Margaretha A. Skowron

Phenotype plasticity rather than repopulation from CD90/CK14+ cancer stem cells leads to cisplatin resistance of urothelial carcinoma cell lines

BackgroundTumour heterogeneity and resistance to systemic treatment in urothelial carcinoma (UC) may arise from cancer stem cells (CSC). A recent model describes cellular differentiation states within UC based on corresponding expression of surface markers (CD) and cytokeratins (CK) with CD90 and CK14 positive cells representing the least differentiated and most tumourigenic population. Based on the fact that this population is postulated to constitute CSCs and the origin of cisplatin resistance, we enriched urothelial carcinoma cell lines (UCCs) for CD90 and studied the tumour-initiating potential of these separated cells in vitro.MethodsMagnetic- and fluorescence-activated- cell sorting were used for separation of CD90+ and CD90− UCCs. Distribution of cell surface markers CD90, CD44, and CD49f and cytokeratins CK14, CK5, and CK20 as well as the effects of short- and long-term treatment with cisplatin were assessed in vitro and measured by qRT-PCR, immunocytochemistry, reporter assay and flow cytometry in 11 UCCs.ResultsWe observed cell populations with surface markers according to those reported in tumour xenografts. However, expression of cytokeratins did not concord regularly with that of the surface markers. In particular, expression of CD90 and CK14 diverged during enrichment of CD90+ cells by immunomagnetic sorting or following cisplatin treatment. Enriched CD90+ cells did not exhibit CSC-like characteristics like enhanced clonogenicity and cisplatin resistance. Moreover, selection of cisplatin-resistant sublines by long-term drug treatment did not result in enrichment of CD90+ cells. Rather, these sublines displayed significant phenotypic plasticity expressing EMT markers, an altered pattern of CKs, and WNT-pathway target genes.ConclusionsOur findings indicate that the correspondence between CD surface markers and cytokeratins reported in xenografts is not maintained in commonly used UCCs and that CD90 may not be a stable marker of CSC in UC. Moreover, UCCs cells are capable of substantial phenotypic plasticity that may significantly contribute to the emergence of cisplatin resistance.

The New Immortalized Uroepithelial Cell Line HBLAK Contains Defined Genetic Aberrations Typical of Early Stage Urothelial Tumors

Background: Cell culture models of normal urothelial cells are important for studying differentiation, disease mechanisms and anticancer drug development. Beyond primary cultures with their limitations in lifespan, interindividual heterogeneity and supply, few conditionally immortalized cell lines with limited applicability due to partial transformation or impaired differentiation capacity are available. We describe characteristics of the new spontaneously immortalized cell line HBLAK derived from a primary culture of uroepithelial cells. Objective: To characterize utility and limitations of HBLAK cells as an urothelial cell culture model. Methods: Differentiation markers were investigated by immunofluorescence and RT-PCR, genetic changes by standard karyotyping, array-CGH, PCR, RT-PCR and exome sequencing; expression of p53 and p21 by Western blotting. Results: HBLAK cells proliferated for >50 passages without senescing. They expressed cytokeratins of basal urothelial cells. Terminal differentiation markers appeared only after induction of differentiation by specific protocols. The karyotype was stable, with few chromosomal changes, especially gains of chromosomes 5 and 20 and a chromosome 9p21 deletion resulting in p16INK4A loss. A C228T TERT promoter mutation was present, but no other mutation typical of urothelial carcinoma. TP53 was wild-type and the cell cycle was arrested in response to genomic stress. Conclusions: HBLAK cells retain some differentiation potential and respond to cytotoxic agents similar to normal urothelial cells, but contain genetic changes contributing to immortalization in urothelial tumors. HBLAK may be valuable for evaluating the tumor specificity of novel cancer drugs, but may also be applied as an urothelial in vitro carcinogenesis model.

Blocking 17β-hydroxysteroid dehydrogenase type 1 in endometrial cancer: a potential novel endocrine therapeutic approach: 17β-HSD-1 inhibition in endometrial cancer

The enzyme type 1 17β‐hydroxysteroid dehydrogenase (17β‐HSD‐1), responsible for generating active 17β‐estradiol (E2) from low‐active estrone (E1), is overexpressed in endometrial cancer (EC), thus implicating an increased intra‐tissue generation of E2 in this estrogen‐dependent condition. In this study, we explored the possibility of inhibiting 17β‐HSD‐1 and impairing the generation of E2 from E1 in EC using in vitro, in vivo, and ex vivo models. We generated EC cell lines derived from the well‐differentiated endometrial adenocarcinoma Ishikawa cell line and expressing levels of 17β‐HSD‐1 similar to human tissues. In these cells, HPLC analysis showed that 17β‐HSD‐1 activity could be blocked by a specific 17β‐HSD‐1 inhibitor. In vitro, E1 administration elicited colony formation similar to E2, and this was impaired by 17β‐HSD‐1 inhibition. In vivo, tumors grafted on the chicken chorioallantoic membrane (CAM) demonstrated that E1 upregulated the expression of the estrogen responsive cyclin A similar to E2, which was impaired by 17β‐HSD‐1 inhibition. Neither in vitro nor in vivo effects of E1 were observed using 17β‐HSD‐1‐negative cells (negative control). Using a patient cohort of 52 primary ECs, we demonstrated the presence of 17β‐HSD‐1 enzyme activity (ex vivo in tumor tissues, as measured by HPLC), which was inhibited by over 90% in more than 45% of ECs using the 17β‐HSD‐1 inhibitor. Since drug treatment is generally indicated for metastatic/recurrent and not primary tumor, we next demonstrated the mRNA expression of the potential drug target, 17β‐HSD‐1, in metastatic lesions using a second cohort of 37 EC patients. In conclusion, 17β‐HSD‐1 inhibition efficiently blocks the generation of E2 from E1 using various EC models. Further preclinical investigations and 17β‐HSD‐1 inhibitor development to make candidate compounds suitable for the first human studies are awaited. Copyright

Distinct mechanisms contribute to acquired cisplatin resistance of urothelial carcinoma cells

Cisplatin (CisPt) is frequently used in the therapy of urothelial carcinoma (UC). Its therapeutic efficacy is limited by inherent or acquired drug resistance. Here, we comparatively investigated the CisPt-induced response of two different parental urothelial carcinoma cell lines (RT-112, J-82) with that of respective drug resistant variants (RT-112R, J-82R) obtained upon month-long CisPt selection. Parental RT-112 cells were ~2.5 fold more resistant to CisPt than J-82 cells and showed a different expression pattern of CisPt-related resistance factors. CisPt resistant RT-112R and J-82R variants revealed a 2–3-fold increased CisPt resistance as compared to their corresponding parental counterparts. Acquired CisPt resistance was accompanied by morphological alterations resembling epithelial mesenchymal transition (EMT). RT-112R cells revealed lower apoptotic frequency and more pronounced G2/M arrest following CisPt exposure than RT-112 cells, whereas no differences in death induction were observed between J-82 and J-82R cells. CisPt resistant J-82R cells however were characterized by a reduced formation of CisPt-induced DNA damage and related DNA damage response (DDR) as compared to J-82 cells. Such difference was not observed between RT-112R and RT-112 cells. J-82R cells showed an enhanced sensitivity to pharmacological inhibition of checkpoint kinase 1 (Chk1) and, moreover, could be re-sensitized to CisPt upon Chk1 inhibition. Based on the data we suggest that mechanisms of acquired CisPt resistance of individual UC cells are substantially different, with apoptosis- and DDR-related mechanisms being of particular relevance. Moreover, the findings indicate that targeting of Chk1 might be useful to overcome acquired CisPt resistance of certain subtypes of UC.

Targeting urothelial carcinoma cells by combining cisplatin with a specific inhibitor of the autophagy-inducing class III PtdIns3K complex

BACKGROUND Cisplatin-based regimens are routinely employed for the treatment of urothelial carcinoma. However, therapeutic success is hampered by the primary presence of or the development of cisplatin resistance. This chemoresistance is executed by multiple cellular pathways. In recent years, the cellular process of autophagy has been identified as a prosurvival pathway of cancer cells. On the one hand, autophagy enables cancer cells to survive conditions of low oxygen or nutrient supply, frequently found in tumors. On the other hand, autophagy supports chemoresistance of cancer cells. Here, we aimed at investigating the involvement of autophagy for cisplatin resistance in different urothelial carcinoma cell lines. MATERIALS & METHODS We analyzed the expression levels of different autophagy-related proteins in cisplatin-sensitive and cisplatin-resistant urothelial carcinoma cell lines. Furthermore, we performed cell viability assays and caspase activity assays with cells treated with cisplatin, non-specific or specific autophagy inhibitors (chloroquine, 3-methyladenine, SAR405) or combinations thereof. RESULTS We found that autophagy-related proteins are up-regulated in different cisplatin-resistant urothelial carcinoma cells compared to the sensitive parental cell lines. Furthermore, inhibition of autophagy, in general, or of the autophagy-inducing class III PtdIns3K complex, in particular, sensitized both sensitive and resistant urothelial carcinoma cells to cisplatin-induced cytotoxic effects. CONCLUSION We propose that targeting the autophagic machinery might represent a suitable approach to complement or even increase cisplatin efficacy in order to overcome cisplatin resistance in urothelial carcinoma.

Applying the chicken embryo chorioallantoic membrane assay to study treatment approaches in urothelial carcinoma

BACKGROUND Rapid development of novel treatment options demands valid preclinical screening models for urothelial carcinoma (UC). The translational value of high-throughput drug testing using 2-dimensional (2D) cultures is limited while for xenograft models handling efforts and costs often become prohibitive for larger-scale drug testing. Therefore, we investigated to which extent the chicken chorioallantoic membrane (CAM) assay might provide an alternative model to study antineoplastic treatment approaches for UC. METHODS The ability of 8 human UC cell lines (UCCs) to form tumors after implantation on CAMs was investigated. Epithelial-like RT-112 and mesenchymal-like T-24 UCCs in cell culture or as CAM tumors were treated with cisplatin alone or combined with histone deacetylase inhibitors (HDACi) romidepsin and suberanilohydroxamic acid. Tumor weight, size, and bioluminescence activity were monitored; tumor specimens were analyzed by histology and immunohistochemistry. Western blotting and quantitative real time polymerase chain reaction were used to measure protein and mRNA expression. RESULTS UCCs were reliably implantable on the CAM, but tumor development varied among cell lines. Expression of differentiation markers (E-cadherin, vimentin, CK5, CK18, and CK20) was similar in CAM tumors and 2D cultures. Cellular phenotypes also remained stable after recultivation of CAM tumors in 2D cultures. Bioluminescence images correlated with tumor weight. Cisplatin and HDACi decreased weight and growth of CAM tumors in a dose-dependent manner, but HDACi treatment acted less efficiently as in 2D cultures, especially on its typically associated molecular markers. Synergistic effects of HDACi and subsequent cisplatin treatment on UCCs were neither detected in 2D cultures nor detected in CAM tumors. CONCLUSION Our results demonstrate that the CAM assay is a useful tool for studying tumor growth and response to conventional anticancer drugs under 3D conditions, especially cytotoxic drugs as cisplatin. With some limitations, it might serve as a cost- and time-effective preclinical screening assay for novel therapeutic approaches before further assessment in expensive and cumbersome animal models.

Various Mechanisms Involve the Nuclear Factor (Erythroid-Derived 2)-Like (NRF2) to Achieve Cytoprotection in Long-Term Cisplatin-Treated Urothelial Carcinoma Cell Lines.

Therapeutic efficacy of cisplatin-based chemotherapy for advanced-stage urothelial carcinoma (UC) is limited by drug resistance. The nuclear factor (erythroid-derived 2)-like 2 (NRF2) pathway is a major regulator of cytoprotective responses. We investigated its involvement in cisplatin resistance in long-term cisplatin treated UC cell lines (LTTs). Expression of NRF2 pathway components and targets was evaluated by qRT-PCR and western blotting in LTT sublines from four different parental cells. NRF2 transcriptional activity was determined by reporter assays and total glutathione (GSH) was quantified enzymatically. Effects of siRNA-mediated NRF2 knockdown on chemosensitivity were analysed by viability assays, γH2AX immunofluorescence, and flow cytometry. Increased expression of NRF2, its positive regulator p62/SQSTM1, and elevated NRF2 activity was observed in 3/4 LTTs, which correlated with KEAP1 expression. Expression of cytoprotective enzymes and GSH concentration were upregulated in some LTTs. NRF2 knockdown resulted in downregulation of cytoprotective enzymes and resensitised 3/4 LTTs towards cisplatin as demonstrated by reduced IC50 values, increased γH2AX foci formation, and elevated number of apoptotic cells. In conclusion, while LTT lines displayed diversity in NRF2 activation, NRF2 signalling contributed to cisplatin resistance in LTT lines, albeit in diverse ways. Accordingly, inhibition of NRF2 can be used to resensitise UC cells to cisplatin, but responses in patients may likewise be variable.

Tumor immunotherapy—the potential of epigenetic drugs to overcome resistance

Immune checkpoint inhibitors recently introduced into clinical practice have enriched available treatment options for a number of solid cancers. The efficacy of these treatments may however be impaired by tumor cell-intrinsic factors and tumor cell-extrinsic factors resulting in repressed tumor immunogenicity and host immune response. Accordingly, only a subgroup of patients responds to this treatment. Epigenetic drugs may offer an exciting novel approach to reverse immune suppression and to ‘prime’ tumors for immunotherapy, as DNA methyltransferase (DNMT) inhibitors, histone deacetylase (HDAC) inhibitors and bromodomain and extra-terminal motif (BET) inhibitors show immunomodulatory properties by affecting both cancer cells directly as well as the tumor microenvironment. Upregulation of tumor associated antigens (TAAs), ligands for natural killer (NK) cell receptors, chemokine expression, altered immune checkpoint molecules, antigen procession and presentation as well as induction of viral mimicry improve recognition of cancer cells. In the tumor microenvironment, suppression of myeloid-derived suppressor cells (MDSCs), modulation of regulatory T cells function and activation of NK cells may improve immunogenic efficacy. In conclusion, current preclinical data supports translation of combinatorial treatment approaches into clinical trials and practice.

Multifaceted Mechanisms of Cisplatin Resistance in Long-Term Treated Urothelial Carcinoma Cell Lines

Therapeutic efficacy of cisplatin-based treatment of late stage urothelial carcinoma (UC) is limited by chemoresistance. To elucidate underlying mechanisms and to develop new approaches for overcoming resistance, we generated long-term cisplatin treated (LTT) UC cell lines, characterised their cisplatin response, and determined the expression of molecules involved in cisplatin transport and detoxification, DNA repair, and apoptosis. Inhibitors of metallothioneins and Survivin were applied to investigate their ability to sensitise towards cisplatin. Cell growth, proliferation, and clonogenicity were examined after cisplatin treatment by MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, EdU (5-ethynyl-2’-deoxyuridine) incorporation assay, and Giemsa staining, respectively. Cell cycle distribution and apoptosis were quantified by flow cytometry. mRNA and protein expressions were measured by real-time quantitative (qRT)-PCR, western blot, or immunofluorescence staining. LTTs recovered rapidly from cisplatin stress compared to parental cells. In LTTs, to various extents, cisplatin exporters and metallothioneins were induced, cisplatin adduct levels and DNA damage were decreased, whereas expression of DNA repair factors and specific anti-apoptotic factors was elevated. Pharmacological inhibition of Survivin, but not of metallothioneins, sensitised LTTs to cisplatin, in an additive manner. LTTs minimise cisplatin-induced DNA damage and evade apoptosis by increased expression of anti-apoptotic factors. The observed diversity among the four LTTs highlights the complexity of cisplatin resistance mechanisms even within one tumour entity, explaining heterogeneity in patient responses to chemotherapy.