Prostaglandin E2 receptor EP1 expression in vulvar cancer

Purpose In recent years, incidence of vulvar cancer has been on the rise, whereas therapeutic options are still restricted. Therefore, new prognosticators and therapeutic targets are essential. Chronic inflammation plays an important role in carcinogenesis and COX-2, and its product prostaglandin E2 and its receptors EP1–4 are known to be important mediators in cancer initiation and progression. Methods EP1 expression in vulvar cancer specimens (n = 129) was investigated via immunohistochemistry and evaluated using the well-established immunoreactive score (IRS). Subsequently, the values were correlated with clinicopathological parameters. Results Our analysis did not reveal EP1 expression as a negative prognostic factor in overall and disease-free survival. However, in the subgroup of patients with lymph-node metastasis, overall survival was significantly shorter in tumors with high EP1 expression. Moreover, EP1 expression correlated positively with good differentiation of the tumor, but not with p16 status or COX-2 expression. Conclusions This study shed first light on EP1 expression in vulvar carcinoma. EP1 expression correlated significantly with the grading of the tumor, suggesting that it influences cell differentiation. Further research on EP1 signaling may lead to a deeper understanding of the molecular mechanisms of carcinogenesis.


Introduction
Vulvar carcinoma is a relatively rare disease, being the fifth most common gynecological tumor worldwide (International Agency for Research on Cancer 2020). Squamous cell carcinoma (SCC) is its most common histologic subgroup, representing around 90% of all tumors of the vulva (Kang et al. 2017). Incidence rates have been on the rise in recent years, possibly caused partly due to an increasing prevalence of human papilloma virus (HPV) infection likely promoted by a social change in sexual behavior (Bray et al. 2020). The HPV-related pathway leading to the development of around 40% of all vulvar squamous cell carcinoma (VSCC) can be distinguished from an HPV independent pathway often associated with chronic inflammatory dermatological diseases such as lichen sclerosis or lichen planus (Carlson et al. 1998;Regauer et al. 2014;Smith et al. 2009). Radical local surgical resection often accompanied by lymphonodectomy and adjuvant radiotherapy is the predominantly applied therapy for both tumor entities, while systemic therapies are only marginally administered resulting from a lack of randomized controlled trials (Mahner et al. 2015). Despite limited literature regarding long-term effects of vulvar cancer therapy, radical surgery often involving large resection areas up to vulvectomy can lead to reduced quality of life with impaired sexual function, lymph edema, or urinary difficulties (Gitas et al. 2021;Pilger et al. 2012;Ramaseshan et al. 2018). Although HPV vaccination gives the opportunity for primary prevention of a smaller proportion of VSCC, further risk factors such as smoking habits and an aging population additionally support the need for new predictive biomarkers and target-based therapies to ameliorate the clinical outcome for patients, especially in late stages of the disease (Daling et al. 1992;Joura et al. 2007). Currently, possible therapy targets are vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) (Mahner et al. 2015). VEGF is an important mediator for angiogenesis and when overexpressed has a negative impact on the prognosis of vulvar carcinoma patients (Obermair et al. 1996). EGFR is a transmembrane protein and belongs to the family of receptor tyrosine kinases. It is often overexpressed in a vulvar carcinoma, and is associated with decreased survival, high tumor stage, lymph-node metastasis, and HPV-negativity (Growdon et al. 2008;Woelber et al. 2012). There are few case reports describing clinical benefits from the treatment with monoclonal antibody Cetuximab (Bergstrom et al. 2015;Matsuzawa et al. 2016). Also, EGFR inhibitor Erlotinib showed overall clinical benefit in 67.5% of patients in a phase II clinical trial with 41 patients. Nevertheless, therapy with the EGFR inhibitor implicated significant grade 3/4 toxicity including allergic reaction, diarrhea with electrolyte abnormalities, ischemic colitis, and renal failure, and often is often accompanied by early resistance to therapy (Horowitz et al. 2012). Therefore, the search for new prognostic markers and possible targets is necessary.
As chronic inflammation is known to play an important role in cancer initiation, progression, angiogenesis, and metastasis, prostanoid metabolism presents an interesting research topic in different tumor entities (Shacter and Weitzman 2002). Previous studies investigated the function of cyclooxygenase enzyme 2 (COX-2), prostaglandin E2 (PGE2), and its receptors (EP) in many different tumor entities like colon cancer, prostate and lung cancers, as well as in breast cancer and gynecological tumors, such as ovarian, cervical, and endometrial cancers (Howe 2007;Dubois 2010a, 2010b;Ye et al. 2020). PGE2 is produced from arachidonic acid by cyclooxygenase enzyme 2 (COX-2) and mediates its effects through its specific G-protein coupled receptors EP1-EP4, whereas EP1 is studied the least (Ye et al. 2020). Affinity for PGE2 binding is substantially different between the EP receptor subtypes with the following rank order of affinities: EP3 > EP4 > > EP2 > EP1 (Abramovitz et al. 2000). Each receptor induces different signaling cascades, e.g., through the intracellular increase of Ca 2+ and subsequently activation of protein kinase C (EP1) or the increase of secondary messenger cAMP levels and activation of protein kinase A (EP2 and EP4) (O'Callaghan and Houston 2015). EP1 is involved in several different signaling pathways. Previous research indicates the involvement of EP1 in an EGFR independent activation of the MAPK/ERK pathway in non-small cell cancer of the lung (Krysan et. al. 2005). Pan et al. demonstrated the regulatory role of EP1 on β1-integrin through the COX-2/EP1/MAPK/E2F-1 pathway (Pan et al. 2016). Moreover, EP1 seems to mediate enhanced matrix metalloproteinase-2 (MMP-2) expression by cAMP independent CREB phosphorylation, illustrating its importance in cancer invasion (Sun et. al. 2013). A study on hepatocellular carcinoma cells shows that EP1 promotes cell adhesion and migration by inducing phosphorylation of focal adhesion kinase (FAK) . Furthermore, in a in vivo study using a prostate cancer mouse model, mice that were treated with an EP1 antagonist showed a significantly lower incidence of cancer at simultaneously a higher percentage of apoptotic cells compared to control (Masato et. al. 2021).
The expression and function of COX-2, PGE 2, and EPs in vulvar cancer have been subject to little research to date. Results from previous studies identified combined cytoplasmatic COX-2 expression and EP4 expression as negative prognostic factors for survival in vulvar cancer patients. Furthermore, positive EP4 expression correlated significantly with higher FIGO stage and tumor size (Ansorge et al. 2021;Buchholz et al. 2021). The aim of this study is to examine the expression of EP1 in vulvar cancer via immunohistochemistry and to analyze its correlation with clinicopathologic variables and its effect on patients' survival to possibly find new prognostic markers and possible therapeutic targets.

Patients collective
The patients' collective was composed of 177 patients who were treated at the Department of Gynecology and Obstetrics of the Ludwig-Maximilians-University in Munich, Germany between 1990 and 2008. During surgery, collected tissue material was histopathological processed and specified. Munich Cancer Registry (MCR) from the Munich Tumor Centre (TZM-Munich Tumor Centre, Munich, Germany) provided the follow-up and survival data. For immunohistochemical staining, 157 of these 177 tissue samples were accessible. During microscopic analysis, another 28 samples were excluded, as the specific tissue slices did not contain cancer tissue. In the end, 129 patients were included in the statistical analysis. Patients' median age was 69.5 years (range 20-96 years) and overall median survival was 7.03 years. Other relevant patient characteristics are displayed in Table 1.

Immunohistochemistry
Formalin fixated and paraffin-embedded samples were cut to 4 µm slices, before being mounted on SuperFrost Plus microscope slides (Menzel Glaeser, Braunschweig, Germany). Staining was conducted as previously similarly described (Buchholz et al. 2021). First, sample slides were deparaffined in xylol for 20 min and washed in 100% alcohol. To stop the activity of endogen peroxidases, samples were incubated in methanol with 3% H 2 O 2 for 20 min. Afterward, the slides were rehydrated in descending alcohol (100%, 70%, and 50%) and washed with distilled water. In the use of a pressure cooker, samples were heated in sodium citrate buffer (pH = 6.0) (0.1 M citric acid and 0.1 M sodium citrate in distilled water) to unmask antigens, which agglomerate during formalin fixation. After cooling and washing in PBS, slides were prepared with a blocking solution (Power Block, Biogenex Laboratories, Fremont, CA, USA) for 5 min to saturate electrostatic charges and avoid nonspecific hydrophobic binding of the primary antibodies. In the next step, the primary anti-EP1 antibody dilution (1:300 in PBS) (polyclonal rabbit IgG; AB217925, Abcam, Cambridge, UK) was applied and the slides were incubated for 16 h at 4 °C in a humidity chamber. ZytoChem Plus HRP Polymer System (Zytomed, Berlin, Germany) was used for detection via secondary complex. It contained the application of a post-block reagent for 20 min and an HRP-polymer for 30 min thereafter, both at room temperature in the humidity chamber. In the end, coloration of the substrate was catalyzed by the chromogen diaminobenzidine (Dako, Hamburg, Germany) and stopped with H 2 O. In a last step, slides were counterstained with hemalaun for 2 min and washed in ascending alcohol and covered with glass. EP1 expression was evaluated with a Leitz microscope (Wetzlar, Germany) using the wellestablished semiquantitative immunoreactivity score (IRS). Therefore, a product of two factors is formed: the intensity of the staining (0 = no, 1 = weak, 2 = moderate, and 3 = strong staining) multiplied by the percentage of stained cells (0 = no staining, 1 = 10% positive cells, 2 = 11-50% positive cells, and 3 = 50% positive cells) (Remmele and Stegner 1987). Ultimately, the IRS score of the samples was correlated with clinicopathological parameters and groups with high and low EP1 expression were compared for progression-free and overall survival. Placenta tissue from the LMU Department of Gynecology and Obstetrics in Munich was utilized as negative and positive control.

Statistical analysis
Data analysis was performed with the Statistical Product and Service Solutions 28 (PASW Statistic, SPSS Inc., IBM, Chicago, IL, USA). Spearmen's test was used to test for correlations between immunohistochemically staining and clinicopathological parameters. Nonparametric tests (Mann-Whitney U) were used for group comparisons regarding the IRS of the prostaglandin receptors between independent clinical and pathological subgroups and are displayed as boxplot graphs. Survival times were analyzed by Kaplan-Meier curves and log-rank testing (Mantel-Cox). Cut-off points were acquired by the receiver operator curve (ROC). We considered p values ≤ 0.05 as statistically significant.

Correlation of enhanced EP1 expression with shorter overall survival in lymph-node positive patients
Regarding all samples, we could not detect any association between EP1 expression and overall survival (p = 0.441) or progression-free survival (p = 0.680). However, in the subgroup of patients with lymph-node metastasis, patients with enhanced EP1 expression (IRS > 3) showed significantly worse overall survival than patients with low EP1 expression (IRS ≤ 3) (median estimate: 2.6 years vs. 8.6 years; p = 0.028) (Fig. 2). In this study, multivariate analysis identified age at diagnosis (p = 0.006) and grading (p = 0.022) as independent prognostic factors for overall survival. Enhanced EP1 expression, nodal status, tumor size, FIGO classification, or p16 status did not prove to be independent prognosticators ( Table 2).

Discussion
This study shed first light on the expression of G-coupled prostaglandin receptor EP1 in vulvar carcinoma and analyzed its correlation with clinicopathologic parameters and survival. To date, EP1 expression has been described in numerous tumor entities, including breast cancer, endometrial cancer, colon cancer, and skin cancer (Gustafsson et al. 2007;Lee et al. 2005;Thorat et al. 2008;Zhu et al. 2018). Moreover, prostaglandin receptor EP1 is known to be involved in tumor initiation and progression, cell migration and invasion, and the adaption of cancer cells to hypoxia (O'Callaghan and Houston 2015). In comparison to the other EP receptors, the knowledge on EP1 in cancer metabolism is still limited and discussed controversially.
Our results revealed no influence of EP1 on overall or disease-free survival in vulvar cancer patients. These findings are analogous to a study by Zhu et al. They analyzed EP1 in a large collective of endometrial cancer (n = 140) and could also not detect any correlation with survival or clinicopathological parameters in endometrial cancer (Zhu et al. 2018). Nonetheless, in our subgroup of patients with lymph-node metastasis, patients with elevated EP1 expression in the tumor had a significantly shorter overall survival.
Since EP1 is known to be involved in cell migration and invasion, it could be possible that EP1 becomes upregulated in tumors during the invasive process of metastasis. Unfortunately, we could not be present during the tissue collecting process; consequently, it was not possible to save lymphnode tissue for the evaluation of EP1 in metastatic tissue.
In addition, we found a correlation of EP1 with tumor grading: well-differentiated tumors showed a significantly higher EP1 expression than poorly differentiated tumors. This result is supported by previous research of EP1 expression in SCC of the skin. Lee et al. found higher EP1 expression, especially in the supra-basal layers of the epidermis in SCC of the skin compared to healthy epidermal tissue (Lee et. al. 2005). EP1 seems to be an important mediator for the regulation of keratinocyte differentiation. This idea is supported by a study of Konger et al. in which they described an inhibition of keratinocyte differentiation after EP1-receptor antagonism in vitro and an enhanced expression of EP1 in well-differentiated SCC compared to poorly differentiated SCC. This regulatory effect seemed to be intact in squamous cell carcinoma of the skin, which is in line with the results from our study (Konger et al. 2009). Furthermore, statistical analysis did surprisingly not reveal any correlation between COX-2 expression [archive data from a previous study by Ansorge et al. (2021)] and EP1 expression. The affinity of EP1 for PGE2 is relatively low; therefore, one could expect an upregulation of COX-2 in EP1-positive tumor samples (Abramovitz et al. 2000). However, previous research showed a posttranslational negative feedback mechanism of EP1 on COX-2 by proteasomal degradation and showed on the other side increased levels of EP1 at COX-2 overexpression, both through transient complex formation (Haddad et al. 2012;Sood et al. 2014).
Finally, some limitations of this study are worth to be noted: on account of the retrospective approach of our study, only one semiquantitative method (immunohistochemistry) was used to evaluate EP1 expression in the patients collective. As we could not be present during the tissue collecting process, it was not possible to save lymph-node tissue for evaluation of EP1 expression and fresh tissue to apply further methods for the analysis of protein activity. Nevertheless, a strength of this study is the large size of the patient collective, considering the rarity of the disease. This study gave first insight in the expression of EP1 in vulvar cancer and its association with clinical data.  Ethical approval This study is in accordance with the ethical standards of the institutional research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Patient data were anonymized. The study was approved by the ethics committee of the Ludwig-Maximilian-University Munich (reference number 19-261). During experimental and statistical analyses, the authors were blinded for clinicopathological parameters and survival data. The cancer tissue was no longer needed for clinical tests as histopathological diagnostics after surgery had been completed prior to this study.
Consent to participate When the current study was performed, all diagnostic procedures were completed, and the patients' data were anonymized. The ethical principles adopted in the Declaration of Helsinki 1975 have been respected. As per the declaration of our ethics committee, no written informed consent of the participants or permission to publish is needed given the circumstances described above. Researchers were blinded from patient data during experimental and statistical analysis.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.