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 prostate cancer (PC)
M. Puchinskaya1, D. Salavei2
1Out-patient Dept., Minsk City Clinical Oncologic Dispensary, Minsk, Belarus,2General Ecology, Biology, and Environmental Genetics, International Sakharov Environmental Institute of Belarusian State University, Minsk, Belarus
Background:Vimentin (Vim) is one of the mesenchymal markers, that are often upre- gulated in cancer cells during epithelial-mesenchymal transition (EMT) – a process endowing tumor cells with invasiveness, pro-metastatic potential and, arguably, cancer stem cells (CSC) properties. It is normally seen in cytoplasm, but cell surface Vim was also 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 our method of semiquantitative staining assessment.
Methods:Vim expression was assessed in 48 cases of PCa using immunohistochemistry with primary rabbit polyclonal antibodies (ThermoFischer, 1:1000). UnoView Rabbit Detection Kit was used for visualization. Specificity of staining was confirmed by another antibody (mouse monoclonal, DAKO, 1:200) in selected cases. To assess Vim expression semiquantitatively we proposed a weighed staining index (WSI) that takes into account the proportion of stained cells as well as the ratios of staining intensities and can be applied to different cellular compartments staining separately. It combines advantages of simple ad oculus semiquantitative staining evaluation and obtaining numerical characteristics of marker expression.
Results:Median WSI for membranous staining was 5 (0 – 52) (7, if cases without expression were excluded), for cytoplasmic – 20.5 (0 – 64) (22.5, if cases without expression were excluded). In 2 (4.2%) cases marker expression was absent in both membrane 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 that for cytoplasmic. Significant correlation was found between WSI for cytoplasmic and membranous staining (Spearman test, r¼0.51, p<0,05). However, no correlation was found between WSI for both compartments and E-cadherin expression (as assessed by average staining intensity score – a method similar to WSI – using Aperio software previously).
Conclusions:Vim expression as a sign of EMT in the tumor is present in 95.8% cases of PCa and is more prevalent in cytoplasm. WSI is a useful tool to assess Vim (and possibly other 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 regulates androgen-independent prostate cancer development
A. Varga1, B. Batai2, P. Gyulavari1, M.T. Nguyen3, C. S}oti3, T. Vantus3
1MTA SE Pathobiochemistry Research Group, Semmelweis University, Budapest, Hungary,2MTA-SE Momentum Molecular Oncohematology Research Group, First Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary,3Department of Medical Chemistry, Molecular Biology and Pathobiochemistry, Semmelweis University, Budapest, Hungary
Background:Prostate cancer is the second leading cause of cancer related deaths in men worldwide. Heat Shock Protein 90 (Hsp90) is expressed in tumour cells at high levels – 3-5% of total proteins – and regulates the function of oncogenes and other tumour 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 to explore the impact of Hsp90 and PKD3, respectively, on prostate cancer growth and their potential interaction.
Methods:We employed the DU145 and PC3 well-characterized androgen-independent prostate cancer cell lines. Cell viability was determined by Trypan Blue exclusion cell counting. Apoptosis analysis was performed by flow cytometry after AnnexinV and propidium-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 and PC3 cell lines. The pan-PKD inhibitor CRT0066101 also decreased cell viability of the prostate cancer cells in a dose-dependent manner. Further, we demonstrated that gane- tespib reduced PKD3 protein level in a concentration-dependent manner and induced its 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 under
way to characterize the biological significance of our findings. Our results contribute to better understand the pathological signalling of androgen-independent prostate cancer cells 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 NSD2 dependent
K. Ruggero1, A. Martinez1, X. Chen1, R. El Bizri1, S. Farran1, L. Palomero1, J.M. Piulats2, A. Villanueva1, M.A. Pujana1, A. Aytes1
1LRT1, 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, Spain
Background: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 pharmacological blocking 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 cancer cells.
Results:Recently, we showed that activation of the chromatin remodeler Nsd2 is required for PCa metastasis and is strongly associated to tumor progression, and that its silencing markedly reduced the metastatic burden and increased survival. Our cur- rent data indicates that Nsd2 silencing sensitizes PCa cells to anti-AR treatment. Nsd2 KO using CRISPR/Cas9 resulted in a markedly enhanced efficacy of Enzalutamide and a significant reduction of AR transcriptional activity with either Enzalutamide or Abiraterone using two different reporter assays. Next, To identify Nsd2-dependent AR co-regulators we immunoprecipitated chromatin-bound endogenous AR in NPp53 cells and NPp53-Nsd2KO and performed nano-LC-MS/MS mass spectrometry on the purified peptide mix. In particular, data indicates that AR association with SWI/SNF members is stronger in the absence of Nsd2, suggesting that Nsd2 overexpression might impair AR interaction to the SWI/SNF complex. We next tested whether Nsd2 in fact binds SWI/SNF subunits by co-immunoprecipitation in the NPp53 cells and whether BAF155, 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 subunits BAF155, 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 be in part through destabilizing the AR-SWI/SNF interaction and altering the specificity of the AR cistrome. Ongoing work will further elucidate this by analyzing chromatin accessibility data coupled with transcriptomics and ChIPseq data for the AR and the BAF170, 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 by alterations of the TLR9- and autophagy signaling
G. M}uzes1, A. Sebestye´n2,A. Simon1, L. Nagy1, B. Barta1, T. Danko2, A.L. Kiss3, F.J. Sipos1
1Division of Immunology, Semmelweis University, Second Department of Internal Medicine, Budapest, Hungary,2Biology, First Department of Pathology and Experimental Cancer Research, Budapest, Hungary,3Department of Human Morphology and Developmental Biology, Semmelweis University, Budapest, Hungary
Background:In HT29 cells, an interplay between self-DNA-induced TLR9-signaling and autophagy response was found with significant effects on cell survival and kinetic parameters. The IGF/IGF1R system plays a determinant role in the pathogenesis and progression of colorectal cancer. This pathway is upstream of mTORC1, a negative reg- ulator of autophagy. I mammalian systems chronic IGF1R inhibition was shown to attenuate autophagosome formation. The interrelated action of IGF1R inhibition and TLR9/autophagy signaling in cancer cells has not yet been clarified. The present study was 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 viability was 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, while morphology of apoptosis and autophagy by transmission electron microscopy (TEM).
Results:In case of autophagy g- and f-DNAs caused significant upregulation of Beclin1, Atg16L1, LC3 mRNAs, and downregulation of mTORC, and Akt, verified by immunocytochemistry, as well. IGF1R-inhibition alone altered inversely the
abstracts
Annals of Oncologyv806 | Tumour Biology and Pathology Volume 30 | Supplement 5 | October 2019
