2008 Summer Meeting - Leeds - The Pathological Society of Great ...

PL1Chemical carcinogenesis in K-ras exon 4A knockout miceshows that mutationally activated K-ras 4A and 4B bothmediate lung tumour formationMJ Arends 1 , CE Patek 2 , WA Wallace 3 ,FLuo 1 ,SHagan 4 ,DG Brownstein 5 ,SJ Plowman 2 , RL Berry 2 ,WKolch 4 ,OJ Sansom 4 , DJ Harrison 2 , ML Hooper 2 .1 Pathology Departmentt, University of Cambridge, Cambridge, 2 SirAlastair Currie CRUK Lab, Molecular Medicine Centre, University ofEdinburgh, Edinburgh, 3 Division of Pathology, University of Edinburgh,New Royal Infirmary of Edinburgh, Edinburgh, 4 Beatson Institute forCancer Research, Garscube Estate, Switchback Road, Glasgow5 Research Animal Pathology Core Lab, Queens Medical ResearchInstitute, University of Edinburgh, EdinburghMice with K-ras exon 4A deleted have a normal lifespan with no increase insporadic tumour susceptibility. Lung carcinogenesis was induced by N-methyl-N-nitrosourea (MNU) in mice with combinations of K-ras exon 4A-knockoutand K-ras whole gene-knockout, to investigate the roles of K-ras4A and K-ras4B isoforms in lung tumourigenesis. MNU induced K-ras G12D mutationsthat jointly affect both isoforms. Compared with K-ras-/4A- mice (wheretumours can express mutationally activated K-ras4B only), tumour number andsize were significantly higher in K-ras+/- mice (where tumours can expressmutationally activated K-ras4A and K-ras4B), and significantly lower in K-ras4A-/4A- mice (where tumours can express both wild-type and activated K-ras4B). MNU induced significantly more and larger tumours in wild-type thanK-ras4A-/4A- mice which differ in that only tumours in wild-type mice canexpress wild-type and activated K-ras4A. Lung tumours from K-ras+/- and K-ras-/4A- mice exhibited phospho-Erk1/2 and phospho-Akt immunostaining. Weconclude that: (1) mutationally activated K-ras4B is sufficient to activate theRaf/MEK/ERK (MAPK) and PI3-K/Akt pathways and initiate lungtumorigenesis; (2) when expressed with activated K-ras4B, mutationallyactivated K-ras4A further enhances lung tumour formation and growth (both inthe presence and absence of its wild-type isoform); and (3) wild-type K-ras4Bshows tumour suppressor activity by reducing lung tumour number and size.PL3Expression Pattern of DNA Double Strand Break RepairProteins and Response to Therapy in Gastric CancerM Hollings 1 , C Beaumont 1 ,TIvanova 2 , L Ling Cheng 2 ,K Ganesan 2 ,YZhu 2 ,JLee 2 ,PTan 2 , H Grabsch 1 .1 Pathology and Tumour Biology, Leeds Institute of Molecular Medicine,University of Leeds, UK, 2 Cellular and Molecular Research, NationalCancer Centre, SingaporeDNA double strand breaks (DSB) are the most dangerous form of DNAdamage. pH2AX has a crucial role in the immediate cellular response to DSBs.We hypothesised that the expression pattern of pH2AX and downstream DNADSB repair proteins is related to response to therapy in gastric cancer (GC) celllines.26 GC cell lines were either processed into paraffin for tissue microarray(TMA) construction or challenged with oxaliplatin (OX), cisplatin (CIS) or 5-fluorouracil (5FU). GI50 (50% growth inhibition) was used to discriminatebetween sensitive and resistant cell lines. Immunocytochemistry (ICC) withantibodies against pH2AX, H2AX, p53, RAD51, MRE11, BRCA1, ATM,Ku70, Ku80 and DNA-PKc was performed on TMA sections and percentage ofpositive cells was scored.Differential expression was seen for pH2AX, H2AX, p53, Ku70 andRAD51. Cell lines with high pH2AX were more likely resistant to OX and CISwhereas 5FU resistant cell lines were more likely to have low pH2AX and lowp53. Hierarchical clustering of all ICC scores revealed a cluster of cell lineswhich are both, sensitive to OX and resistant to 5FU.This is the first study investigating the association between the expressionpattern of proteins of the DNA DSB repair pathway and response to therapy ina large set of GC cell lines. Further studies are justified to characterise theexpression pattern of all proteins involved in DNA DSB repair, to identify themost important molecular player by functional studies and to validate thefindings in clinical trial material in parallel.PL2Identification of PPM1D as a Novel Therapeutic Target inOvarian Clear Cell AdenocarcinomasDSP Tan 1,2 , MBK Lambros 1 ,SRayter 1 , C Marchio 1 , C Jameson 3 ,A Williams 4 , WG McCluggage 5 ,MEl-Bahrawy 6 ,AJW Paige 6 ,SB Kaye 2 , A Ashworth 1 , JS Reis-Filho 1 .1 The Breakthrough Breast Cancer Research Centre, Institute of CancerResearch, London, 2 Section of Medicine, Institute of Cancer Research,Royal Marsden Hospital, Sutton, Surrey, 3 Department of Pathology RoyalMarsden Hospital, London UK, 4 Department of Pathology, University ofEdinburgh, Edinburgh, 5 Department of Pathology, Royal Group ofHospitals Trust, Belfast, 6 Imperial College School of Medicine,Hammersmith Hospital, LondonThe genetic and transcriptomic profiles of 12 ovarian clear cell adenocarcinoma(OCCA) cell lines were analysed using a 32K tiling path microarray CGHplatform and the Illumina human ref6 gene expression array respectively. Ouraims were to characterise the molecular genetic profiles of OCCAs and identifypotential therapeutic targets from a list of candidate amplicon drivers. Recurrentgains/amplifications containing a number of putative oncogenes were identifiedincluding a focal amplification of 17q23.2, whose smallest region of overlapharbours PPM1D, APPBP2 and CA4, in one cell line (SMOV2). Mann WhitneyU analysis between the levels of gene expression for PPM1D, APPBP2 andCA4 for OCCA cell lines with and without gains/amplifications in this regionrevealed a trend for higher levels of PPM1D expression in cell lines withgains/amplifications of 17q23.2. This was confirmed at the protein level bywestern blot analysis. Colony formation assays using a recently developedchemical inhibitor of PPM1D (CT007093) and a short hairpin RNA (shRNA)against PPM1D demonstrated that PPM1D signalling is selectively essential forthe survival of SMOV2 cells. Chromogenic in situ hybridisation with an inhousegenerated probe for PPM1D revealed high level gain/amplification of thislocus in 6 out of 59 (10.2%) primary OCCAs. Our study suggests that PPM1Dis a potential novel therapeutic target for a subgroup of OCCAs and provides amodel for the integration of high throughput genetic, genomic and shRNA datafor the identification of novel therapeutic targets for these chemotherapyresistant tumours.PL4Inhibition of PKC-zeta Expression in Human Prostate CancerCellsSYao 1 ,YKe 1 , C Gosden 1 , CS Foster 1 .1 University of LiverpoolProtein Kinase C-zeta (PKC-) independently predicts poor clinical outcome inprostate cancer. Several variants of PKC- are potentially encoded by thePKC- gene, although their functions remain unclear. After showing thatantisense inhibition of PKC- gene expression diminished the motility ofprostate cancer cells, we hypothesised that PKC- might be responsible formodulating prostate cancer cell apoptosis, invasion and metastasis. Using RNAidirected to three different exonic sites in variant “b” (wild-type) of the PKC-genome, we have generated both transient and stable transfectants of theprostate cancer cell-line PC3-M. These genetic knockdowns expressed reducedlevels of PKC- at, or below, that of benign PNT-2 prostate cells, as confirmedby qPCR. Cell invasion assays in-vitro confirmed altered morphology of thecells with maintained levels of proliferation but almost complete arrest ofinvasive capacity into collagen. However, apoptosis was not significantlyaffected. We now believe we have strong evidence that expression of PKC-variant “b” in human prostate cancer cells is differentially involved inpromoting cell invasion and hence metastasis. Conversely, expression of thisgene does not affect either cell proliferation or apoptosis. Analysis ofdownstream genes modulated following PKC- knockdown may identify newtherapeutic targets to inhibit prostate cancer cell invasion and metastasis.28 Summer Meeting (194 th ) 1–4 July 2008 Scientific Programme