Significance of the expression of carbonic anhydrase IX in models of ovarian cancer, gastric cancer and small cell lung cancer

Sowohl das Magenkarzinom, Ovarialkarzinom und das kleinzellige Lungenkarzinom (SCLC) sprechen auf eine Platinum-basierte Ersttherapie sehr gut an, jedoch entsteht in den meisten Fällen binnen kurzer Zeit ein therapieresistenter Rückfall. In der vorliegenden Arbeit wurde die Expression der Carboanhydrase 9 (CAIX) und des Hitzeschockproteins 90 (HSP90) in Tumorproben und Tumorzelllinien untersucht und deren Potenzial als mögliche Therapieziele evaluiert. Besonders CAIX ist von Interesse in der Krebstherapie, da dieses Protein unter physiologischen Konditionen nur im Gastrointestinaltrakt vorkommt, jedoch regelmäßig in verschiedenen soliden Tumoren überexprimiert wird. HSP90 interagiert mit mutierten Onkogenen, inklusive mutiertem p53, und trägt somit zu deren Stabilisierung und Tumorprogression bei. In der Klarzell-Ovarialkarzinom Zelllinie ES2 wurde eine konstitutive Expression von CAIX festgestellt, welche mit einer hohen Konzentration von löslichem CAIX (sCAIX) im Zellkulturüberstand einhergeht. Eine Behandlung dieser Zellen mit Cisplatin veränderte die CAIX Expression in einer dosisabhängigen Weise, jedoch nicht auf eine konsistente Art. Des Weiteren wurden hohe sCAIX Konzentration in Aszitesproben von Ovarialkarzinompatientinnen mit verschiedener Histologie detektiert aber nur in einer Subpopulation von Patienten wurden diese auch in Serumproben gefunden. Nachdem die bestehende Kombinationstherapie aus einem CAIX Inhibitor, C207A, und Carboplatin antagonistisch in einem erhöhten Zellüberleben resultierte, kann CAIX nicht als mögliches Ziel für eine Therapie in Betracht gezogen, aber möglicherweise als Marker für die Prognose herangezogen werden. Magenkarzinome weisen in hoher Frequenz p53 Mutationen auf. Nachdem bekannt ist, das HSP90 solch mutierte Onkogene stabilisieren kann, wurde die Inhibition von HSP90 via Ganetespib in verschieden Magenkarzinomzelllinien mit verschiedenen p53 Mutationen untersucht: MKN1 (Mutation im Kodon 143), MKN28 (Mutation im Kodon 257) und MKN45 (Wildtyp). Zytotoxische Effekte von Ganetespib konnten nur in der rasch proliferierenden Zelllinie MKN28 festgestellt werden, jedoch nicht in MKN1 und MKN45 Zellen. In Kombination mit Oxoplatin (cis, cis, trans-diammine-dichlorido-dihydroxido-platinum(IV)), einer oral aktiven Platinumsubstanz, wurden synergistische Effekte mit Ganetespib beobachtet. Des Weiteren reduzierte sich die Phosphorylierung von p53, Akt1/2/3, PRAS40, und WNK1 nach Behandlung mit Ganetespib. Diese stellen daher potentielle neue Ziele für eine Therapie des Magenkarzinoms dar. Außerdem wurde die CAIX Expression in der Anwesenheit von Ganetespib nahezu komplett unterdrückt. Unsere Gruppe war in der Lage stabile zirkulierende Tumor (CTC) Zelllinien von metastasierten SCLC Patienten zu etablieren. In dieser Arbeit wurde der neuroendokrine Ursprung dieser Zellen durch die Bestätigung der mRNA Expression von Chromogranin A, Synaptophysin und Enolase 2 festgestellt. In allen CTC Zelllinien wurde die spontane Formation von Sphäroiden, sogenannten Tumorosphäroide, beobachtet. Diese Tumorosphäroide zeigen eine deutlich höhere Resistenz gegenüber den bei SCLC verwendeten Chemotherapeutika im Vergleich zu Einzelzellkulturen. Des Weiteren besitzen diese Strukturen einen hypoxischen/nekrotischen Kern mit einhergehender CAIX Expression. Deswegen kann die Therapieresistenz des SCLC durch eine limitierte Diffusion der Wirkstoffe, sowie durch das Vorliegen von ruhenden und hypoxischen Zellsubpopulationen erklärt werden. Zusammengefasst stellt CAIX einen guten Marker für Hypoxie dar, jedoch ist die effektive Verwendung als Therapieziel durch die Anwesenheit hoher Konzentration von sCAIX und den limitieren Zugang zu hypoxischen Tumorarealen eingeschränkt.Gastric cancer, ovarian cancer and small cell lung cancer (SCLC) show a very good initial response to platinum-based first-line therapy, but chemoresistant relapse is seen in most cases within a short time after treatment onset. In the thesis at hand, we studied expression of carbonic anhydrase IX (CAIX) and heat-shock protein 90 (HSP90) in tumor-derived samples and evaluated their potential as novel targets for improvement of patient survival. CAIX was of particular interest since its physiological expression is restricted to few tissues of the gastrointestinal tract, but it is frequently found to be overexpressed in various solid tumors. HSP90 is known to stabilize mutated oncogenes, including p53, and thereby contributes to tumor progression. In the clear cell ovarian cancer cell line ES2 we found constitutive expression of CAIX with high levels of soluble CAIX (sCAIX) in the supernatant. Cisplatin treatment of these cells altered expression of CAIX in a dose-dependent manner, but not in a consistent way. Furthermore, high levels of sCAIX could be detected in ascites of ovarian cancer patients of various histology, while serum sCAIX levels were found in only in a subpopulation of patients. Because combinational therapy of the CAIX inhibitor C207A and carboplatin resulted antagonistically in increased cancer cell survival, CAIX is not a suitable target for therapy but may be used as prognostic marker. In gastric cancer, p53 is one of the most frequently mutated genes. Since HSP90 is thought to stabilize such mutated oncogenes, inhibition of HSP90 with ganetespib was evaluated in gastric cancer cell lines comprising different p53 mutations: MKN1 (mutation at codon 143), MKN28 (mutation at codon 257) and MKN45 (wild type). Ganetespib treatment alone resulted in cytotoxic effects in fast proliferating MKN28 cells but not in MKN1 and MKN45 cells. In contrast, synergistic effects of ganetespib in combination with the orally active oxoplatin (cis,cis,trans-diaminodichlorido-dihydroxido platinum(IV)) could be observed in all cell lines. Phosphorylation of p53, Akt1/2/3 and PRAS40, as well as of WNK1 was reduced in response to ganetespib treatment such identifying new targets in gastric cancer. CAIX expression was almost completely abolished by ganetespib. Our group had previously established five stable circulating tumor cell (CTC) cell lines from extended stage SCLC patients. In this work, neuroendocrine origin of these cells was shown by demonstrating mRNA expression of chromogranin A, synaptophysin and enolase 2. All CTC cell lines display spontaneous formation of large spheroids, termed tumorospheres. Compared to single cell cultures, tumorospheres exhibit severalfold higher resistance to standard SCLC chemotherapeutics. These structures comprise hypoxic/necrotic cores with adjacent CAIX expression and, thereby, chemoresistance seems to be caused by limited drug penetration and the presence of quiescent and hypoxic cell populations. In conclusion, CAIX represents a hypoxic marker for tumor cells but targeting of CAIX may be not possible due to high concentrations of soluble fragments and limited access of hypoxic tumor regions.submitted by Lukas Klameth, MScZusammenfassung in deutscher SpracheAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersMedizinische Universität Wien, Dissertation, 2017Medizinische Universität Wien, Diss., 2017OeB

In vitro Cytotoxic Activities of the Oral Platinum(IV) Prodrug Oxoplatin and HSP90 Inhibitor Ganetespib against a Panel of Gastric Cancer Cell Lines

Gastric cancer exhibits a poor prognosis and is the third most common cause of cancer death worldwide. Chemotherapy of metastatic gastric cancer is based on combinations of platinum drugs and fluoropyrimidines, with added agents. Oxoplatin is a stable oral platinum(IV) prodrug which is converted to a highly active tetrachlorido(IV) complex under acidic conditions. In the present work, we studied the cytotoxic effects of oxoplatin against a panel of four gastric cancer cell lines in vitro. Furthermore, the role of HSP90 in chemoresistance of these lines was investigated using the specific inhibitor ganetespib. The KATO-III, MKN-1, MKN-28, MKN-45 lines were used in MTT chemosensitivity, cell cycle and apoptosis assays. KATO-III is a signet ring diffuse cell type, MKN-1 an adenosquamous primary, MKN-28 a well-differentiated intestinal type and the MKN-45 a poorly differentiated, diffuse type gastric carcinoma line. Cytotoxicity was tested in MTT assays and intracellular signal transduction with proteome profiler Western blot arrays. Interactions of platinum drugs and ganetespib were calculated with help of the Chou-Talalay method. The prodrug oxoplatin revealed low activity against the four gastric cancer cell lines, whereas the platinum tetrachlorido(IV) complex and cisplatin gave IC values of 1-3 g/ml with increasing chemoresistance observed in the order of MKN-1, KATO-III, MKN-28 to MKN-45. With exception of KATO-III and MKN-28/oxoplatin, all other cell lines featured marked synergistic toxicity with clinically achievable concentrations of ganetespib. Oral administration of a platinum agent such as oxoplatin would be of great value for patients and care providers alike. These results suggest that the oncogene-stabilizing HSP90 chaperone represents an important mediator of chemoresistance in gastric cancer. Ganetespib reduced the phosphorylation of p53, Akt1/2/3 and PRAS40, as well as of WNK1, a kinase which regulates intracellular chloride concentrations. Intracellular chloride was reported to control proliferation of gastric cancer cell lines. Expression of MUC1 was not downregulated in contrast to the expression of CAIX, a prognostic marker in gastric cancer. In conclusion, the HSP90 inhibitor ganetespib synergizes with platinum anticancer drugs and modulates intracellular signal transduction in direction of a less proliferative and aggressive phenotype.(VLID)486417

Synergism of Cyclin-Dependent Kinase Inhibitors with Camptothecin Derivatives in Small Cell Lung Cancer Cell Lines

Advanced small cell lung cancer (SCLC) has a dismal prognosis. Modulation of the camptothecin topotecan, approved for second-line therapy, may improve response. Our recent finding of synergistic enhancement of the cytotoxic activity of camptothecin (CPT) by cyclin-dependent kinase 4 inhibitors is extended here to a panel of camptothecin analogs comprising 10-hydroxy-CPT (HOCPT), topotecan (TPT; 9-[(dimethylamino)-methyl]-10-hydroxy-CPT), 9-amino-CPT (9AC), 9-nitrocamptothecin (rubitecan), SN38 (7-ethyl-10-hydroxycamptothecin) and 10-hydroxy-9-nitrocamptothecin (CPT109) in combination with PD0332991, CDK4I, roscovitine and olomoucine. SCLC cell lines employed are chemoresistant NCI-H417 and DMS153 and the chemosensitive SCLC26A line established at our institution. The CPT analogs exhibiting highest cytotoxicity towards the three SCLC lines tested were SN38 and 9AC, followed by rubitecan, HOCPT, TPT and CPT109. NCI-H417 and DMS153 revealed an approximately 25-fold and 7-fold higher resistance compared to the chemosensitive SCLC26A cell line. Whereas the CDK4/6 inhibitor PD0332991 proved less effective to chemosensitize SCLC cells to CPT analogs, the CDK inhibitors CDK4I, roscovitine and olomoucine gave comparable chemosensitization effects in combination with 9AC, SN38, rubitecan and to a lesser extent with TPT and CPT109, not directly related with topoisomerase mRNA expression. In conclusion, small chemical modifications of the parent CPT structure result in differing cytotoxicities and chemomodulatory effects in combination with CDKIs of the resulting analogs

Expression of Proteolytic Enzymes by Small Cell Lung Cancer Circulating Tumor Cell Lines

Small cell lung cancer (SCLC) is an aggressive type of lung cancer which disseminates vigorously and has a dismal prognosis. Metastasis of SCLC is linked to an extremely high number of circulating tumor cells (CTCs), which form chemoresistant spheroids, termed tumorospheres. Intravasation and extravasation during tumor spread requires the activity of a number of proteases to disintegrate the stroma and vascular tissue. Generation of several permanent SCLC CTC lines allowed us to screen for the expression of 35 proteases using Western blot arrays. Cell culture supernatants of two CTC lines, namely BHGc7 and 10, were analyzed for secreted proteases, including matrix metalloproteinases (MMPs), ADAM/TS, cathepsins, kallikreins, and others, and compared to proteases expressed by SCLC cell lines (GLC14, GLC16, NCI-H526 and SCLC26A). In contrast to NCI-H526 and SCLC26A, MMP-9 was highly expressed in the two CTC lines and in GLC16 derived of a relapse. Furthermore, cathepsins (S, V, X/Z/P, A and D) were highly expressed in the CTC lines, whereas ADAM/TS and kallikreins were not detectable. In conclusion, SCLC CTCs express MMP-9 and a range of cathepsins for proteolysis and, aside from tissue degradation, these enzymes are involved in cell signaling, survival, and the chemoresistance of tumor cells

Scientific Reports / Small cell lung cancer : model of circulating tumor cell tumorospheres in chemoresistance

Small cell lung cancer (SCLC) represents 15% of lung cancers and is characterized by early dissemination, development of chemoresistance and a poor prognosis. A host of diverse drugs failed invariably and its mechanisms of global chemoresistance have not been characterized so far. SCLC represents the prototype of an aggressive and highly metastatic tumor which is ultimately refractory to any treatment. High numbers of circulating tumor cells (CTCs) allowed us to establish 5 CTC cell lines (BHGc7, 10, 16, 26 and UHGc5) from patients with recurrent SCLC. These cell lines exhibit the typical SCLC markers and CTCs of all patients developed spontaneously large multicellular aggregates, termed tumorospheres. Ki67 and carbonic anhydrase 9 (CAIX) staining of tumorosphere sections revealed quiescent and hypoxic cells, respectively. Accordingly, comparison of the chemosensitivity of CTC single cell suspensions with tumorospheres demonstrated increased resistance of the clusters against chemotherapeutics commonly used for treatment of SCLC. Therefore, global chemoresistance of relapsing SCLC seems to rely on formation of large tumorospheres which reveal limited accessibility, lower growth fraction and hypoxic conditions. Since similar tumor spheroids were found in other tumor types, SCLC seems to represent a unique tumor model to study the association of CTCs, metastasis and drug resistance.(VLID)460735

The Sulfatase Pathway for Estrogen Formation: Targets for the Treatment and Diagnosis of Hormone-Associated Tumors

The extragonadal synthesis of biological active steroid hormones from their inactive precursors in target tissues is named “intracrinology.” Of particular importance for the progression of estrogen-dependent cancers is the in situ formation of the biological most active estrogen, 17beta-estradiol (E2). In cancer cells, conversion of inactive steroid hormone precursors to E2 is accomplished from inactive, sulfated estrogens in the “sulfatase pathway” and from androgens in the “aromatase pathway.” Here, we provide an overview about expression and function of enzymes of the “sulfatase pathway,” particularly steroid sulfatase (STS) that activates estrogens and estrogen sulfotransferase (SULT1E1) that converts active estrone (E1) and other estrogens to their inactive sulfates. High expression of STS and low expression of SULT1E1 will increase levels of active estrogens in malignant tumor cells leading to the stimulation of cell proliferation and cancer progression. Therefore, blocking the “sulfatase pathway” by STS inhibitors may offer an attractive strategy to reduce levels of active estrogens. STS inhibitors either applied in combination with aromatase inhibitors or as novel, dual aromatase-steroid sulfatase inhibiting drugs are currently under investigation. Furthermore, STS inhibitors are also suitable as enzyme–based cancer imaging agents applied in the biomedical imaging technique positron emission tomography (PET) for cancer diagnosis