Die Metalloprotease ADAM17 - potentieller Tumormarker und Schlüsselregulator in der Epidermal-growth factor receptor-Expression von Pankreaskarzinomzellen

Die Metalloprotease ADAM17 besitzt ein großes Spektrum an Substraten, deren Prozessierung zahlreiche physiologische und pathophysiologische Prozesse beeinflussen kann. Eine besondere Bedeutung kommt ADAM17 in der Entstehung und Progression von Tumoren zu. Diese wurde bisweilen hauptsächlich mit einer gesteigerten ADAM17-abhängigen Freisetzung von EGFR-Liganden erklärt. Neueste Erkenntnisse zeigten neben einer ADAM17-assoziierten EGFR-Aktivierungssteigerung auch eine ADAM17-abhängige Erhöhung der EGFR-Expression, welche mit einer Notch1-Rezeptor-Aktivierung begründet wurde. Diese Tatsache macht ADAM17 als therapeutische Zielstruktur insbesondere für eine target-gerichtete Krebs-Therapie zunehmend bedeutender. In der vorliegenden Arbeit konnte die erwähnte ADAM17-induzierte EGFR-Expressionssteigerung, welche bisher lediglich für eine Krebsart - den non-small-cell lung cancer - beschrieben wurde auch für Zellen des Pankreaskarzinoms beobachtet werden. In den verwendeten Panc 89-Zellen wurde eine gesteigerte Notch1-Rezeptoraktivierung als Ursache für die ADAM17 induzierte EGFR-Expressionssteigerung ermittelt. Des Weiteren konnte in diesen Pankreaskrebszellen eine ADAM17-abhängige Migrationssteigerung gezeigt werden. Eine Kolokalisierung von ADAM17 mit der Zellmembran sowie eine Häufung von ADAM17 in Zellausläufern wurden ebenfalls detektiert. Aufgrund der Bedeutsamkeit von ADAM17 in Bezug auf verschiedene Krankheiten, insbesondere Tumoren, wurden die im Rahmen des SFB877 generierten, monoklonalen Anti-ADAM17-Antikörper auf ihre diagnostischen und therapeutischen Fähigkeiten hin getestet. Antikörper, welche gegen die Disintegrin- bzw. die membran-proximale Domäne gerichtet waren, konnten ADAM17 spezifisch mittels verschiedener biochemischer und molekularbiologischer Methoden wie Westernblot, Durchflusszytometrie, Immunzytochemie und ELISA detektieren. Für eine mögliche diagnostische Anwendung zeigte sich der gegen die membran-proximale Domäne gerichtete Antikörper A300E als besonders geeignet, da dieser in verschiedenen Assays die native Form von ADAM17 erkennen konnte. Geeignete Überprüfungen der Sensitivität und Spezifität desselbigen müssten durch Verwendung in immunhistochemischen Ansätzen überprüft werden. Der funktionellen Testung der Antikörper vorangehend wurden zunächst geeignete Testverfahren etabliert, die Zellfunktionen wie Proliferation und Migration überprüfen sollten. Obwohl nach Einsatz von Antikörperüberständen im Proliferations-Assay ein inhibierender Einfluss, insbesondere des Anti-Membran-proximale-Antikörpers E20-6, festgestellt wurde, konnten die gereinigten Antikörper keinen gleichbleibenden inhibitorischen Effekt erzielen. Folgeexperimente, welche die therapeutischen Anwendungsmöglichkeiten der Anti-ADAM17-Antikörper überprüfen sollten, verliefen hingegen erfolgreich. Der an Doxorubizin gekoppelte Anti-ADAM17-Antikörper A300E wurde von ADAM17 überexprimierenden NCI-H292-Zellen und MDA-MB-231-Zellen internalisiert und entfaltete zytotoxische Effekte (Trad et al., 2012). Könnten die Zytotoxizität dieses Toxin gekoppelten Antikörpers auf andere Zellarten dosisabhängig minimiert und die Effekte des A300E auch in vivo verifiziert werden, stellt der Einsatz eines solchen Toxin gekoppelten Anti-ADAM17-Antikörpers eine ernstzunehmende und interessante Perspektive in der Zielmolekül orientierten Krebstherapie dar. Diese gewinnt insbesondere vor dem Hintergrund einer Chemotherapie-induzierten ADAM17-Aktivitätssteigerung und den daraus resultierenden Kompensationsmechanismen zunehmend an Bedeutung

Combined PARP and Dual Topoisomerase Inhibition Potentiates Genome Instability and Cell Death in Ovarian Cancer

Although ovarian cancer is a rare disease, it constitutes the fifth leading cause of cancer death among women. It is of major importance to develop new therapeutic strategies to improve survival. Combining P8-D6, a novel dual topoisomerase inhibitor with exceptional anti-tumoral properties in ovarian cancer and compounds in preclinical research, and olaparib, a PARP inhibitor targeting DNA damage repair, is a promising approach. P8-D6 induces DNA damage that can be repaired by base excision repair or homologous recombination in which PARP plays a major role. This study analyzed benefits of combining P8-D6 and olaparib treatment in 2D and 3D cultures with ovarian cancer cells. Measurement of viability, cytotoxicity and caspase activity were used to assess therapy efficacy and to calculate the combination index (CI). Further DNA damage was quantified using the biomarkers RAD51 and γH2A.X. The combinational treatment led to an increased caspase activity and reduced viability. CI values partially show synergisms in combinations at 100 nM and 500 nM P8-D6. More DNA damage accumulated, and spheroids lost their membrane integrity due to the combinational treatment. While maintaining the same therapy efficacy as single-drug therapy, doses of P8-D6 and olaparib can be reduced in combinational treatments. Synergisms can be seen in some tested combinations. In summary, the combination therapy indicates benefits and acts synergistic at 100 nM and 500 nM P8-D6

ADAM17 Inhibition Increases the Impact of Cisplatin Treatment in Ovarian Cancer Spheroids

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 CelltoxTM 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

Newly developed dual topoisomerase inhibitor P8-D6 is highly active in ovarian cancer

Background: Ovarian cancer (OvCa) constitutes a rare and highly aggressive malignancy and is one of the most lethal of all gynaecologic neoplasms. Due to chemotherapy resistance and treatment limitations because of side effects, OvCa is still not sufficiently treatable. Hence, new drugs for OvCa therapy such as P8-D6 with promising antitumour properties have a high clinical need. The benzo[c]phenanthridine P8-D6 is an effective inductor of apoptosis by acting as a dual topoisomerase I/II inhibitor. Methods: In the present study, the effectiveness of P8-D6 on OvCa was investigated in vitro. In various OvCa cell lines and ex vivo primary cells, the apoptosis induction compared with standard therapeutic agents was determined in two-dimensional monolayers. Expanded by three-dimensional and co-culture, the P8-D6 treated cells were examined for changes in cytotoxicity, apoptosis rate and membrane integrity via scanning electron microscopy (SEM). Likewise, the effects of P8-D6 on non-cancer human ovarian surface epithelial cells and primary human hepatocytes were determined. Results: This study shows a significant P8-D6-induced increase in apoptosis and cytotoxicity in OvCa cells which surpasses the efficacy of well-established drugs like cisplatin or the topoisomerase inhibitors etoposide and topotecan. Non-cancer cells were affected only slightly by P8-D6. Moreover, no hepatotoxic effect in in vitro studies was detected. Conclusion: P8-D6 is a strong and rapid inductor of apoptosis and might be a novel treatment option for OvCa therapy

DataSheet_1_Vdelta1 T cells are more resistant than Vdelta2 T cells to the immunosuppressive properties of galectin-3.pdf

Ovarian carcinomas have the highest lethality amongst gynecological tumors. A problem after primary resection is the recurrence of epithelial ovarian carcinomas which is often associated with chemotherapy resistance. To improve the clinical outcome, it is of high interest to consider alternative therapy strategies. Due to their pronounced plasticity, γδ T cells are attractive for T-cell-based immunotherapy. However, tumors might escape by the release of lectin galectin-3, which impairs γδ T-cell function. Hence, we tested the effect of galectin-3 on the different γδ T-cell subsets. After coculture between ovarian tumor cells and Vδ1 or Vδ2 T cells enhanced levels of galectin-3 were released. This protein did not affect the cytotoxicity of both γδ T-cell subsets, but differentially influenced the proliferation of the two γδ T-cell subsets. While increased galectin-3 levels and recombinant galectin-3 inhibited the proliferation of Vδ2 T cells, Vδ1 T cells were unaffected. In contrast to Vδ1 T cells, the Vδ2 T cells strongly upregulated the galectin-3 binding partner α3β1-integrin after their activation correlating with the immunosuppressive properties of galectin-3. In addition, galectin-3 reduced the effector memory compartment of zoledronate-activated Vδ2 T cells. Therefore, our data suggest that an activation of Vδ1 T-cell proliferation as part of a T-cell-based immunotherapy can be of advantage.</p

High Antitumor Activity of the Dual Topoisomerase Inhibitor P8-D6 in Breast Cancer

Breast cancer constitutes the leading cause of cancer deaths among females. However, numerous shortcomings, including low bioavailability, resistance and significant side effects, are responsible for insufficient treatment. The ultimate goal, therefore, is to improve the success rates and, thus, the range available treatment options for breast cancer. Consequently, the identification, development and evaluation of potential novel drugs such as P8-D6 with seminal antitumor capacities have a high clinical need. P8-D6 effectively induces apoptosis by acting as a dual topoisomerase I/II inhibitor. This study provides an overview of the effectiveness of P8-D6 in breast cancer with both 2D monolayers and 3D spheroids compared to standard therapeutic agents. For this drug effectiveness review, cell lines and ex vivo primary cells were used and cytotoxicity, apoptosis rates and membrane integrity were examined. This study provides evidence for a significant P8-D6-induced increase in apoptosis and cytotoxicity in breast cancer cells compared to the efficacy of standard therapeutic drugs. To sum up, P8-D6 is a fast and powerful inductor of apoptosis and might become a new and suitable therapeutic option for breast cancer in the future

Tetraspanin 8 Subfamily Members Regulate Substrate-Specificity of a Disintegrin and Metalloprotease 17

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

ADAM17—A Potential Blood-Based Biomarker for Detection of Early-Stage Ovarian Cancer

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 &lt; 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

Nectin-4 as Blood-Based Biomarker Enables Detection of Early Ovarian Cancer Stages

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&rsquo; 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 &lt; 0.0001, p = 0.016). Strikingly, significantly higher Nectin-4 and HB-EGF levels were detected in early-stage FIGO I/II (p &lt;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

Chemotherapy‐induced release of ADAM17 bearing EV as a potential resistance mechanism in ovarian cancer

Abstract 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