EOC. Interestingly, it was discovered that levels of both activating and inhibitory immune checkpoint receptors correlate to a higher percentage of immune cell populations, indicating overall immune content is important in predicting improved outcomes. Future directions include repeating this assay in a larger patient cohort to validate the predictive threshold determined for ICOS and CTLA-4. Citation Format: Nicole James, Matthew Oliver, Joyce Ou, Jenna Emerson, Katherine Miller, Erin Lips, Ashley Borgstadt, Paul DiSilvestro, Jennifer Ribeiro. Immune modeling analysis identifies ICOS and CTLA-4 as predictive biomarkers in serous epithelial ovarian cancer [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B04.