Loss of stearoyl-CoA desaturase expression is a frequent event in prostate carcinoma

Int. J. Cancer: 114, 563–571 (2005) © 2004 Wiley-Liss, Inc.

Loss of stearoyl-CoA desaturase expression is a frequent event in prostate carcinoma Stacy Moore1, Beatrice Knudsen2, Lawrence D. True3, Sarah Hawley2, Ruth Etzioni2, Christian Wade1, David Gifford1, Ilsa Coleman1 and Peter S. Nelson1* 1 Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA 2 Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, WA, USA 3 Department of Pathology, University of Washington, Seattle, WA, USA Prostate carcinogenesis is influenced by genetic alterations resulting in a biochemical condition that favors cell proliferation and survival. Studies of prostate carcinoma using comparative genomic hybridization and cDNA microarray analysis indicate that numerous biochemical processes may be affected during cellular transformation and progression to an invasive phenotype. Among the consistently observed tumor-associated changes are alterations in fatty acid metabolism that influence diverse cellular activities such as signaling, energy utilization, and membrane fluidity. Increases in fatty acid synthase (FAS) levels have been shown to be one of the earliest and most frequent molecular alterations in prostate carcinogenesis. We sought to identify tumor-associated changes in the expression of genes with functional roles associated with lipid metabolism. Defined populations of normal and neoplastic prostate epithelium were acquired by laser capture microdissection and transcript levels were measured by cDNA microarray hybridization. We determined that stearoylCoA desaturase (SCD) transcripts were downregulated in cancer relative to normal epithelium. These results were confirmed by quantitative PCR. Further analysis by immunohistochemical evaluation of radical prostatectomy samples employed a quantitative scoring system with a range of 0 –300. The median SCD expression levels were 150, 45 and 10 for normal, PIN and carcinoma samples, respectively. Statistically significant differential SCD expression between normal and cancerous epithelium was determined at the p ⴝ 0.001 level, and between PIN and prostate carcinoma at the p ⴝ 0.03 level. Of these cases, 92% overexpressed fatty acid synthase (FAS) in cancerous cells and 84.7% exhibited the signature of FAS overexpression and SCD loss in prostate carcinoma as compared to normal prostate epithelium. These results indicate that loss of SCD expression is a frequent event in prostate adenocarcinoma, and further supports a role for altered lipid metabolism as a factor in the process of carcinogenesis. © 2004 Wiley-Liss, Inc. Key words: prostate cancer; microarray; microdissection; lipid metabolism; stearoyl-CoA desaturase

The development of prostate carcinoma is driven by specific genetic alterations resulting in a biochemical state that favors cell proliferation and survival. The diversity and complexity of tumor cell aberrations is exemplified by the numerous chromosomal rearrangements and gene expression changes identified using comprehensive analytical technologies such as comparative genomic hybridization and microarray analysis.1–3 The extent of these genetic alterations suggests that numerous biochemical processes may be influenced during cellular transformation and the subsequent progression to an invasive phenotype. The identification of consistent neoplasia-associated alterations involving specific gene products or molecular pathways may provide important insights into essential events associated with carcinogenesis. Fatty acid metabolism represents a key process that influences several diverse cellular pathways and characteristics including cell signaling, energy processing and membrane fluidity.4 – 6 The enzymatic and transporter activities that modulate concentrations of lipid substrate molecules represent important regulatory elements that modulate cellular proliferation and apoptosis. Several proteins involved in the metabolism of fatty acids have been determined to be altered in neoplastic prostate cells relative to their normal counterparts. For example, increases in fatty acid synthase (FAS) levels have been shown to be one of the earliest and most frequent molecular alterations in prostate carcinogenesis.3,7 FAS plays a Publication of the International Union Against Cancer

pivotal role in the de novo biosynthesis of fatty acids.8 FAS expression in primary prostate carcinomas is predictive of cancer progression,9 and the inhibition of FAS activity induces cell death in FAS-overexpressing cancer cell lines and prolongs survival of host animals implanted with tumor xenografts.10 –13 Increased levels of transcripts encoding the ␣-methylacyl-CoA-racemase (AMACR) enzyme have also been shown to occur frequently in prostate carcinoma.14 –17 AMACR regulates the peroxisomal ␤-oxidation of dietary branched chain fatty acids, a biochemical process that is plausibly related to prostate carcinogenesis through the production of pro-carcinogenic oxidative intermediates.17 In vitro studies have shown that branched fatty acids in dairy and beef products, food sources with epidemiological associations with prostate cancer risk, markedly enhance AMACR expression in prostate cancer cells.18 Immunohistochemical studies of AMACR protein expression have demonstrated a relationship between AMACR protein expression and cancer grade.19 Due to the importance of lipid metabolism in prostate carcinogenesis, we sought to identify alterations in the expression levels of additional genes with functional roles associated with lipid metabolism that had consistent alterations in mRNA levels between primary prostate carcinomas and corresponding non-neoplastic secretory cells. Defined populations of normal and neoplastic prostate epithelium were acquired through laser capture microdissection and transcript levels were assessed by cDNA microarray analysis after a linear amplification procedure. In addition to increased expression levels of AMACR and decreased levels of GSTP1 in cancerous epithelium, we determined that transcripts encoding the stearoyl-CoA desaturase (SCD) gene were downregulated. SCD encodes a key rate-limiting enzyme involved in the synthesis of monounsaturated fatty acids.20 Quantitative PCR measured a reduction of SCD transcripts in 10 of 12 microdissected carcinoma samples relative to benign epithelium. Immunohistochemical evaluation of 69 radical prostatectomy cases confirmed a significant reduction or complete lack of SCD protein expression in prostate carcinoma relative to benign epithelium and an overexpression of FAS in the majority of cancers. Our results indicate that loss of SCD expression is a frequent event in prostate adenocarcinoma.

Abbreviations: aRNA, amplified antisense RNA; AMACR, ␣-methylacyl-CoA racemase; CaP, prostate cancer; FAS, fatty acid synthase; GSTP1, glutathione-S-transferase ␲; H&E, hematoxylin and eosin; LCM, laser capture microdissection; PPAR, peroxisome proliferator activated receptor; SCD, stearoyl-CoA desaturase. Grant sponsor: NIH; Grant number: CA97186, DK65204; Basic Sciences Training; Grant number: T32-DK007779. *Correspondence to: Division of Human Biology, Mailstop D4-100, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue, Seattle, WA 98109. Fax: 206-667-3377. E-mail: [email protected]. Received 23 March 2004; Accepted 4 October 2004 DOI 10.1002/ijc.20773 Published online 17 December 2004 in Wiley InterScience (www. interscience.wiley.com).

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Material and methods Prostate tissue acquisition, microdissection, RNA extraction and aRNA amplification Prostate tissue was obtained from patients undergoing radical prostatectomy in accordance with an IRB-approved collection protocol. Tissue was snap-frozen in a liquid nitrogen/isopentane bath and cryopreserved in embedding medium at ⫺80°C. Pathological grading was assigned using H&E-stained sections. Seven micron thick sections were cut from frozen tissue blocks, applied to uncharged microscope slides, fixed in 95% ethanol/5% diethylpyrocarbonate (DEPC)-treated water, and stored desiccated at ⫺80°C. Before microdissection, sections were rehydrated in RNase-free water and stained with HistoGene Stain Solution (Arturus, Mountain View, CA) for 30 sec. After rinsing in RNase-free water, sections were dehydrated using 4 steps of 10 sec each in 100% ethanol, 100% ethanol, xylene and xylene, followed by air-drying. Microdissection was carried out using a PixCell II laser capture microdissection (LCM) system, following the manufacturer’s protocols (Arcturus). Approximately 2,000 neoplastic and normal prostate epithelial cells were separately collected from each patient sample and placed into 30 ␮l of sample lysis buffer (Arcturus). Total RNA was extracted and DNase-treated from each LCM sample using a spin column isolation method, according to the manufacturer’s protocols (Nanoprep kit, Stratagene, La Jolla, CA). RNA was eluted in 20 ␮l of RNase-free water and vacuumevaporated to a volume of 10 ␮l. T7-based RNA amplification was carried out using a MessageAmp aRNA amplification kit (Ambion, Austin, TX) according to the manufacturer’s suggested protocol. The entire first round of amplified RNA (aRNA) was used as a template for a second round of amplification. Two rounds of amplification yielded 20 –100 ␮g of aRNA. Gene expression analysis by cDNA microarray hybridization A non-redundant set of approximately 6,200 cDNA clones was identified from the Prostate Expression DataBase (PEDB), a public sequence repository of Expressed Sequence Tag (EST) data derived from human prostate cDNA libraries.21 Microarrays were constructed as we have described previously; each cDNA is represented twice per array.22,23 cDNA probes were synthesized from 2 ␮g of aRNA amplified from normal and neoplastic epithelium using Cy3-dCTP or Cy5-dCTP (Amersham Bioscience, Piscataway, NJ) fluorescent dyes. Probes were competitively hybridized to microarrays under a coverslip for 16 hr at 63°C. Fluorescent array images were collected for Cy3 and Cy5 using a GenePix 4000A fluorescent scanner (Axon Instruments, Foster City, CA), and image intensity data were extracted and analyzed using GENEPIX PRO 3.0 microarray analysis software. Each experiment was repeated with a switch in fluorescent labels to account for dye effects. Data analysis was carried out as we have described previously.23 Briefly, data were filtered to remove values from poorly hybridized cDNAs either flagged by the GenePix software, or exhibiting intensity levels ⬍2 SD above the background local to each spot. Intensity ratios for each cDNA hybridized with probes derived from the neoplastic and normal samples were calculated as log2 (neoplastic/normal). Data from the 4 replicate cDNAs for each experiment were combined and the average log2 ratios were used for comparative analyses. cDNAs with average log2 ratios of ⬎1 or ⬍⫺1 (2-fold change) were classified as differentially-expressed between normal and neoplastic epithelium. Quantitative PCR cDNA was generated from 1–5 ␮g aRNA, or 30 ␮g total control RNA using 2 ␮g random hexamers for priming reverse transcription by SuperScript II (400 U per reaction; Invitrogen, Carlsbad, CA). Primers and salts were removed using a Microcon 30 filter (Millipore, Bedford, MA). Quantitative PCR (QPCR) reactions were done in triplicate per experiment, using approximately 5 ng

of cDNA, 0.1– 0.3 ␮M of each primer, and SYBR Green PCR master mix (Applied Biosystems, Foster City, CA) in a 50 ␮L reaction volume. cDNA generation and PCR amplifications were repeated three times. Reactions were carried out and analyzed using an Applied Biosystems 7700 sequence detector. Samples were normalized to the cycle threshold value obtained during the exponential amplification of GAPDH. Expression levels of AMACR, GSTP1 and SCD were calculated. Values are reported as the ratio of gene expression in neoplastic to normal epithelium. Error bars represent the error of the mean from the 3 experiments. Primer sets were tested using serial 10-fold dilutions of template. For the 10-fold dilutions, the difference in threshold cycle number was approximately 3.2, indicating high PCR efficiency. Control reactions with RNA or water as template did not produce significant amplification products. Amplification of a single PCR-product per reaction was monitored by generation of a single dissociation curve. The sequences of primers used in our study were: GAPDH forward, 5⬘-CCTCAACGACCACTTTGTCA-3⬘; GAPDH reverse, 5⬘-TTACTCCTTGGAGGCCATGT-3⬘; AMACR forward, 5⬘-AGTAACTCGGGGCCTGTTTC-3⬘; AMACR reverse, 5⬘-CTGGATGTTGCTGTGTGTTG-3⬘; GSTP1 forward, 5⬘-AGGCAAGACCTTCATTGTGG-3⬘; GSTP1 reverse, 5⬘-TCATGGATCAGCAGCAAGTC3⬘; SCD forward, 5⬘-TTGGAGAAGCGGTGGATAAC-3⬘; SCD reverse, 5⬘-AAAAATCCCACCCAATCACA-3⬘. Northern analysis A human multiple tissue Northern blot was obtained from CLONTECH and hybridized with SCD cDNA probe labeled with [␣-32P]dCTP by random priming using the Rediprime II random primer labeling system (Amersham) according to the manufacturer’s protocol. Filters were imaged and quantitated using a phosphor-capture screen and ImageQuant software (Molecular Dynamics). Tissue microarray immunohistochemistry A tissue microarray (TMA) was constructed with 0.6 mm diameter triplicate cores of tumor and normal tissue from 69 prostatectomy cases containing Gleason sum score of 6, 7 or 8 cancer using an ATA-27 tissue arrayer (Beecher Instruments, Sun Prairie, WI). The tissue was obtained under an IRB approved protocol after removal of patient identifiers. For immunohistochemical analysis, the TMA slide was deparaffinized and rehydrated. For SCD antigen retrieval, the tissue was first treated with 0.0005% proteinase K (Boehringer Mannheim, Indianapolis, IN) in 50 mM Tris buffer pH 8.0 and then placed in a steamer for 20 minutes. FAS antigen was retrieved in the microwave for 15 min in citrate buffer pH 6.0. The SCD polyclonal rabbit antibody (Alpha Diagnostic International Inc., San Antonio, TX ) was diluted 1:50 in PBS/1% BSA and the FAS monoclonal antibody (clone 23, BD Bioscience, Pharmingen, San Diego, CA) was diluted 1:2,000. Both antibodies were incubated with slides at room temperature for 60 min. Antibody staining was developed with the VECTOR ABC kit. Stained slides were imaged using the BLISS system (Bachus Laboratories, Lombard, IL). Cores were visualized with a Web based image display at 20⫻ magnification. Tumor and normal tissue was assessed on each core using an H&E-stained sequential array slide. The tumor was graded and the normal tissue was evaluated for the degree of atrophy. During initial phases of the analysis we noted maximal SCD expression in fully differentiated secretory epithelium. Atrophic epithelium demonstrated decreased or absent SCD immunoreactivity presumably due to the absence of differentiated secretory epithelial cells. Cores containing partially or fully atrophic epithelium were therefore excluded from the final analysis. SCD expression was scored as the percentage of epithelial cells with granular cytoplasmic SCD expression. Basal epithelial cells were considered internal negative controls.

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FIGURE 1 – Analysis of SCD expression in microdissected normal and neoplastic prostate epithelium. (a) cDNA microarray expression values for glutathione-S-transferase ␲ (GSTP1), ␣-methylacyl-CoA racemase (AMACR) and stearoyl-CoA-desaturase (SCD) in 12 matched microdissected pairs of normal and neoplastic prostate epithelium. The scale represents fold-change in cancer epithelium relative to normal epithelium. (b) Quantitative PCR expression values for GSTP1 (hatched bar), AMACR (open bar), and SCD (solid bar) in the same 12 matched pairs of normal and neoplastic prostate epithelium used in the microarray studies.

Interpretation and statistical analysis of immunohistochemistry staining A scoring system incorporating staining intensity parameters and percentage of cell reactivity was used to facilitate the comparison of SCD and FAS expression in normal epithelium, PIN and invasive carcinoma. Each tissue core was used as an independent sample. Cores comprised of both normal epithelium and PIN or cancer were given a separate score for each histology. Four categories of staining were defined: no stain, faint, moderate and intense. The percent of cells falling into each of these categories was estimated by the study pathologist and used to compute a single score as follows: Score ⫽ 0 ⫻ (percent with no stain) ⫹ 1 ⫻ (percent with faint stain) ⫹ 2 ⫻ (percent with moderate stain) ⫹ 3 ⫻ (percent with intense stain). The minimal score was 0 and the maximal score was 300. When 2 tissue types (e.g., PIN, cancer) were present in the same TMA score, a score was calculated for each tissue type.

The box plots summarize the distribution of Score for each group of cores, Normal, PIN, Cancer. The “box” shows the median Score as a dashed line and the 25th percentile and 75th percentile of the Score distribution as the lower and upper parts of the box. The median is the Score at the 50% percentile: half of all cores get a Score higher than the median, and half get a Score lower. The 25th (75th) percentile is the point at which 25% (75%) of the cores score lower (and 75% or 25% score higher). Thus, the area in the “box” represents the middle 50% of the cores. The “whiskers” shown above and below the boxes represent the largest and smallest observed Scores that are ⬍1.5 box lengths from the end of the box. Circles (outliers) represent cores with Scores ⬎1.5 box lengths from the end of the box. We used generalized estimating equations (GEE) to conduct a regression analysis of the association between score (dependent variable) and tissue type (normal epithelium, PIN, cancer). The models fit were of the form: Scope ⫽ Intercept ⫹ Slope*(tissue

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type).24,25 This technique accounts for the correlation that arises among observations from the same patient. Results cDNA array expression profiling of microdissected prostate epithelium identifies decreased SCD expression in prostate carcinoma The cellular architecture and composition of the human prostate gland is complex, comprising several different cell populations present in varying numbers in any particular region. The bulk tissue preparations commonly used in studying epithelial tumors, such as the prostate, often contain large numbers of ‘contaminating’ cells, including fibromuscular stromal components, leukocytes and endothelial cells. To facilitate the comparative analysis of gene expression between normal and neoplastic prostate epithelium, we used laser capture microdissection methods to acquire pure populations of epithelial cells. Approximately 2,000 epithelial cells with normal or neoplastic morphology were captured from each of 12 radical prostatectomy specimens. After total RNA isolation, a linear RNA amplification procedure was employed to generate sufficient RNA for labeling and cDNA microarray hybridization.26 Several genes previously identified to be differentially expressed between normal prostate epithelium and prostate carcinoma were found to be altered in this analysis. These included increased expression of AMACR in carcinoma, and decreased expression of GSTP1 in cancer relative to benign cells (Fig. 1a). Due to the recognized importance of enzymes regulating lipid metabolism, we searched the expression profiles for genes with functional roles known to be involved in lipid biosynthesis or utilization and identified the decreased expression of transcripts encoding stearoyl-coenzyme A desaturase (SCD) in 8 of 12 carcinoma samples with 5 of 12 exhibiting more than a 50% reduction in expression (Fig. 1a). QPCR analysis of genes differentially expressed in neoplastic prostate epithelium To confirm the differential expression of SCD, GSTP1 and AMACR in normal and neoplastic prostate epithelium, we carried out quantitative PCR analysis on total RNA samples obtained by microdissection. The QPCR analysis confirmed the microarray results, though in general the differential expression measurements were greater as measured by QPCR relative to microarray values (Fig. 1b). GSTP1 expression was reduced in cancer epithelium relative to normal epithelium in all 12 samples analyzed. AMACR expression was increased in cancer epithelium relative to normal epithelium in all cases. Transcripts encoding SCD were downregulated in 10 of 12 carcinoma samples relative to normal epithelium confirming the microarray results. SCD transcript levels were generally reduced 3– 4-fold in neoplastic epithelium with a differential expression range between 2-fold and 30-fold (Fig. 1b). Northern analysis of stearoyl-CoA desaturase 1 expression The distribution of SCD transcripts in normal human tissues was determined by Northern analysis. Of 8 adult tissues assessed, the SCD cDNA probe hybridized to transcripts of approximately 4 kb and 5.5 kb in prostate and testis (Fig. 2). Minimal expression was observed in thymus, ovary, small bowel and colon and no expression was detected in spleen and peripheral blood leukocytes. Human SCD has been shown previously to be expressed in 2 isoforms of 3.9 and 5.2 kb due to alternative use of poly-adenylation signals. Immunohistochemical analysis of stearoyl-CoA desaturase expression in prostate carcinoma The levels of SCD were evaluated by immunohistochemistry using a tissue microarray comprised of representative normal and neoplastic prostate samples from 69 radical prostatectomy cases. Each case was represented by 3 normal and 3 neoplastic cores. H&E staining was carried out to facilitate the classification of normal glands, PIN, and invasive carcinoma. Glands with a lumi-

FIGURE 2 – Northern analysis of SCD expression in normal human tissues. SCD expression was evaluated using a commercial blot comprised of 2␮g of poly(A)⫹ RNA from each of 8 normal human tissues. Equivalent loading of each lane was determined using a ␤-actin control probe (data not shown).

nal layer that consisted of differentiated secretory epithelial cells adjacent to a basal cell layer embedded in fibromuscular stroma were classified as normal. Cores with benign glands in which the luminal layer was incompletely developed were annotated as atrophic, and were excluded from further analysis. PIN was characterized by nuclear crowding and prominent nucleoli with or without interruption of the basal cell layer. Invasive carcinoma was graded using the Gleason grading scheme. A major and minor Gleason grade was assigned to each core if more than 3 cancer glands were present in a core. Because TMA design only involved prostate cancers with a sum of 6 or 7 on final pathological evaluation, the Gleason sum score within cores ranged from 6 to 8. After eliminating cores that were not readable or that did not contain any prostate epithelium, there were 123 normal observations, 147 tumor observations and 11 PIN observations available for analysis. A semi-quantitative scoring system was used that provided a linear scale of 0 –300 based upon staining intensity parameters and the percentage of cells showing reactivity. Abundant SCD immunoreactivity was found in the secretory epithelium of normal glands, and not detected in basal epithelium (Fig. 4a– d). SCD protein localized to granules within the cytoplasm of secretory cells (Fig. 4c). Less intense immunoreactivity was observed in PIN lesions, though only a limited number of samples were available for evaluation. SCD staining in cancerous glands was decreased significantly compared to normal glands. The number of cancer cells expressing SCD was diminished and in addition, fewer SCD positive granules per cell were identified (Fig. 4c– d). The median

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SCD expression measured 150, 45 and 10 for normal prostate, PIN and cancer, respectively (Fig. 4e). In this limited sample set, there was no correlation between SCD expression and the Gleason sum score in cancer-containing cores. Statistically significant differential SCD expression between normal prostate and prostate carcinoma was determined at the p ⫽ 0.001 level, and between PIN and prostate carcinoma at the p ⫽ 0.03 level. The comparison between normal prostate and PIN and between PIN and prostate carcinoma was statistically significant at the p ⫽ 0.05 level. We observed weak stromal cell reactivity with the SCD-1 antibody in most tissue samples. It is likely that this reactivity represents non-specific binding of the SCD-1 antibody. A control, leaving out the primary antibody was negative. We cannot completely exclude the possibility that a small a mount of SCD-1 expression occurs in stromal cells. If this is the case, there is no difference between normal and cancer stroma. It is therefore unlikely that SCD-1 in the stroma is related to the presence or the biologic behavior of prostate cancer. To assess the association between SCD and FAS expression, a parallel TMA slide was stained with the FAS antibody. Data for FAS and SCD expression were available for 85 cancer-containing cores. In 78 of 85 cores (92%), over expression of FAS (total score⫽ ⱖ100) was observed. Of the 78 FAS overexpressing cores, 72 (92%) demonstrated decreased SCD expression (total score ⫽ ⬍100) occurred in cancerous compared to normal glands (Fig. 4f). A 95% confidence interval for the percent of cores staining positive for FAS and negative for SCD extends from 0.83– 0.97. These results demonstrated that the vast majority of the tumor samples studied exhibited FAS overexpression and loss of SCD. Discussion Stearoyl-CoA desaturase (SCD; EC 1.14.99.5) is an iron-containing enzyme involved in mediating the rate-limiting step of unsaturated fatty acid biosynthesis. SCD catalyzes the ⌬-9-cis desaturation of palmitic acid (C16:0) and stearic acid (C18:0) to the monounsaturated fatty acids palmitoleic acid (C16:1) and oleic acid (C18:1) respectively (Fig. 3).27,28 These monounsaturated fatty acids are principle components of membrane phospholipids, triglycerides and cholesterol esters.29 The ratio of saturated and unsaturated fatty acids has been implicated in the regulation of cell growth and differentiation through effects on the physical properties of cell-membrane fluidity and signal transduction.4,30 Small changes in this ratio, and in the amount of available unsaturated fatty acids can affect the ability of the cell to respond to external stimuli. Alterations in cellular fatty acid composition have been associated with a range of pathologies including neurological and vascular diseases, diabetes, obesity, hypertension and cancer.29 In humans, a single functional gene encoding SCD has been identified that maps to chromosome 10 and produces 2 transcripts of 3.9 and 5.2kb through the use of alternative polyadenylation signals.31 The mouse expresses 4 SCD isoforms designated Scd1, Scd2, Scd3 and Scd4.32 Murine Scd1 and Scd2 share 85% and 82% nucleotide sequence identity with human SCD, respectively. In vivo and in vitro studies have demonstrated that SCD gene expression is regulated normally by dietary constituents, hormonal factors and developmental processes. SCD expression is negatively regulated by polyunsaturated fatty acids, cholesterol and arachidonic acid as measured by decreased SCD promoter activity, mRNA and protein levels.33 The expression of SCD is positively regulated by a lipogenic diet through induction of sterol response element binding protein (SREBP), a major coordinate regulator of multiple enzymes involved in fatty acid metabolism.33 TGF-␤, retinoids and peroxisome proliferator activated receptor (PPAR) agonists such as Clofibrate and Gemfibrozil have also been shown to induce SCD expression.34 –36 Consistent with these observations, a sterol response element (SRE) and peroxisome proliferator response element (PPRE) have been identified in the SCD promoter region.36 In vitro studies of SCD overexpression resulted an

FIGURE 3 – Biochemical pathway of fatty acid metabolism involving stearoyl-CoA-desaturase. Stearoyl-CoA-desaturase (SCD) catalyzes the desaturation of the unsaturated fatty acids palmitate (C16:0) and stearate (C18:0) to the 9 monounsaturated fatty acids palmitoleate (C16:1) and oleate (C18:1), respectively.59 In prostate carcinoma, increased expression of FAS (vertical dashed arrows) and decreased expression of SCD (vertical dashed arrow) would produce increased levels of palmitate. FAS, fatty acid synthase; SCD, stearoyl-CoAdesaturase; GPAT, glycerol-3-phosphate acyltransferase.

increase in the plasma membrane content of monounsaturated fatty acids relative to saturated fatty acids in addition to alterations in membrane organization with decreases in membrane-ordered regions.37 Recent studies describing the molecular and physiological effects of SCD deficiency in animal models are enlightening. Ntambi et al.38 demonstrated that mice with a targeted disruption of Scd1 exhibited reduced body adiposity, increased insulin sensitivity, and resistance to diet-induced weight gain. The protection from obesity involved increased energy expenditure and increased oxygen consumption. The expression of several genes involved in the oxidation of lipids was upregulated, whereas lipid synthesis genes were downregulated. These observations suggest that a consequence of Scd1 deficiency is an activation of lipid oxidation in addition to reduced triglyceride synthesis and storage. Studies of murine models with leptin deficiency demonstrated that leptin specifically represses Scd1 transcript levels and enzymatic activity resulting in lean and hypermetabolic animals.39 In the context of our results, an increased incidence of prostate carcinoma or other malignancies in mice with germline alterations in Scd expression was not described. Mice express 4 isoforms of Scd, however, and redundant effects may mask some pathological features of Scd1 loss in the prostate. How might a reduction of SCD expression contribute to the development of human prostate carcinoma? At this time, several possible mechanisms are intriguing, but speculative. One cellular attribute implicated in neoplastic growth involves the regulation of cellular signaling pathways through targeted par-

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FIGURE 4.

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FIGURE 4 – Immunohistochemical analysis of SCD protein expression. (a) Section of formalin fixed paraffin embedded tissue stained with the SCD antibody. Normal epithelial glands (NE) are embedded in a fibromuscular stroma and express SCD in secretory epithelial cells, as indicated by a brown immunohistochemical staining reaction (100⫻). (b) High power view of a normal gland showing SCD expression in the cytoplasm and localized to cytoplasmic granules (600⫻). (c) Decreased SCD expression in prostate cancer. Low power view of an SCD negative prostate cancer with a GS of 6 (CE) infiltrating in between SCD positive normal glands (NE). Magnification ⫽ 100⫻. (d) Higher power view of granular SCD immunoreactivity predominantly in the normal secretory epithelium with loss of expression in adjacent cancer epithelium. Magnification ⫽ 400⫻. (e) Quantitative analysis of SCD expression in normal and neoplastic prostate tissue. Boxes show the 25–75th percentiles and median level of expression (center dashed line). The whiskers extend 1.5 times the Inter-quartile range from the end of the box. (f) FAS expression in prostate cancer cores. FAS and SCD expression were scored using the same scoring scheme. A score of ⱖ100 indicated FAS overexpression and a score of ⬍ 100 the loss of SCD expression.

titioning of signaling molecules to regions of the plasma membrane. Several signal transducers responding to receptorgenerated signals are localized to specialized plasma membrane domains comprised of cholesterol and glycosphingolipids.40 These domains are detergent insoluble and have been termed rafts.41 The spatial organization and concentration of selected proteins that partition to rafts may serve to increase the efficiency and specificity of signal transduction by facilitating protein interactions and preventing inappropriate cross-talk between pathways.40 Importantly, a significant characteristic dictating the partitioning of signaling proteins to lipid raft microdomains involves the addition of palmitate to cysteine residues, a reaction termed palmitoylation.42,43 Numerous signaling proteins have been shown to undergo regulation by palmitoylation including Wnt, RAS and regulators of G protein signaling (RGS proteins).44 – 47 Loss of SCD expression would be expected to increase cellular palmitate levels. Further enhancement of the palmitate pool in neoplastic prostate epithelium would also result from overexpression of the fatty acid synthase enzyme (Fig. 3), a finding observed in a majority of primary prostate carcinomas.7,48 It has been shown that a cell survival signal mediated by epidermal growth factor receptor (EGFR) activation of the PI3K/Akt pathway is operative in prostate cancer cells, and is dependent upon localization to lipid rafts.49 Pharmacological disruption of lipid rafts inhibited signaling via EGFR and led to apoptosis. Overexpression of SCD also leads to a reduction of detergent insoluble lipid rafts,37 and thus could result in loss of cell survival signals. Conversely, a loss of SCD expression might be expected to provide an increase in lipid rafts with the ability to activate anti-apoptotic pathways. Additional evidence supporting a role for the influence of cellular lipid composition on signaling pathways is provided by studies of tumor cell sensitivity to cytotoxicity mediated by tumor necrosis factor (TNF). Murine tumor cell lines engineered to express the yeast ⌬-9-desaturase gene, Ole 1, contained increased plasma membrane levels of unsaturated oleic acid and decreased levels of saturated stearic acid.30 These altered fatty acid ratios were associated with increased membrane fluidity and a markedly enhanced sensitivity to killing by TNF. One mechanism pro-

posed for TNF cytotoxicity involves lipid peroxidation resulting from increased production of oxygen free radicals in the mitochondria of TNF-sensitive cells.50 Membranes containing extra double bonds in the form of unsaturated fatty acids may become more accessible to the action of free radicals. It follows that tumor cells expressing SCD at a low level, with saturated rather than unsaturated fatty acids comprising membrane structure, may have a survival advantage toward host anti-tumor mechanisms including TNF-mediated cell killing. Several biochemical regulators of tumor cell growth have also been shown to modulate SCD expression. TGF-␤ and retinoids increase cellular levels of SCD and also inhibit tumor growth through effects on cell cycle, cell differentiation and apoptosis.51,52 Our results open the possibility that the loss of SCD expression in tumor cells may serve to modulate these growth inhibitory effects. Ligands of the PPAR family of nuclear receptors also upregulate SCD expression, and show growth inhibitory activities toward a wide variety of tumor types in vitro and in vivo.53 Ligands of PPAR ␥ in particular have been the focus of study in prostate carcinoma and demonstrate substantial growth inhibitory effects toward prostate cancer cell lines.54 Phase II clinical trials of troglitazone, a PPAR ␥ ligand used for treating Type 2 diabetes, exhibited measurable biological activities in patients with advanced prostate carcinoma.54 It is plausible that a component of PPAR ␥ affects may be mediated through SCD activity, and downregulation of SCD expression in neoplastic cells could abrogate growth suppression. The significance of PPARs in prostate carcinogenesis remains controversial, 55 however, and this mechanism may represent only one of several pathways influencing SCD activity. Although signaling regulators of tumor cell growth have been shown to modulate SCD expression, alterations in SCD levels influence signaling pathways important for cell growth and metabolism. SCD deficiency enhances signaling through the insulin receptor (IR) pathway as demonstrated by an increase in basal phosphorylation of the IR and insulin receptor substrates-1 and -2 (IRS-1 and IRS-2), increased association of IRS-1 and IRS-2 with PI3-kinase, and increased phosphorylation of Akt.56 Activation of the PI3kinase/Akt pathway has been shown to be an important pathway regulating proliferation, apoptosis and growth in many cancers including prostate carcinoma.57 Our observation that reduced SCD expression is associated with prostate carcinoma adds another member to a growing list of cancer-associated alterations in components of lipid metabolism that includes FAS,48 AMACR15 and sterol regulatory element binding protein (SREBP).58 The relationships between these proteins in the context of prostate carcinogenesis remains to be determined. Our findings offer many testable hypothesis regarding the role of known SCD substrates and products in regulating cellular growth. It is also possible that the desaturase activity of SCD may operate on additional substrates not yet known to be associated with carcinogenesis. Acknowledgements We wish to thank K. Adolphson for assistance with immunohistochemistry. This work was supported by NIH grants CA97186 and DK65204 to P.S.N. S.M. was supported by a Basic Sciences Training Grant in Urology (T32-DK007779).

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