1. Investigation Of Cell-Adhesion Molecules In Prostate Cancer Progression Abstract. Lakshana Sreenivasan 4,5, Edward Abril 4,5, Kathleen McDaniel 4,5, Raymond B. Nagle, M.D., Ph.D. 2,4,5 and Anne E. Cress, Ph.D. 1,3,5 Departments of 1 Radiation Oncology, 2 Pathology, 3 Cellular and Molecular Medicine, 4 TACMASS and 5 The University of Arizona Cancer Center. Tucson, AZ The current study was designed to determine the distribution of integrin β4, and Plectin in normal, PIN and invasive PIN lesions using an automated staining platform by Ventana Medical Systems. Immunohistochemical techniques are performed on paraffin fixed human prostate tissues to map the expression of α6β4 and Plectin. Our results for α6β4 expression on formalin fixed and paraffin embedded human prostate tissue confirm previous findings that α6β4 expression is observed in normal and PIN prostate tissue but completely lost in invasive prostate carcinoma. Of new interest, is the observed expression of Plectin within the lateral sides of both basal and luminal cells of normal prostate and invasive prostate cancer. The expression of Plectin within the invasive prostate cancer without integrin α6β4, suggests that a switch to an actin based invasion occurred involving the α6β1 integrin. Future studies will be to determine whether the forced suppression of α6β4 in normal prostate cells will cause redistribution of Plectin to α6β1 integrin to promote the invasion phenotype. ( Supported in part by NIH CA159406 and CA23074 ). Methods Conclusions 5% IMDM Future Directions 5% IMDM Results Citations Figure 3 : Plectin is able to bind cytokeratin without competing with the other molecules. In the absence of β4, Plectin binds to actin. ( PMID: 17457303 ) Figure 1 : Pathway for Human Prostate Cancer Progression. Stages of prostate cancer progression correspond primarily to the loss or alteration of the basal cells and basal lamina. ( PMID: 11018010 ) Figure 2 : Tumors arise from PIN lesions where the basal cells are lost and basal lamina is altered. Prostate cancer progression occurs through collective cell migration via a process of tubulogenesis. ( PMID: 3177701) Figure 4 : Figure A and B are stained with anti- β4. Figure A shows staining of normal glands and basal cells. Note: Staining adjacent to basal cells and intracellular staining of luminal cells is observed in normal prostate glands. Figure B contrasts normal gland staining on the left and cancer glands on the right. Note: There is absence of β4 staining in the cancer glands. Figure C and D are stained with anti-plectin antibody. Figure C shows plectin staining in the basal cells and intracellular staining in the luminal cells in normal prostate glands and apical staining in the prostate cancer glands. Figure D is a higher magnification showing apical staining of the luminal cells. Expression of β4 is present in normal prostate glands but it is completely lost in invasive carcinoma. β4 is observed in basal cell-hemidesmosome complex as well as the lateral sides of the luminal cells. Expression of β4 in the lateral membranes is a novel finding and to date it has not been associated with a ligand. Expression of plectin is observed in normal prostate glands and invasive carcinoma. Distribution of Plectin in cancer glands is apical, which is different from normal prostate glands where it is on the lateral and basement membrane. Introduction Prostate cancer progression to invasive and metastatic disease occurs through a budding process similar to embryonic tubulogenesis. Acquiring the invasive and metastatic phenotype is the lethal form of the disease. Primary prostate carcinoma arises from prostate intraepithelial neoplasia (PIN) lesions where the basal cells are altered or lost. Previous studies performed on frozen human prostate tissues using immunohistochemical techniques demonstrated that α6β4 integrin and its ligand, Laminin 332 were completely lost in invasive human prostate carcinomas, resulting in continued expression of the α6β1 integrin. The α6β4 integrin and plectin are essential for the assembly and stability of the normal hemidesmosome (HD) complex. HDs are dominant adhesion complexes that adhere the stratified epithelium to the basal lamina providing glandular stability, a process disrupted in cancer. Plectin is a cytoplasmic linker type protein that connects the α6β4 subunit and the intermediate filaments (cytokeratin 5, 14) within the basal cells of the epithelium. Alternatively, plectin can link the cytoplasmic microfilaments (actin) to the α6β1 integrin. In experimental model systems, the plectin interaction with the α6β4 integrin acts as a tumor suppressor. Normal prostate glands, PIN lesions and invasive cancer was selected from H&E stained sections obtained from radical prostatectomy specimens. From FFPE selected blocks 3-4um sections were obtained and H&E staining was performed. Sections were stained with anti- β4 and anti- plectin. The Anti-Integrin beta 4 antibody [EPR8558(2)] from Abcam is a rabbit monoclonal antibody. Anti-plectin [HPA025967] from Sigma Aldrich is a rabbit polyclonal antibody. Immunohistochemistry is performed using an automated staining platform from Ventana Medical Systems. After deparraffinization and heat-induced epitope retrieval using standard Cell Conditioning 1, prostate tissues are incubated for 30 minutes. The samples are incubated with 1:75 dilution of primary antibodies for 60 minutes. ChromoMap DAB detection kit from Vetana Medical Systems is used to stain the slides and visualize the signal. The samples are counterstained with Hematoxylin and Bluing agent for 4 minutes each. The stained prostate tissues are dehydrated with graded alcohol and xylene and cover slipped. Future studies will determine the post transcriptional regulation of ITGβ4 expression during the progression to invasive carcinoma. We will determine if the presence of ITGβ4 isoforms explain the cell-cell distribution within luminal cells of normal glands. Improved imaging methods will be used to observe the switch from a cytokeratin based to actin based cytoskeleton in aggressive disease. 5% IMDM + 5ug/ml J8H 5% IMDM + 5ug/mL J8H A B C D Normal Cancer Normal Cancer Abate-Shen, C. "Molecular Genetics of Prostate Cancer." Genes & Development 14.19 (2000): 2410-434. Marinkovich, M. Peter. "Tumour Microenvironment: Laminin 332 in Squamous-cell Carcinoma." Nature Reviews Cancer 7.5 (2007): 370-80. Nagle, Raymond B., and Anne E. Cress. "Metastasis Update: Human Prostate Carcinoma Invasion via Tubulogenesis." Prostate Cancer 2011 (2011): 1-10.