- Email: [email protected]
Fatty Acid Synthase Over Expression is an Indicator of Tumor Aggressiveness and Poor Prognosis in Renal Cell Carcinoma
Fatty Acid Synthase Over Expression is an Indicator of Tumor Aggressiveness and Poor Prognosis in Renal Cell Carcinoma Akio Horiguchi,* Tomohiko Asano, Takako Asano, Keiichi Ito, Makoto Sumitomo and Masamichi Hayakawa From the Department of Urology, National Defense Medical College, Tokorozawa-City, Saitama, Japan
Purpose: Fatty acid synthase is a key enzyme in the de novo biosynthesis of fatty acids. Increased fatty acid synthase expression and its association with tumor aggressiveness and poor prognosis have been demonstrated in various human malignant tumors. We investigated fatty acid synthase expression in patients with renal cell carcinoma and its impact on clinicopathological parameters. Materials and Methods: Fatty acid synthase expression in 120 patients with renal cancer was examined by immunohistochemistry. The relationship between fatty acid synthase expression status and various clinicopathological parameters was analyzed. Survival analysis was performed using the log rank test and a Cox multivariate hazard model. Results: Of 120 tumors 18 (15%) showed positive fatty acid synthase expression, which was significantly associated with advanced pathological T stage (pT3-4, p ⫽ 0.0009), regional lymph node metastasis (p ⫽ 0.0429), distant metastasis (p ⫽ 0.0042), higher histological grade (G3, p ⫽ 0.0017) and microvascular invasion (p ⫽ 0.0357). Patients with positive fatty acid synthase expression had significantly shorter cancer specific survival than those with negative FAS expression (p ⬍0.0001). Multivariate Cox proportional hazards model analysis demonstrated that positive fatty acid synthase expression was an independent predictor of shortened cancer specific survival (p ⫽ 0.0363, HR 3.736). Conclusions: Increased FAS expression could be an indicator of tumor aggressiveness and poor prognosis of renal cell carcinoma. Patients with fatty acid synthase positive tumors should be followed closely and carefully, and adjuvant therapy should be considered. Key Words: kidney; carcinoma, renal cell; fatty acid synthase; mortality; tumor markers, biological
ammalian FAS is a complex multifunctional enzyme that contains 7 catalytic domains and a phosphopantetheine prosthetic group on a single polypeptide, and in the presence of NADPH (reduced nicotinamide adenine dinucleotide phosphate) it catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA.1 This enzyme also has a pivotal role in energy homeostasis by converting excess carbon intake into fatty acids that, when energy is needed, can be broken down to acetyl-CoA via -oxidation.1 FAS has specialized physiological functions, including the production of lecithin in fetal lungs, the synthesis of milk lipids in the lactating breast, and the conversion and storage of energy in the liver and adipose tissue,1 but the endogenous synthesis of fatty acid is usually minimal because diet supplies most of the fatty acids in cells. Consequently FAS is expressed at a low or undetectable level in most normal human tissues other than those of the lactating breast and cycling endometrium.1 However, in various types of cancer FAS is over expressed and increased FAS expression correlates with tumor aggressiveness and poor prognosis.1–7 It has also been suggested that such up-regulation of FAS in cancer provides a selective advantage to tumor cells by promoting proliferation and
M
Submitted for publication December 5, 2007. * Correspondence and requests for reprints: Department of Urology, National Defense Medical College, 3-2 Namiki, TokorozawaCity, Saitama 359-8513, Japan (telephone: ⫹81-4-2995-1676; FAX: ⫹81-4-2996-5210; e-mail: [email protected]).
0022-5347/08/1803-1137/0 THE JOURNAL OF UROLOGY® Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION
inhibiting apoptosis.1 FAS over expression in cancer cells has recently been exploited as a target for anticancer chemotherapy.1,4,8 Chemical inhibitors of FAS have been shown to induce apoptosis in cultured cancer cells and decrease tumor size in animal models of various cancers.1,4,8 Because to our knowledge neither the expression status of FAS in renal cell carcinoma nor the impact of FAS expression on clinicopathological parameters in renal cell carcinoma has yet been clarified, we performed immunohistochemical analysis to investigate this status and this impact. We found increased FAS expression in more aggressive forms of renal cell carcinoma and noted that it was associated with a poorer cancer specific survival rate.
MATERIALS AND METHODS Patients The study included 120 consecutive patients who underwent surgical treatment for renal cell carcinoma at our institution between December 1994 and December 2006. There were 83 men and 37 women 36 to 81 years old (median age 64). Followup from the date of operation to the last recorded followup was 2 to 141 months (median 24). Disease progression was defined as evidence of recurrence or metastasis on radiological or physical examinations. All tumor tissues were evaluated for pathological staging and histological grading according to the 2002 TNM classification. Microvascular invasion was considered to have occurred when rou-
1137
Vol. 180, 1137-1140, September 2008 Printed in U.S.A. DOI:10.1016/j.juro.2008.04.135
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FATTY ACID SYNTHASE EXPRESSION IN RENAL CELL CANCER
tine pathological examination revealed neoplastic cells in at least 1 endothelium lined space. Table 1 lists patient characteristics. Immunohistochemical Analysis of FAS Expression Immunohistochemistry was performed as described previously.9 Briefly, formalin fixed, paraffin embedded 4 m tumor sections were deparaffinized in xylene and rehydrated through graded ethanols. The slides were placed in Target Retrieval Solution High pH (Dako, Carpinteria, California) and heated for 40 minutes for antigen retrieval. Endogenous peroxidase activity was quenched with Peroxidase Blocking Reagent (Dako) for 10 minutes. The sections were incubated in 10% normal goat serum in phosphate buffered saline for 60 minutes at room temperature. After the sections were incubated overnight at 4C in a 1:200 dilution of rabbit polyclonal anti-FAS antibody (Immuno-Biological Laboratories, Fujioka, Japan) in phosphate buffered saline the slides were stained using a Simple Stain Max PO kit (Nichirei, Tokyo, Japan) according to manufacturer instructions. Specificity of the FAS antibody used in this study has been reported previously.2,5 Reaction products were visualized by immersing the slides in diaminobenzidine tetrahydrochloride for 5 minutes. The cytoplasmic membrane of normal fat cells served as an internal positive control2 and negative controls were made using nonimmunized rabbit serum instead of primary antibody during the procedure. The slides were analyzed by 3 independent observers (AH, TA and TA) blinded to any clinical data. As described previously,2,5 immunostaining was graded as low—less than 5% immunostained cancer cells, intermediate—5% to 50% or high— greater than 50%. Tumors with high immunostaining were considered FAS positive and other tumors were considered FAS negative. Statistical Analysis All statistical analysis was performed using StatView® 5.0 for Windows®. The chi-square test was used to assess the association between pathological features and FAS expres-
TABLE 1. Patient characteristics Characteristic
No. Pts (%)
Overall Median age (range) Sex: M F Pathological T stage: T1 T2 T3 T4 Regional lymph node metastasis: N0 N1 N2 Distant metastasis: M0 M1 Histological grade: G1 G2 G3 Histological type: Conventional Papillary Chromophobe
120 64 (36–81) 83 37
(69.2) (30.8)
80 13 24 3
(66.7) (10.8) (20.0) (2.5)
114 3 3
(95.0) (2.5) (2.5)
103 17
(85.8) (14.2)
22 83 15
(18.3) (69.2) (12.5)
112 6 2
(93.3) (5.0) (1.7)
sion levels. Kaplan-Meier survival curves for cancer specific death were estimated. The log rank test was used for univariate survival comparison and the multivariate Cox proportional hazards model was used for multivariate analysis. Statistical significance was considered at p ⬍0.05. RESULTS Relationship Between FAS Expression and Pathological Parameters of Renal Cell Carcinoma FAS immunoreactivity was located in the cytoplasm and it showed a granular pattern in tumor tissue but staining was heterogeneous in any tumor and staining intensity varied among tumors (fig. 1). FAS immunoreactivity was also observed in adjacent nonneoplastic kidney tissue but its intensity was modest (fig. 1, B). No immunostaining was observed in the negative control (data not shown). FAS expression of 18 of 120 tumors (15%) was positive with immunostaining in greater than 50% of cells, while that in the other 102 was negative with 16 (13%) showing intermediate immunostaining with immunostaining in 5% to 50% of the cells and 86 showing low immunostaining with immunostaining in less than 5% of the cells. Table 2 lists p values for the associations between FAS status and pathological parameters. Positive FAS expression was significantly associated with advanced pathological T stage (pT3-4, p ⫽ 0.0009), regional lymph node metastasis (p ⫽ 0.0429), distant metastasis (p ⫽ 0.0042), higher histological grade (G3, p ⫽ 0.0017) and microvascular invasion (p ⫽ 0.0357, table 2). Impact of FAS Expression on Clinical Outcome We next evaluated the impact of FAS expression on the clinical outcome in patients with renal cell carcinoma. Cancer specific survival in patients with positive FAS expression was significantly shorter than that in patients with negative FAS expression (p ⬍0.0001, fig. 2). Other factors shown by univariate analysis to be significantly associated with shortened cancer specific survival were advanced pathological stage (T3-4, p ⬍0.0001), regional lymph node metastasis (N1 or greater, p ⫽ 0.0063), distant metastasis (M1, p ⬍0.0001), higher histological grade (G3, p ⫽ 0.0148) and microvascular tumor invasion (p ⬍0.0001). We further performed multivariate Cox proportional hazards analysis using all of these significant prognostic factors, and found that distant metastasis (M1), venous invasion and positive FAS expression were independent predictors of shortened cancer specific survival (p ⬍0.0001, 0.0497 and 0.0363, respectively, table 3). DISCUSSION Recent studies have demonstrated that various human malignant tumors, especially a biologically aggressive subset, express increased levels of FAS.1–7 FAS was first identified as an oncogenic antigen 519 in patients with breast cancer and a markedly worsened prognosis.10 Increased FAS expression has been found in 82% of lethal androgen independent prostate tumors examined at autopsy and it appears to increase the risk of death from prostate cancer 4.45-fold.4,11 In addition, increased FAS expression was observed in 31.3% of soft tissue sarcomas, and it was found to correlate with larger tumors and decreased survival.2 The results of
FATTY ACID SYNTHASE EXPRESSION IN RENAL CELL CANCER
1139
Proportion surviving
1
D
C
.8 .6 FAS negative FAS positive
.4
Log-rank test p <0.0001
.2 0 0
FIG. 1. Representative immunostaining for FAS in renal cell carcinoma. A, high FAS expression. B, high FAS expression invading adjacent normal kidney. Note that immunoreactivity in cancer cells is higher than in nonneoplastic kidney tissue. C, intermediate FAS expression. D, low FAS expression. Reduced from ⫻200.
the current study of FAS expression, and its impact on pathological parameters and clinical outcome in patients with renal cell carcinoma are consistent with the results of previous studies of other types of cancer, in that they indicate that increased FAS expression is an indicator of tumor aggressiveness and poor prognosis. To our knowledge this is the first report showing the status of FAS expression in renal cell carcinoma and demonstrating that FAS expression has a potential role in determining pathological features and clinical outcome. The almost universal up-regulation of FAS in many human cancers and its association with poor clinical outcome indicate that FAS is involved in the development, maintenance and enhancement of the malignant phenotype. It has been suggested that FAS is involved in the production of phospholipids participating in reactions occurring in detergent resistant membrane microdomains and it has been implicated in such key biological processes as the signal transduction of Her2 and EGFR.12 Menendez et al found that the pharmacological FAS inhibitors cerulenin and C75 suppress Her2 expression and tyrosine kinase activity in
TABLE 2. FAS expression and pathological parameters
Pathological stage: T1–T2 T3–T4 Regional lymph node metastasis: N0 N1 or Greater Distant metastasis: M0 M1 Histological grade: G1–G2 G3 Microvascular invasion: Negative Positive
No. FAS Expression
20
40
60 80 100 120 140 160 Time (months)
FIG. 2. Overall cancer specific survival curves in patients with positive and negative FAS expression (log rank test p ⬍0.0001).
breast and ovarian cells by impairing the correct localization and function of Her2.13 Pharmacological FAS inhibitors also have cytotoxic and cytostatic effects on various cancer cells.4,8 These studies suggest that FAS provides some survival benefit to cancer cells. Although not all of the mechanisms responsible for tumor associated FAS over expression are known, several have been demonstrated.1,11,14 –16 Two of the major contributors to FAS over expression in cancer are aberrant growth factor expression and increased levels of messengers on downstream signaling pathways, such as Akt, which promotes FAS transcription.1,11,14,15 FAS expression has been shown to be stimulated by EGFR and Her2 signaling pathways through modulation of the expression and nuclear maturation of the transcriptional factor sterol regulatory elementbinding protein 1c, which binds to and activates sterol regulatory elements in the promoter region of FAS.1,14 FAS expression has been shown to be increased in LNCaP prostate cancer cells, in which FAS expression is increased by inherently active Akt signaling and can be pharmacologically reduced by PI3K inhibition.17 Furthermore, increased Akt activation has been shown to be associated with FAS over expression in prostate and ovarian cancer tissues.11,15 Increased expression of EGFR and Her2, and aberrant activation of Akt are frequently observed in renal cell carcinoma, and they are associated with higher tumor grade, metastasis and poor prognosis.18,19 These aberrant signaling processes might also be responsible for the over expression of FAS in renal cell carcinoma. A recent study found increased FAS levels in the blood of patients with breast cancer.20 Serum FAS levels were significantly higher in patients with breast cancer than in healthy subjects and serum FAS levels increased with tumor stage. Therefore, serum FAS levels might be a useful tumor marker for breast cancer and they might have a role in
No. Pts
Neg
Pos
p Value
93 27
85 17
8 10
0.0009
114 6
99 3
15 3
0.0429
103 17
92 10
11 7
0.0042
105 15
94 8
11 7
0.0017
Variable
HR
95% CI
p Value
0.0357
Metastasis (M1) Microvascular invasion (pos) FAS expression (pos)
19.99 5.415 3.736
4.662–85.789 1.002–29.259 1.087–12.837
⬍0.0001 0.0497 0.0363
70 50
64 38
6 12
TABLE 3. Cox proportional hazards model multivariate prognostic analysis of cancer specific survival
1140
FATTY ACID SYNTHASE EXPRESSION IN RENAL CELL CANCER
determining cancer virulence. Whether serum FAS levels as well as the levels of FAS expression in tumor specimens could be useful clinical markers is renal cell carcinoma should be examined in future studies. CONCLUSIONS Increased FAS expression could be a novel indicator of tumor aggressiveness and poor prognosis of renal cell carcinoma. Patients with FAS positive tumors should be followed closely and carefully, and adjuvant therapy should be considered.
9.
10.
11.
12.
Abbreviations and Acronyms CoA ⫽ coenzyme A EGFR ⫽ epidermal growth factor FAS ⫽ fatty acid synthase
13.
REFERENCES 1.
2.
3.
4.
5.
6.
7.
8.
Menendez JA and Lupu R: Fatty acid synthase and the lipogenic phenotype in cancer pathogenesis. Nat Rev Cancer 2007; 7: 763. Takahiro T, Shinichi K and Toshimitsu S: Expression of fatty acid synthase as a prognostic indicator in soft tissue sarcomas. Clin Cancer Res 2003; 9: 2204. Gabrielson EW, Pinn ML, Testa JR and Kuhajda FP: Increased fatty acid synthase is a therapeutic target in mesothelioma. Clin Cancer Res 2001; 7: 153. Pizer ES, Pflug BR, Bova GS, Han WF, Udan MS and Nelson JB: Increased fatty acid synthase as a therapeutic target in androgen-independent prostate cancer progression. Prostate 2001; 47: 102. Kusakabe T, Nashimoto A, Honma K and Suzuki T: Fatty acid synthase is highly expressed in carcinoma, adenoma and in regenerative epithelium and intestinal metaplasia of the stomach. Histopathology 2002; 40: 71. Swinnen JV, Roskams T, Joniau S, Van Poppel H, Oyen R, Baert L et al: Overexpression of fatty acid synthase is an early and common event in the development of prostate cancer. Int J Cancer 2002; 98: 19. Esslimani-Sahla M, Thezenas S, Simony-Lafontaine J, Kramar A, Lavaill R, Chalbos D et al: Increased expression of fatty acid synthase and progesterone receptor in early steps of human mammary carcinogenesis. Int J Cancer 2007; 120: 224. Pizer ES, Chrest FJ, DiGiuseppe JA and Han WF: Pharmacological inhibitors of mammalian fatty acid synthase suppress DNA replication and induce apoptosis in tumor cell lines. Cancer Res 1998; 58: 4611.
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Horiguchi A, Sumitomo M, Asakuma J, Asano T, Zheng R, Nanus DM et al: Increased serum leptin levels and over expression of leptin receptors are associated with the invasion and progression of renal cell carcinoma. J Urol 2006; 176: 1631. Kuhajda FP, Jenner K, Wood FD, Hennigar RA, Jacobs LB, Dick JD et al: Fatty acid synthesis: a potential selective target for antineoplastic therapy. Proc Natl Acad Sci U S A 1994; 91: 6379. Bandyopadhyay S, Pai SK, Watabe M, Gross SC, Hirota S, Hosobe S et al: FAS expression inversely correlates with PTEN level in prostate cancer and a PI 3-kinase inhibitor synergizes with FAS siRNA to induce apoptosis. Oncogene 2005; 24: 5389. Swinnen JV, Van Veldhoven PP, Timmermans L, De Schrijver E, Brusselmans K, Vanderhoydonc F et al: Fatty acid synthase drives the synthesis of phospholipids partitioning into detergent-resistant membrane microdomains. Biochem Biophys Res Commun 2003; 302: 898. Menendez JA, Vellon L, Mehmi I, Oza BP, Ropero S, Colomer R et al: Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells. Proc Natl Acad Sci U S A 2004; 101: 10715. Swinnen JV, Heemers H, Deboel L, Foufelle F, Heyns W and Verhoeven G: Stimulation of tumor-associated fatty acid synthase expression by growth factor activation of the sterol regulatory element-binding protein pathway. Oncogene 2000; 19: 5173. Wang HQ, Altomare DA, Skele KL, Poulikakos PI, Kuhajda FP, Di Cristofano A et al: Positive feedback regulation between AKT activation and fatty acid synthase expression in ovarian carcinoma cells. Oncogene 2005; 24: 3574. Graner E, Tang D, Rossi S, Baron A, Migita T, Weinstein LJ et al: The isopeptidase USP2a regulates the stability of fatty acid synthase in prostate cancer. Cancer Cell 2004; 5: 253. Van de Sande T, De Schrijver E, Heyns W, Verhoeven G and Swinnen JV: Role of the phosphatidylinositol 3=-kinase/ PTEN/Akt kinase pathway in the overexpression of fatty acid synthase in LNCaP prostate cancer cells. Cancer Res 2002; 62: 642. Horiguchi A, Oya M, Uchida A, Marumo K and Murai M: Increased Akt activation and its impact on clinicopathological features of renal cell carcinoma. J Urol 2003; 169: 710. Stumm G, Eberwein S, Rostock-Wolf S, Stein H, Pomer S, Schlegel J et al: Concomitant overexpression of the EGFR and erbB-2 genes in renal cell carcinoma (RCC) is correlated with dedifferentiation and metastasis. Int J Cancer 1996; 69: 17. Wang Y, Kuhajda FP, Li JN, Pizer ES, Han WF, Sokoll LJ et al: Fatty acid synthase (FAS) expression in human breast cancer cell culture supernatants and in breast cancer patients. Cancer Lett 2001; 167: 99.
Purpose: Fatty acid synthase is a key enzyme in the de novo biosynthesis of fatty acids. Increased fatty acid synthase expression and its association with tumor aggressiveness and poor prognosis have been demonstrated in various human malignant tumors. We investigated fatty acid synthase expression in patients with renal cell carcinoma and its impact on clinicopathological parameters. Materials and Methods: Fatty acid synthase expression in 120 patients with renal cancer was examined by immunohistochemistry. The relationship between fatty acid synthase expression status and various clinicopathological parameters was analyzed. Survival analysis was performed using the log rank test and a Cox multivariate hazard model. Results: Of 120 tumors 18 (15%) showed positive fatty acid synthase expression, which was significantly associated with advanced pathological T stage (pT3-4, p ⫽ 0.0009), regional lymph node metastasis (p ⫽ 0.0429), distant metastasis (p ⫽ 0.0042), higher histological grade (G3, p ⫽ 0.0017) and microvascular invasion (p ⫽ 0.0357). Patients with positive fatty acid synthase expression had significantly shorter cancer specific survival than those with negative FAS expression (p ⬍0.0001). Multivariate Cox proportional hazards model analysis demonstrated that positive fatty acid synthase expression was an independent predictor of shortened cancer specific survival (p ⫽ 0.0363, HR 3.736). Conclusions: Increased FAS expression could be an indicator of tumor aggressiveness and poor prognosis of renal cell carcinoma. Patients with fatty acid synthase positive tumors should be followed closely and carefully, and adjuvant therapy should be considered. Key Words: kidney; carcinoma, renal cell; fatty acid synthase; mortality; tumor markers, biological
ammalian FAS is a complex multifunctional enzyme that contains 7 catalytic domains and a phosphopantetheine prosthetic group on a single polypeptide, and in the presence of NADPH (reduced nicotinamide adenine dinucleotide phosphate) it catalyzes the synthesis of palmitate from acetyl-CoA and malonyl-CoA.1 This enzyme also has a pivotal role in energy homeostasis by converting excess carbon intake into fatty acids that, when energy is needed, can be broken down to acetyl-CoA via -oxidation.1 FAS has specialized physiological functions, including the production of lecithin in fetal lungs, the synthesis of milk lipids in the lactating breast, and the conversion and storage of energy in the liver and adipose tissue,1 but the endogenous synthesis of fatty acid is usually minimal because diet supplies most of the fatty acids in cells. Consequently FAS is expressed at a low or undetectable level in most normal human tissues other than those of the lactating breast and cycling endometrium.1 However, in various types of cancer FAS is over expressed and increased FAS expression correlates with tumor aggressiveness and poor prognosis.1–7 It has also been suggested that such up-regulation of FAS in cancer provides a selective advantage to tumor cells by promoting proliferation and
M
Submitted for publication December 5, 2007. * Correspondence and requests for reprints: Department of Urology, National Defense Medical College, 3-2 Namiki, TokorozawaCity, Saitama 359-8513, Japan (telephone: ⫹81-4-2995-1676; FAX: ⫹81-4-2996-5210; e-mail: [email protected]).
0022-5347/08/1803-1137/0 THE JOURNAL OF UROLOGY® Copyright © 2008 by AMERICAN UROLOGICAL ASSOCIATION
inhibiting apoptosis.1 FAS over expression in cancer cells has recently been exploited as a target for anticancer chemotherapy.1,4,8 Chemical inhibitors of FAS have been shown to induce apoptosis in cultured cancer cells and decrease tumor size in animal models of various cancers.1,4,8 Because to our knowledge neither the expression status of FAS in renal cell carcinoma nor the impact of FAS expression on clinicopathological parameters in renal cell carcinoma has yet been clarified, we performed immunohistochemical analysis to investigate this status and this impact. We found increased FAS expression in more aggressive forms of renal cell carcinoma and noted that it was associated with a poorer cancer specific survival rate.
MATERIALS AND METHODS Patients The study included 120 consecutive patients who underwent surgical treatment for renal cell carcinoma at our institution between December 1994 and December 2006. There were 83 men and 37 women 36 to 81 years old (median age 64). Followup from the date of operation to the last recorded followup was 2 to 141 months (median 24). Disease progression was defined as evidence of recurrence or metastasis on radiological or physical examinations. All tumor tissues were evaluated for pathological staging and histological grading according to the 2002 TNM classification. Microvascular invasion was considered to have occurred when rou-
1137
Vol. 180, 1137-1140, September 2008 Printed in U.S.A. DOI:10.1016/j.juro.2008.04.135
1138
FATTY ACID SYNTHASE EXPRESSION IN RENAL CELL CANCER
tine pathological examination revealed neoplastic cells in at least 1 endothelium lined space. Table 1 lists patient characteristics. Immunohistochemical Analysis of FAS Expression Immunohistochemistry was performed as described previously.9 Briefly, formalin fixed, paraffin embedded 4 m tumor sections were deparaffinized in xylene and rehydrated through graded ethanols. The slides were placed in Target Retrieval Solution High pH (Dako, Carpinteria, California) and heated for 40 minutes for antigen retrieval. Endogenous peroxidase activity was quenched with Peroxidase Blocking Reagent (Dako) for 10 minutes. The sections were incubated in 10% normal goat serum in phosphate buffered saline for 60 minutes at room temperature. After the sections were incubated overnight at 4C in a 1:200 dilution of rabbit polyclonal anti-FAS antibody (Immuno-Biological Laboratories, Fujioka, Japan) in phosphate buffered saline the slides were stained using a Simple Stain Max PO kit (Nichirei, Tokyo, Japan) according to manufacturer instructions. Specificity of the FAS antibody used in this study has been reported previously.2,5 Reaction products were visualized by immersing the slides in diaminobenzidine tetrahydrochloride for 5 minutes. The cytoplasmic membrane of normal fat cells served as an internal positive control2 and negative controls were made using nonimmunized rabbit serum instead of primary antibody during the procedure. The slides were analyzed by 3 independent observers (AH, TA and TA) blinded to any clinical data. As described previously,2,5 immunostaining was graded as low—less than 5% immunostained cancer cells, intermediate—5% to 50% or high— greater than 50%. Tumors with high immunostaining were considered FAS positive and other tumors were considered FAS negative. Statistical Analysis All statistical analysis was performed using StatView® 5.0 for Windows®. The chi-square test was used to assess the association between pathological features and FAS expres-
TABLE 1. Patient characteristics Characteristic
No. Pts (%)
Overall Median age (range) Sex: M F Pathological T stage: T1 T2 T3 T4 Regional lymph node metastasis: N0 N1 N2 Distant metastasis: M0 M1 Histological grade: G1 G2 G3 Histological type: Conventional Papillary Chromophobe
120 64 (36–81) 83 37
(69.2) (30.8)
80 13 24 3
(66.7) (10.8) (20.0) (2.5)
114 3 3
(95.0) (2.5) (2.5)
103 17
(85.8) (14.2)
22 83 15
(18.3) (69.2) (12.5)
112 6 2
(93.3) (5.0) (1.7)
sion levels. Kaplan-Meier survival curves for cancer specific death were estimated. The log rank test was used for univariate survival comparison and the multivariate Cox proportional hazards model was used for multivariate analysis. Statistical significance was considered at p ⬍0.05. RESULTS Relationship Between FAS Expression and Pathological Parameters of Renal Cell Carcinoma FAS immunoreactivity was located in the cytoplasm and it showed a granular pattern in tumor tissue but staining was heterogeneous in any tumor and staining intensity varied among tumors (fig. 1). FAS immunoreactivity was also observed in adjacent nonneoplastic kidney tissue but its intensity was modest (fig. 1, B). No immunostaining was observed in the negative control (data not shown). FAS expression of 18 of 120 tumors (15%) was positive with immunostaining in greater than 50% of cells, while that in the other 102 was negative with 16 (13%) showing intermediate immunostaining with immunostaining in 5% to 50% of the cells and 86 showing low immunostaining with immunostaining in less than 5% of the cells. Table 2 lists p values for the associations between FAS status and pathological parameters. Positive FAS expression was significantly associated with advanced pathological T stage (pT3-4, p ⫽ 0.0009), regional lymph node metastasis (p ⫽ 0.0429), distant metastasis (p ⫽ 0.0042), higher histological grade (G3, p ⫽ 0.0017) and microvascular invasion (p ⫽ 0.0357, table 2). Impact of FAS Expression on Clinical Outcome We next evaluated the impact of FAS expression on the clinical outcome in patients with renal cell carcinoma. Cancer specific survival in patients with positive FAS expression was significantly shorter than that in patients with negative FAS expression (p ⬍0.0001, fig. 2). Other factors shown by univariate analysis to be significantly associated with shortened cancer specific survival were advanced pathological stage (T3-4, p ⬍0.0001), regional lymph node metastasis (N1 or greater, p ⫽ 0.0063), distant metastasis (M1, p ⬍0.0001), higher histological grade (G3, p ⫽ 0.0148) and microvascular tumor invasion (p ⬍0.0001). We further performed multivariate Cox proportional hazards analysis using all of these significant prognostic factors, and found that distant metastasis (M1), venous invasion and positive FAS expression were independent predictors of shortened cancer specific survival (p ⬍0.0001, 0.0497 and 0.0363, respectively, table 3). DISCUSSION Recent studies have demonstrated that various human malignant tumors, especially a biologically aggressive subset, express increased levels of FAS.1–7 FAS was first identified as an oncogenic antigen 519 in patients with breast cancer and a markedly worsened prognosis.10 Increased FAS expression has been found in 82% of lethal androgen independent prostate tumors examined at autopsy and it appears to increase the risk of death from prostate cancer 4.45-fold.4,11 In addition, increased FAS expression was observed in 31.3% of soft tissue sarcomas, and it was found to correlate with larger tumors and decreased survival.2 The results of
FATTY ACID SYNTHASE EXPRESSION IN RENAL CELL CANCER
1139
Proportion surviving
1
D
C
.8 .6 FAS negative FAS positive
.4
Log-rank test p <0.0001
.2 0 0
FIG. 1. Representative immunostaining for FAS in renal cell carcinoma. A, high FAS expression. B, high FAS expression invading adjacent normal kidney. Note that immunoreactivity in cancer cells is higher than in nonneoplastic kidney tissue. C, intermediate FAS expression. D, low FAS expression. Reduced from ⫻200.
the current study of FAS expression, and its impact on pathological parameters and clinical outcome in patients with renal cell carcinoma are consistent with the results of previous studies of other types of cancer, in that they indicate that increased FAS expression is an indicator of tumor aggressiveness and poor prognosis. To our knowledge this is the first report showing the status of FAS expression in renal cell carcinoma and demonstrating that FAS expression has a potential role in determining pathological features and clinical outcome. The almost universal up-regulation of FAS in many human cancers and its association with poor clinical outcome indicate that FAS is involved in the development, maintenance and enhancement of the malignant phenotype. It has been suggested that FAS is involved in the production of phospholipids participating in reactions occurring in detergent resistant membrane microdomains and it has been implicated in such key biological processes as the signal transduction of Her2 and EGFR.12 Menendez et al found that the pharmacological FAS inhibitors cerulenin and C75 suppress Her2 expression and tyrosine kinase activity in
TABLE 2. FAS expression and pathological parameters
Pathological stage: T1–T2 T3–T4 Regional lymph node metastasis: N0 N1 or Greater Distant metastasis: M0 M1 Histological grade: G1–G2 G3 Microvascular invasion: Negative Positive
No. FAS Expression
20
40
60 80 100 120 140 160 Time (months)
FIG. 2. Overall cancer specific survival curves in patients with positive and negative FAS expression (log rank test p ⬍0.0001).
breast and ovarian cells by impairing the correct localization and function of Her2.13 Pharmacological FAS inhibitors also have cytotoxic and cytostatic effects on various cancer cells.4,8 These studies suggest that FAS provides some survival benefit to cancer cells. Although not all of the mechanisms responsible for tumor associated FAS over expression are known, several have been demonstrated.1,11,14 –16 Two of the major contributors to FAS over expression in cancer are aberrant growth factor expression and increased levels of messengers on downstream signaling pathways, such as Akt, which promotes FAS transcription.1,11,14,15 FAS expression has been shown to be stimulated by EGFR and Her2 signaling pathways through modulation of the expression and nuclear maturation of the transcriptional factor sterol regulatory elementbinding protein 1c, which binds to and activates sterol regulatory elements in the promoter region of FAS.1,14 FAS expression has been shown to be increased in LNCaP prostate cancer cells, in which FAS expression is increased by inherently active Akt signaling and can be pharmacologically reduced by PI3K inhibition.17 Furthermore, increased Akt activation has been shown to be associated with FAS over expression in prostate and ovarian cancer tissues.11,15 Increased expression of EGFR and Her2, and aberrant activation of Akt are frequently observed in renal cell carcinoma, and they are associated with higher tumor grade, metastasis and poor prognosis.18,19 These aberrant signaling processes might also be responsible for the over expression of FAS in renal cell carcinoma. A recent study found increased FAS levels in the blood of patients with breast cancer.20 Serum FAS levels were significantly higher in patients with breast cancer than in healthy subjects and serum FAS levels increased with tumor stage. Therefore, serum FAS levels might be a useful tumor marker for breast cancer and they might have a role in
No. Pts
Neg
Pos
p Value
93 27
85 17
8 10
0.0009
114 6
99 3
15 3
0.0429
103 17
92 10
11 7
0.0042
105 15
94 8
11 7
0.0017
Variable
HR
95% CI
p Value
0.0357
Metastasis (M1) Microvascular invasion (pos) FAS expression (pos)
19.99 5.415 3.736
4.662–85.789 1.002–29.259 1.087–12.837
⬍0.0001 0.0497 0.0363
70 50
64 38
6 12
TABLE 3. Cox proportional hazards model multivariate prognostic analysis of cancer specific survival
1140
FATTY ACID SYNTHASE EXPRESSION IN RENAL CELL CANCER
determining cancer virulence. Whether serum FAS levels as well as the levels of FAS expression in tumor specimens could be useful clinical markers is renal cell carcinoma should be examined in future studies. CONCLUSIONS Increased FAS expression could be a novel indicator of tumor aggressiveness and poor prognosis of renal cell carcinoma. Patients with FAS positive tumors should be followed closely and carefully, and adjuvant therapy should be considered.
9.
10.
11.
12.
Abbreviations and Acronyms CoA ⫽ coenzyme A EGFR ⫽ epidermal growth factor FAS ⫽ fatty acid synthase
13.
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