Chemotherapy resistance is a major challenge in ovarian cancer (OvCa). Thus, novel treatment combinations are highly warranted. However, many promising drug candidates tested in two-dimensional (2D) cell culture have not proved successful in the clinic. For this reason, we analyzed our drug combination not only in monolayers but also in three-dimensional (3D) tumor spheroids. One potential therapeutic target for OvCa is A disintegrin and metalloprotease 17 (ADAM17). ADAM17 can be activated by chemotherapeutics, which leads to enhanced tumor growth due to concomitant substrate cleavage. Therefore, blocking ADAM17 during chemotherapy may overcome resistance. Here, we tested the effect of the ADAM17 inhibitor GW280264X in combination with cisplatin on ovarian cancer cells in 2D and 3D. In 2D, the effect on five cell lines was analyzed with two readouts. Three of these cell lines formed dense aggregates or spheroids (HEY, SKOV-3, and OVCAR-8) in 3D and the treatment effect was analyzed with a multicontent readout (cytotoxicity, viability, and caspase3/7 activation). We tested the combined therapy on tumor spheroids derived from primary patient cells. In 2D, we found a significant reduction in the half minimal (50%) inhibitory concentration (IC₅₀) value of the combined treatment (GW280264X plus cisplatin) in comparison with cisplatin monotherapy in all five cell lines with both 2D readout assays (viability and caspase activation). In contrast, the combined treatment only showed an IC₅₀ reduction in HEY and OVCAR-8 3D tumor spheroid models using caspase3/7 activity or Celltox^TM Green as the readout. Finally, we found an improved effect of GW280264X with cisplatin in tumor spheroids derived from patient samples. In summary, we demonstrate that ADAM17 inhibition is a promising treatment strategy in ovarian cancer.

Ovarian cancer (OvCa) is the gynaecological disorder with the poorest prognosis due to the fast development of chemoresistance. We sought to connect chemoresistance and cancer cell‐derived extracellular vesicles (EV). The mechanisms of how chemoresistance is sustained by EV remained elusive. One potentially contributing factor is A Disintegrin and Metalloprotease 17 (ADAM17)—itself being able to promote chemoresistance and inducing tumour cell proliferation and survival via the Epidermal Growth Factor Receptor (EGFR) pathway by shedding several of its ligands including Amphiregulin (AREG). We now demonstrate that upon chemotherapeutic treatment, proteolytically active ADAM17 is released in association with EV from OvCa cells. In terms of function, we show that patient‐derived EV induce AREG shedding and restore chemoresistance in ADAM17‐deficient cells. Confirming that ADAM17‐containing EV transmit chemoresistance in OvCa, we propose that ADAM17 levels (also on EV) might serve as an indicator for tumour progression and the chemosensitivity status of a given patient.

Chemotherapeutic resistance evolves in about 70 % of ovarian cancer patients and is a major cause of death in this tumor entity. Novel approaches to overcome these therapeutic limitations are therefore highly warranted. A disintegrin and metalloprotease 17 (ADAM17) is highly expressed in ovarian cancer and required for releasing epidermal growth factor receptor (EGFR) ligands like amphiregulin (AREG). This factor has recently been detected in ascites of advanced stage ovarian cancer patients. However, it is not well understood, whether and how ADAM17 might contribute to chemo resistance of ovarian cancer. In this study, we identified ADAM17 as an essential upstream regulator of AREG release under chemotherapeutic treatment in ovarian cancer cell lines and patient derived cells. In the majority of ovarian cancer cells cisplatin treatment resulted in enhanced ADAM17 activity, as shown by an increased shedding of AREG. Moreover, both mRNA and the protein content of AREG were dose-dependently increased by cisplatin exposure. Consequently, cisplatin strongly induced phosphorylation of ADAM17-downstream mediators, the EGFR and extracellular signal-regulated kinases (ERK). Phorbol 12-myristate 13-acetate (PMA), similarly to cisplatin, mediated AREG shedding and membrane fading of surface ADAM17. Inhibition of ADAM17 with either GW280264X or the anti-ADAM17 antibody D1 (A12) as well as silencing of ADAM17 by siRNA selectively reduced AREG release. Thus, ADAM17 inhibition sensitized cancer cells to cisplatin-induced apoptosis, and significantly reduced cell viability. Based on these findings, we propose that targeting of ADAM17 in parallel to chemotherapeutic treatment suppresses survival pathways and potentially diminish evolving secondary chemo resistance mechanisms.

Ovarian cancer has the highest mortality rate among gynecological tumors. This is based on late diagnosis and the lack of early symptoms. To improve early detection, it is essential to find reliable biomarkers. The metalloprotease ADAM17 could be a potential marker, as it is highly expressed in many solid tumors, including ovarian and breast cancer. The aim of this work is to evaluate the relevance of ADAM17 as a potential diagnostic blood-based biomarker in ovarian cancer. Ovarian cancer cell lines IGROV-1 and A2780, as well as primary patient-derived tumor cells obtained from tumor tissue and ascitic fluid, were cultured to analyze ADAM17 abundance in the culture supernatant. In a translational approach, a cohort of 117 well-characterized ovarian cancer patients was assembled and ADAM17 levels in serum and corresponding ascitic fluid were determined at primary diagnosis. ADAM17 was quantified by enzyme-linked immunosorbent assay (ELISA). In the present study, ADAM17 was detected in the culture supernatant of ovarian cancer cell lines and primary cells. In addition, ADAM17 was found in serum and ascites of ovarian cancer patients. ADAM17 level was significantly increased in ovarian cancer patients compared to an age-matched control group (p < 0.0001). Importantly early FIGO I/II stages, which would not have been detected by CA-125, were associated with higher ADAM17 concentrations (p = 0.007). This is the first study proposing ADAM17 as a serum tumor marker in the setting of a gynecological tumor disease. Usage of ADAM17 in combination with CA-125 and other markers could help detect early stages of ovarian cancer.

Ovarian cancer is the third most common gynecological malignancy and has the highest mortality rate. Owing to unspecific symptoms, ovarian cancer is not detected until an advanced stage in about two-thirds of cases. Therefore, it is crucial to establish reliable biomarkers for the early stages to improve the patients’ prognosis. The aim of this study is to investigate whether the ADAM17 substrates Nectin-4, Heparin-binding EGF-like growth factor (HB-EGF) and Amphiregulin (AREG) could function as potential tumor markers for ovarian cancer. In this study a set of 231 sera consisting of 131 ovarian cancer patients and 100 healthy age-matched controls were assembled. Nectin-4, HB-EGF and AREG levels of preoperatively collected sera were determined by enzyme-linked immunosorbent assay (ELISA). Our analysis revealed that Nectin-4 and HB-EGF were significantly increased compared to the age-matched control group (p < 0.0001, p = 0.016). Strikingly, significantly higher Nectin-4 and HB-EGF levels were detected in early-stage FIGO I/II (p <0.001; p = 0.025) compared to healthy controls. Eighty-four percent (16/19) of patients with low Ca-125 levels showed increased Nectin-4 levels. Our study proposes Nectin-4 and HB-EGF as promising blood-based biomarkers for the detection of early stages of ovarian cancer patients that would not have been detected by Ca-125.

Ectodomain shedding is an irreversible process to regulate inter- and intracellular signaling. Members of the a disintegrin and metalloprotease (ADAM) family are major mediators of ectodomain shedding. ADAM17 is involved in the processing of multiple substrates including tumor necrosis factor (TNF) α and EGF receptor ligands. Substrates of ADAM17 are selectively processed depending on stimulus and cellular context. However, it still remains largely elusive how substrate selectivity of ADAM17 is regulated. Tetraspanins (Tspan) are multi-membrane-passing proteins that are involved in the organization of plasma membrane micro-domains and diverse biological processes. Closely related members of the Tspan8 subfamily, including CD9, CD81 and Tspan8, are associated with cancer and metastasis. Here, we show that Tspan8 subfamily members use different strategies to regulate ADAM17 substrate selectivity. We demonstrate that in particular Tspan8 associates with both ADAM17 and TNF α and promotes ADAM17-mediated TNF α release through recruitment of ADAM17 into Tspan-enriched micro-domains. Yet, processing of other ADAM17 substrates is not altered by Tspan8. We, therefore, propose that Tspan8 contributes to tumorigenesis through enhanced ADAM17-mediated TNF α release and a resulting increase in tissue inflammation.