[CANCER RESEARCH 60, 7099 –7105, December 15, 2000] Heat Shock Protein Expression Independently Predicts Clinical Outcome in Prostate Cancer 1 Philip A. Cornford, 2 Andrew R. Dodson, Keith F. Parsons, Anthony D. Desmond, Alan Woolfenden, Mark Fordham, John P. Neoptolemos, Youqiang Ke, and Christopher S. Foster 3 Departments of Surgery [P. A. C., J. P. N.] and Pathology [A. R. D., Y. K., C. S. F.], The University of Liverpool, and Department of Urology, The Royal Liverpool University Hospital [P. A. C., K. F. P., A. D. D., A. W., M. F.], Liverpool, L69 3GA, United Kingdom ABSTRACT Heat shock proteins (hsps) occupy a central role in the regulation of intracellular homeostasis, and differential expression of individual hsps occurs in a broad range of neoplastic processes. This study was performed to test the hypothesis that the particular patterns by which individual hsps become specifically modulated in human prostate cancers are correlated with behavioral phenotype and hence may be of value in determining the most appropriate clinical management of individual patients. Monoclonal antibodies specific for each hsp protein were used to assess expression of hsp27, hsp60, and hsp70 in formalin-fixed, paraffin wax-embedded, ar- chival tissue specimens of early prostatic adenocarcinomas (pT 1–2 N o M o ) removed at radical prostatectomy (n  25) and in advanced cancers (n  95) identified at transurethral resection of prostate (TURP). These findings were compared with similar data from control prostates (n  10) removed at primary cystectomy for urinary bladder neoplasia not involv- ing the prostate and also at TURP for benign prostatic hyperplasia (n  50). Western blotting of whole cell lysates derived from established human prostatic epithelial cell lines PNT2, LNCaP, DU145, and PC3 was compared with expression of hsps by the primary human tissues. This study found that early in situ neoplastic transformation of normal pros- tatic epithelium was consistently associated with loss of hsp27 expression and that the level of hsp27 expression by individual prostate cancers was correlated with their Gleason grade. In advanced cancers, hsp27 expres- sion was invariably associated with poor clinical outcome (P  0.0001). Data from cell lines supported the primary tissue findings, with elevated hsp27 expression only in aggressive malignant cell lines and androgen- insensitive cell lines. Expression of hsp60 was significantly increased in both early and advanced prostate cancer when compared with nonneo- plastic prostatic epithelium (P < 0.0001), as well as in malignant prostate cancer cell lines. Expression of hsp70 was unaltered in early prostate cancers when compared with nonneoplastic prostatic epithelium but showed a diminished expression in morphologically advanced cancers (P  0.0029). No consistent correlation was found between levels of hsp60 or hsp70 expression and phenotypic behavior of individual primary pros- tatic cancers. Thus, patterns of hsp expression have been confirmed to be specifically and consistently modulated in both early and advanced human prostate cancers. Whereas absence of hsp27 is a reliable objective marker of early prostatic neoplasia, reexpression of this protein by an individual invasive prostatic carcinoma invariably heralds poor clinical prognosis. Because this protein has been shown to alter the balance between prolif- eration and apoptosis, understanding the mechanism(s) by which individ- ual hsps regulate intracellular homeostasis may assist in explaining some key processes that occur during evolution of human prostate cancers. We suggest that hsp27 expression provides novel diagnostic and prognostic information on individual patient survival which, if obtained at the time of primary diagnosis, would assist in determining tumor-specific manage- ment strategies. Development of techniques to therapeutically modulate hsp27 expression raises the possibility of novel targeted approaches to regulate this homeostatic mechanism, thus allowing better control over tumor cell proliferation and hence patient survival. INTRODUCTION hsps 4 are highly conserved throughout evolution. They mediate and modulate a diverse range of intracellular activities which, according to their relative levels, fulfill protective functions (1, 2), ensure meta- bolic homeostasis (3, 4), and participate in a diverse range of patho- genic processes (5, 6). Some are normal cellular proteins expressed under nonstressed conditions and in a cell cycle-dependent manner (7, 8), whereas others are chaperonins of nascent proteins (9). In neopla- sia, hsps have been implicated in multidrug resistance (10), in regu- lation of apoptosis (11), and as modulators of p53 function (12). hsp27 is constitutively expressed at low levels in the cytosol of most human cells (13). After induction, the protein becomes phos- phorylated while simultaneously translocated from the cytoplasm to within or around the nucleus (14 –16). Phosphorylation is a key regulator of hsp27 function occurring at serine residues 78 and 82 (17) through interaction with a specific kinase, but it may be activated by several different signal transduction mechanisms. The presence of the protein contributes to cell survival after diverse stress insults (14, 16, 18). In murine L929 (19) and in human HT-29 and CaCo2 colorectal cancer cell lines (20), hsp27 inhibits apoptosis induced by a variety of different stimuli. It has been proposed that hsp27 modulates reactive oxygen species via a glutathione-dependent pathway (21), thereby protecting intracellular proteins and explaining, in part, the protective effect of hsp27 against chemotherapeutic agents (22, 23). hsp60 is abundant in most mammalian cells under normal condi- tions (24), where its major functions are protein chaperoning and protein folding (25). Both processes are coregulated by hsp10 (26). Whereas aberrant expression of hsp60 has been associated with au- toimmune disease, hsp60 has a role together with hsp70 in antigen presentation in malignant diseases (27), with enhanced hsp60 expres- sion reported in breast carcinoma (28) and myeloid leukemia (29). hsp70 regulates a wide range of protein-associated activities (13, 30 –33). Expression of hsp70 is enhanced after transformation by oncogenes (34, 35). Elevated levels of hsp70 protect cells from apoptotic death induced by TNF- and TNF- (36). hsp70 interacts with p53 to stabilize mutant but not the wild-type protein (37). Conversely, wild-type (but not mutant) p53 down-regulates hsp70 expression (38). Our recent studies demonstrated that expression of other homeo- static regulator proteins, including PKC isoenzymes (39), Ca 2+ -bind- ing proteins (40), and ion channels (41, 42), are differentially modu- lated in prostate cancers. When enhanced, these proteins promote the metastatic phenotype, and their expression predicts poor clinical out- come (39). Because some members of the PKC and hsp families are Received 9/17/99; accepted 10/16/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by grants from the Trust Research Fund, Research and Develop- ment Office, Royal Liverpool and Broadgreen Hospitals NHS Trust, The Prostate Cancer Charity, and the Merseyside Prostate Cancer Trust (to C. S. F.). 2 P. A. C. is the current holder of the Insole Award and Tomkinson Award, British Medical Association, United Kingdom. 3 To whom requests for reprints should be addressed, at Department of Cellular and Molecular Pathology, University of Liverpool, Duncan Building, Daulby Street, Liver- pool, L69 3GA, United Kingdom. 4 The abbreviations used are: hsp, heat shock protein; PIN, prostatic intraepithelial neoplasia; PSA, prostate-specific antigen; BPH, benign prostatic hyperplasia; TURP, transurethral resection of prostate; TNF, tumor necrosis factor; PKC, protein kinase C; BSCO, bilateral subcapsular orchidectomy; TBS, Tris-buffered saline. 7099 Research. on March 18, 2016. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from