Batai, B.

Gyulavari, P.

Nguyen, M. T.

Soti, C.

Vantus, T.

Varga, A.

Semmelweis Repository

Legal entity responsible for the study: National University of Singapore.Funding: National Medical Research Council of Singapore.Disclosure: All authors have declared no conflicts of interest.2011P Semiquantitative assessment of vimentin expression in prostatecancer (PC)M. Puchinskaya1, D. Salavei21Out-patient Dept., Minsk City Clinical Oncologic Dispensary, Minsk, Belarus, 2GeneralEcology, Biology, and Environmental Genetics, International Sakharov EnvironmentalInstitute of Belarusian State University, Minsk, BelarusBackground: Vimentin (Vim) is one of the mesenchymal markers, that are often upre-gulated in cancer cells during epithelial-mesenchymal transition (EMT) – a processendowing tumor cells with invasiveness, pro-metastatic potential and, arguably, cancerstem cells (CSC) properties. It is normally seen in cytoplasm, but cell surface Vim wasalso detected on cells with CSC characteristics. Vim expression may be used as a surro-gate marker of EMT in tumor tissue samples from patients and aid in prognosis evalua-tion. We aimed to characterize Vim expression in prostate cancer (PCa) using ourmethod of semiquantitative staining assessment.Methods: Vim expression was assessed in 48 cases of PCa using immunohistochemistrywith primary rabbit polyclonal antibodies (ThermoFischer, 1:1000). UnoView RabbitDetection Kit was used for visualization. Specificity of staining was confirmed byanother antibody (mouse monoclonal, DAKO, 1:200) in selected cases. To assess Vimexpression semiquantitatively we proposed a weighed staining index (WSI) that takesinto account the proportion of stained cells as well as the ratios of staining intensitiesand can be applied to different cellular compartments staining separately. It combinesadvantages of simple ad oculus semiquantitative staining evaluation and obtainingnumerical characteristics of marker expression.Results: Median WSI for membranous staining was 5 (0 – 52) (7, if cases withoutexpression were excluded), for cytoplasmic – 20.5 (0 – 64) (22.5, if cases withoutexpression were excluded). In 2 (4.2%) cases marker expression was absent in bothmembrane and cytoplasm. WSI in PCa was significantly higher for cytoplasmic stain-ing. However, in 3 (6.25%) cases WSI for membranous staining was higher than thatfor cytoplasmic. Significant correlation was found between WSI for cytoplasmic andmembranous staining (Spearman test, r¼ 0.51, p< 0,05). However, no correlation wasfound between WSI for both compartments and E-cadherin expression (as assessed byaverage staining intensity score – a method similar to WSI – using Aperio softwarepreviously).Conclusions: Vim expression as a sign of EMT in the tumor is present in 95.8% cases ofPCa and is more prevalent in cytoplasm. WSI is a useful tool to assess Vim (and possiblyother markers) expression.Legal entity responsible for the study: M. Puchinskaya.Funding: Belarusian Republican Foundation for Fundamental Research.Disclosure: All authors have declared no conflicts of interest.2012P Heat shock protein 90 chaperones and protein kinase D3 regulatesandrogen-independent prostate cancer developmentA. Varga1, B. Batai2, P. Gyulavari1, M.T. Nguyen3, C. S}oti3, T. Vantus31MTA SE Pathobiochemistry Research Group, Semmelweis University, Budapest,Hungary, 2MTA-SE Momentum Molecular Oncohematology Research Group, FirstDepartment of Pathology and Experimental Cancer Research, Semmelweis University,Budapest, Hungary, 3Department of Medical Chemistry, Molecular Biology andPathobiochemistry, Semmelweis University, Budapest, HungaryBackground: Prostate cancer is the second leading cause of cancer related deaths inmen worldwide. Heat Shock Protein 90 (Hsp90) is expressed in tumour cells at highlevels – 3-5% of total proteins – and regulates the function of oncogenes and othertumour related proteins. Protein kinase D3 (PKD3) has a proven role in the progres-sion of androgen-independent prostate cancer. In the present study we set out toexplore the impact of Hsp90 and PKD3, respectively, on prostate cancer growth andtheir potential interaction.Methods: We employed the DU145 and PC3 well-characterized androgen-independentprostate cancer cell lines. Cell viability was determined by Trypan Blue exclusion cellcounting. Apoptosis analysis was performed by flow cytometry after AnnexinV andpropidium-iodide co-staining. Protein levels were detected by western blot and pro-tein-protein interactions were investigated by co-immunoprecipitation.Results: We found that the clinically used Hsp90 inhibitor ganetespib induced apopto-sis and significantly reduced the viability of the androgen-independent DU145 andPC3 cell lines. The pan-PKD inhibitor CRT0066101 also decreased cell viability of theprostate cancer cells in a dose-dependent manner. Further, we demonstrated that gane-tespib reduced PKD3 protein level in a concentration-dependent manner and inducedits proteasomal degradation. Finally, a co-immunoprecipitation study revealed a physi-cal connection between PKD3 and Hsp90.Conclusions: We identified and confirmed an Hsp90-PKD3 chaperone client interac-tion, which may be important in prostate cancer cell survival. Further studies are underway to characterize the biological significance of our findings. Our results contribute tobetter understand the pathological signalling of androgen-independent prostate cancercells and to find novel treatment strategies.Legal entity responsible for the study: MTA-SE Pathobiochemistry Research Group.Funding: National Research, Development and Innovation Office - Hungary.Disclosure: All authors have declared no conflicts of interest.2013P The SWI/SNF driven reprograming for the AR cistrome is NSD2dependentK. Ruggero1, A. Martinez1, X. Chen1, R. El Bizri1, S. Farran1, L. Palomero1, J.M. Piulats2,A. Villanueva1, M.A. Pujana1, A. Aytes11LRT1, Idibell, Catalan Institute of Oncology. Hospital Druan y Reynals, Barcelona,Spain, 2Department of Medical Oncology, Genitourinary, Melanoma and Sarcoma Unit,Institut Catala d’Oncologia-IDIBELL-CIBERONC, Barcelona, SpainBackground: Resistance to androgen receptor signaling is arguably the principal hall-mark of lethal prostate cancer. Several mechanisms account for this resistance, includ-ing mutations in the AR, restoration of signaling downstream of the pharmacologicalblocking and activation of alternative oncogenic pathways.Methods: We used the Nkx3.1CreERT2/þ; Ptenfloxed/floxed; p53floxed/floxed(NPp53) mice that develop CRPC upon castration and undergo neuroendocrine differ-entiation with anti-AR treatment to isolate Enzalutamide resistance prostate cancercells.Results: Recently, we showed that activation of the chromatin remodeler Nsd2 isrequired for PCa metastasis and is strongly associated to tumor progression, and thatits silencing markedly reduced the metastatic burden and increased survival. Our cur-rent data indicates that Nsd2 silencing sensitizes PCa cells to anti-AR treatment. Nsd2KO using CRISPR/Cas9 resulted in a markedly enhanced efficacy of Enzalutamide anda significant reduction of AR transcriptional activity with either Enzalutamide orAbiraterone using two different reporter assays. Next, To identify Nsd2-dependent ARco-regulators we immunoprecipitated chromatin-bound endogenous AR in NPp53cells and NPp53-Nsd2KO and performed nano-LC-MS/MS mass spectrometry on thepurified peptide mix. In particular, data indicates that AR association with SWI/SNFmembers is stronger in the absence of Nsd2, suggesting that Nsd2 overexpression mightimpair AR interaction to the SWI/SNF complex. We next tested whether Nsd2 in factbinds SWI/SNF subunits by co-immunoprecipitation in the NPp53 cells and whetherBAF155, BAF170 and BRG1 association to AR increases in the absence of Nsd2. As sus-pected, there is a remarkable increase in the binding of AR to SWI/SNF subunitsBAF155, BAF170 and BRG1 in the absence of Nsd2.Conclusions: Together, our data suggests that the mechanisms by which Nsd2 overex-pression drives aggressive castration resistant and androgen independent PCa may bein part through destabilizing the AR-SWI/SNF interaction and altering the specificityof the AR cistrome. Ongoing work will further elucidate this by analyzing chromatinaccessibility data coupled with transcriptomics and ChIPseq data for the AR and theBAF170, BAF155 and Brg1 SWI/SNF subunits.Legal entity responsible for the study: Alvaro Aytes.Funding: EAU.Disclosure: All authors have declared no conflicts of interest.2014P IGF1R inhibition affects the survival of HT29 cancer cells byalterations of the TLR9- and autophagy signalingG. M}uzes1, A. Sebestyén2, A. Simon1, L. Nagy1, B. Barta1, T. Danko2, A.L. Kiss3, F.J. Sipos11Division of Immunology, Semmelweis University, Second Department of InternalMedicine, Budapest, Hungary, 2Biology, First Department of Pathology andExperimental Cancer Research, Budapest, Hungary, 3Department of HumanMorphology and Developmental Biology, Semmelweis University, Budapest, HungaryBackground: In HT29 cells, an interplay between self-DNA-induced TLR9-signalingand autophagy response was found with significant effects on cell survival and kineticparameters. The IGF/IGF1R system plays a determinant role in the pathogenesis andprogression of colorectal cancer. This pathway is upstream of mTORC1, a negative reg-ulator of autophagy. I mammalian systems chronic IGF1R inhibition was shown toattenuate autophagosome formation. The interrelated action of IGF1R inhibition andTLR9/autophagy signaling in cancer cells has not yet been clarified. The present studywas designed to assess this complex network using HT29 colon carcinoma cells.Methods: HT29 cells were incubated for 72 h with genomic (g), hypermethylated (m),and fragmented (f) tumor self-DNAs, and with/without inhibitors of IGF1R (picropo-dophyllin), autophagy (chloroquine) and TLR9 (ODN2088), respectively. Cell viabilitywas measured by MTT assay. Transcriptional changes of TLR9-signaling, IGF1R,mTORC, Akt, and the autophagy process were assayed by Human v3 miRNA Assay(NanoString). Autophagy proteins were detected by immunocytochemistry, whilemorphology of apoptosis and autophagy by transmission electron microscopy (TEM).Results: In case of autophagy g- and f-DNAs caused significant upregulation ofBeclin1, Atg16L1, LC3 mRNAs, and downregulation of mTORC, and Akt, verified byimmunocytochemistry, as well. IGF1R-inhibition alone altered inversely theabstracts Annals of Oncologyv806 | Tumour Biology and Pathology Volume 30 | Supplement 5 | October 2019

Heat shock protein 90 chaperones and protein kinase D3 regulates androgen-independent prostate cancer development

http://repo.lib.semmelweis.hu//bitstream/123456789/8193/1/2012P%20-%20Heat%20shock%20protein%2090%20chaperones%20and%20protein%20kinase%20D3%20regulates%20androgen-independent%20prostate%20cancer%20development.pdf

Heat shock protein 90 chaperones and protein kinase D3 regulates androgen-independent prostate cancer development

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