specific reduction of fas-associated protein with death domain (fadd) in clear cell renal cell...
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Cancer Investigation, 27:836–843, 2009ISSN: 0735-7907 print / 1532-4192 onlineCopyright c© Informa Healthcare USA, Inc.DOI: 10.1080/07357900902849681
ORIGINAL ARTICLECellular and Molecular Biology
Specific Reduction of Fas-Associated Protein withDeath Domain (FADD) in Clear Cell Renal Cell
CarcinomaHui Xu,1 Lizhi He,2,3 Xinchang Feng,2,3 Anil Kapoor,2,4 and Damu Tang2,3
Department of Nephrology, Xiangya Hospital, Central South University, Changsha, Hunan, P.R. China,1 The Hamilton Centre for KidneyResearch (HCKR), Division of Nephrology, St. Joseph’s Healthcare, Hamilton, Ontario, Canada,2 Departments of Medicine3 and Surgery,4
McMaster University, Hamilton, Ontario, Canada
ABSTRACT
Fas-associated protein with death domain (FADD) plays a major role in the execution ofapoptosis. Attenuation of apoptosis is a hallmark of cancer. Through systemic examination ofFADD in renal cell carcinoma (RCC) and adjacent nontumor kidney tissues from 85 patients,we demonstrated a significant reduction of FADD in clear cell RCC (ccRCC) compared to therespective nontumor kidney tissues. In human kidney, FADD is expressed in both the proximaland distal tubules. As ccRCC originates from the proximal tubular epithelium, reduction ofFADD in ccRCC indicates that FADD-mediated apoptosis may inhibit ccRCC tumorigenesis.
INTRODUCTION
Kidney cancer accounts for nearly 3% of human malignan-cies. It affects 32,000 individuals and causes 12,000 cancer-related deaths annually in the United States (1). The diseasecomprises a heterogeneous group of tumors, including primaryneoplasm of the renal pelvis or ureter (7–8%), Wilms tumor(5–6%), and renal cell carcinoma (RCC, 85%). RCC consists ofclear cell RCC (ccRCC) (80%), papillary RCC (pRCC) (15%),and others with different histological features and factors con-tributing to the behavior of the individual RCC (2).
A key mechanism that suppresses tumorigenesis, includingthe formation of RCC, involves apoptosis. Therefore, evadingapoptosis is a characteristic of cancer (3). Cancerous cells atten-uate apoptosis by amplification of anti-apoptotic proteins, suchas Bcl-2 (4). Bcl-2 protein is upregulated in 57% of metastaticccRCC. In comparison, 21% of primary ccRCCs show highlevels of Bcl-2 (5). As most cancer therapeutic reagents induce
Keywords: Renal cell carcinoma (RCC), FADD, ApoptosisCorrespondence to:Damu TangT3310, St. Joseph’s Hospital50 Charlton Ave EastHamilton, ON, Canada L8N 4A6email: [email protected]
apoptosis, attenuation of apoptosis thus contributes to treatment-resistance. RCC is well known for its resistance to chemotherapyand radiotherapy. This is in part attributable to elevated Bcl-2and XIAP (X-linked inhibitor of apoptosis protein) expression(6). RCC also reduces apoptosis by upregulation of growth fac-tor signal pathways, such as VEGF, EGF, TGFα, and c-Met.Activation of these growth factor pathways commonly occurs inRCC and promotes RCC tumorigenesis by activating the PI3K-Akt pathway (7). The PI3K-Akt pathway enhances cell survivalby inhibiting apoptosis via a variety of mechanisms. These in-clude the phosphorylation-mediated inactivation of Bad (8) andcaspase 9 (9), as well as the stimulation of NF-B activity throughthe activation of IKK (10–12) and the inactivation of forkheadtranscription factor pathways (13, 14). Inactivation of von Hip-pel Lindau (VHL) and tuberous sclerosis complex 1/2 (TSC1/2)tumor suppressors and activation of the mammalian target ofrapamycin (mTOR) kinase play critical roles in RCC tumori-genesis (7, 15). While mTOR activates Akt and thus inhibitsapoptosis (16), TSC1/2 suppresses mTOR activation, therebyenhancing apoptosis (16). VHL induces apoptosis in part viathe stabilization of p53 (17).
While apoptosis can be induced by a variety of stimuli, apop-tosis is executed via two major pathways: the death receptor(DR) (extrinsic) pathway and the mitochondrial (intrinsic) path-way (18). Both pathways have been intensively explored to in-duce apoptosis in cancer (19). DRs include Fas (CD95), TNFreceptor 1 (TNFR1), TRAIL (TNF-related apoptosis-inducing
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ligand) receptor 1 and 2 (TRAILR1/DR4 and TRAILR2/DR5),and others (20–22). Because normal tissues express higher levelsof two decoy receptors (DCRs) for TRAIL (TRAILR3/DCR1and TRAILR4/DCR2) than cancers, cancer cells are more sen-sitive to TRAIL-induced apoptosis than normal cells. This prop-erty has been heavily investigated to eradicate cancer cells formore than a decade and numerous reagents have been developed(22). However, clinical trials, although producing some encour-aging results, showed that the impact of TRAIL-targeted therapyon cancer is rather limited (22). This suggests that cancer cellsmay find ways to evade DR-mediated apoptosis.
One of the central components of DR-induced apoptosis isFADD (Fas-associated protein with death domain). Engagementof the DR via binding of its ligand or agonistic monoclonal an-tibody recruits FADD to specific DRs. FADD in turn binds tocaspase 8 or caspase 10, resulting in the activation of both cas-pases. Active caspase 8 and 10 trigger downstream caspasesto execute apoptotic cell death (20, 22). Therefore, a potentialmechanism for cancer cells in reducing DR-induced apoptosisis by compromising FADD’s proapoptotic function. To inves-tigate this possibility, we examined FADD expression in 85RCC patients on their normal kidney and carcinoma tissues. Wedemonstrated that FADD expression is reduced in ccRCC incomparison to the respective normal kidney tissues.
MATERIALS AND METHODS
Collection of primary RCCs
Eighty-five pairs of RCCs and their respective nontumorkidney tissues were collected in compliance with the localethics regulations at St. Joseph’s Healthcare, Hamilton, Ontario,Canada. The pathological diagnoses were performed by the spe-cialists of the hospital. All RCCs and their nontumor kidneytissues were confirmed by H&E staining. Details of the patho-logical characteristics for these patients are presented in Table 1.
Immunohistochemistry
Three-micron-thick slides were prepared from RCCs andtheir respective nontumor kidney tissues at McMaster Univer-sity’s Central Histological Facility. The immunohistochemistry(IHC) procedure was then optimized and did not produce de-tectable artificial staining that might be associated with antigen-retrieval and fixation, as control IgG (negative control) did notgenerate detectable staining (data not shown). IHC staining wascarried out in batches containing 20 individual slides. To over-come signal variations among individual batches, a few over-lapping slides (slides from the same patients) were present inindividual batches. Briefly, tumors and the matched nontumorkidney tissue were mounted on the same slide and deparaffinized(3 × 10 min in xylene, 2 × 2 min in 100% ethanol, and 2 ×2 min in 70% ethanol). After quenching endogenous peroxi-dase using 3% H2O2, antigen retrieval was carried out by heattreatment for 20 min in a 10 mM sodium citrate buffer (pH 6.0)using a food steamer. Primary antibody was incubated with thesections overnight at 4◦C at a concentration of 1:100 for an anti-
FADD antibody (BD Transduction Laboratories, Mississauga,Ontario, Canada). Biotinylated goat antimouse IgG and ExtrA-vidin conjugated peroxidase (Sigma, Oakville, Ontario, Canada)were then sequentially added. The chromogen reaction was car-ried out with diaminobenzidine, and counterstaining was donewith hematoxylin. To reduce signal variations among individ-ual batches of IHC staining, the chromogen reaction was doneusing the same stock solutions. We have noticed that differentslides from the same block, stained with the same antibody be-tween different batches, displayed comparable levels of signalintensity.
Tissue lysate preparation and Western blot
Frozen renal cancer tissue and corresponding normal renaltissue were crushed under liquid nitrogen and resuspended inlysis buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl,1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 25 mM sodiumpyrophosphate, 1 mM NaF, 1 mM β-glycerophosphate, 0.1 mMsodium orthovanadate, 1 mM PMSF, 2 µg/mL leupeptin, and10 µg/mL aprotinin. Cell lysates were prepared and Westernblot was performed according to our published procedure (23).Anti-FADD antibody was used at a 1:1000 dilution.
Quantification of FADD
FADD expression in RCCs and their respective nontumorkidney tissues were determined by Western blot. Actin levels inthese tissues were also examined by Western blot. FADD levelsin RCC and the matched nontumor kidney tissue were normal-ized against their respective actin levels using a densitometrysoftware program (Scion Imaging for Windows). The normal-ized FADD levels in RCCs were then compared to the nor-malized FADD expressions in the respective nontumor kidneytissues to derive changes (in fold, normal kidney tissue/RCC) inFADD expression. The results are summarized in Table 1. Sta-tistical analysis was performed using SPSS 10.0 for Windows.
RESULTS
Expression of FADD in nontumor kidneytissues
To investigate FADD expression in RCC, we collected 85pairs of RCCs, consisting of 62 ccRCCs, 17 pRCCs, and 6 otherRCCs (chromophobe and Sarcomatoid), and their matched non-tumor kidney tissues (Table 1). IHC staining revealed that FADDwas expressed in the proximal and distal tubules (Figure 1, seethe normal kidney portion) with very faint staining occasionallyobserved in glomeruli (Figure 1). FADD localizes largely in thecytosol of kidney tubular epithelial cells (Figure 1). The anti-FADD antibody detected FADD, as control IgG did not produceany detectable signal (data not shown). There was some vari-ation in the levels of FADD staining from case to case. It isunlikely that this resulted from variations associated with theIHC procedure, as variations were observed for slides stainedin the same batches. Furthermore, in a given patient, the levels
Reduction of FADD in ccRCC 837
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Table 1. FADD expression in RCC patients
Patients Age Gender Tumor type Fuhrman Sizea FADD in kidneyb FADD in tumorb Ratio N/Tc
1 54 M ccRCCg 1 15.75 0.811908 0.689609 1.182 70 F ccRCCf 1 1 21.00 0.617194 0.854289 0.723 49 F ccRCCf 2 1 18.00 1.046587 1.281364 0.824 51 M ccRCC 1 25.25 1.516540 0.292890 5.185 54 F ccRCC 1 8.93a 1.344919 0.290072 4.646 41 F ccRCC 1 46.50 1.382048 0.844890 1.647 50 M ccRCC 1 3.83 0.57960 0.482317 1.208 45 F ccRCC 1 7.70 0.482173 0.187852 2.579 48 M ccRCC 1 62.50 0.904558 0.111619 8.1010 53 F ccRCC 1 31.50 0.917125 0.259693 3.5311 61 M ccRCCf 3 1 61.88 0.484855 0.946444 0.5112 46 M ccRCC 2 18.38 0.735997 0.627995 1.1713 38 M ccRCC 2 9.43 1.565692 1.151232 1.3614 77 M ccRCC 2 126.75 1.539861 1.244258 1.2415 56 M ccRCC 2 33.00 0.988612 0.492325 2.0116 50 M ccRCCf 4 2 45.56 0.430385 0.68437 0.6317 63 M ccRCC 2 280.31 1.435316 1.023426 1.4018 71 M ccRCC 2 36.00 1.660704 0.976358 1.7019 75 M ccRCCf 5 2 N/A 0.221988 0.362244 0.6120 58 M ccRCC 2 3.00 1.457779 0.915065 1.5921 58 M ccRCC 2 45.00 1.408431 1.236178 1.1422 58 M ccRCC 2 43.20 0.591818 0.348827 1.7023 77 F ccRCC 2 49.50 0.335103 0.351664 0.9524 76 M ccRCCf 6 2 85.25 2.644654 3.567708 0.7425 59 M ccRCC 2 68.75 0.76467 0.622122 1.2326 49 F ccRCC 2 60.02 0.700510 0.013300 52.6727 43 M ccRCC 2 13.50 1.856262 0.503691 3.6928 59 M ccRCCf 7 2 264.00 0.912056 1.802439 0.5129 83 F ccRCCf 8 2 168.00 0.554688 0.809010 0.6930 N/Ad F ccRCC 2 4.00 1.228290 0.062795 19.5631 91 F ccRCCf 9 2 8.44 0.353174 0.614488 0.5732 72 F ccRCC 2 15.75 0.842530 0.198882 4.2433 75 M ccRCC 2 48.00 2.069292 0.504079 4.1134 74 M ccRCCf 10 2 10.94 0.205122 0.332157 0.6235 62 M ccRCC 2 N/A 0.778849 0.190024 4.1036 54 M ccRCC 3 68.75 1.218416 0.830965 1.4737 60 F ccRCCf 11 3 143.00 0.799383 1.333333 0.6038 62 F ccRCC 3 600.00 1.380842 0.714403 1.9339 48 M ccRCC 3 33.00 1.743647 0.867812 2.0140 75 F ccRCCf 12 3 54.45 0.489540 1.057260 0.4641 63 M ccRCC 3 36.00 1.230901 0.805460 1.5342 55 M ccRCC 3 147.00 0.427344 0.394031 1.0843 63 M ccRCC 3 8.75 1.121590 0.488868 2.2944 62 F ccRCC 3 9.38 1.167044 0.795136 1.4745 48 M ccRCCf 13 3 519.75 0.574992 2.037322 0.2846 45 M ccRCC 3 23.63 0.46749 0.090817 5.1547 68 M ccRCC 3 74.25 1.259496 0.718862 1.7548 57 M ccRCC 3 222.75 0.807059 0.691665 1.1749 74 F ccRCC 3 43.31 1.069811 0.120795 8.8650 46 M ccRCC 3 120.00 1.033512 0.997440 1.0451 35 M ccRCC 3 160.00 0.823404 0.439706 1.8752 68 M ccRCCf 14 3 36.45 0.664873 1.116983 0.6053 71 F ccRCC 3 196.00 0.714580 0.528879 1.3554 56 F ccRCCf 15 3 13.50 0.603599 0.715296 0.8455 71 M ccRCCf 16 3 406.25 0.55048 1.110570 0.5056 N/A F ccRCC 3 13.50 0.747701 0.186011 4.0257 62 M ccRCC 3 12.25 1.340639 0.79615 1.6858 52 M ccRCC 3 18.00 0.859520 0.262523 3.2759 65 M ccRCC 4 132.00 1.626384 1.141673 1.4260 54 F ccRCC 4 N/A 1.351308 1.030947 1.3161 55 M ccRCC 4 468.00 1.087440 0.147521 7.37
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Table 1. FADD expression in RCC patients (Continued)
Patients Age Gender Tumor type Fuhrman Sizea FADD in kidneyb FADD in tumorb Ratio N/Tc
62 N/A N/A ccRCCf 17 N/A N/A 0.641837 1.008658 0.6463 56 M pRCC 2 135.00 0.749022 1.416058 0.5364 59 F pRCC 2 1.76 1.307180 1.22139 1.0765 57 M pRCC 3 48.00 1.440499 1.443498 1.0066 58 F pRCC 3 12.50 1.129196 0.865481 1.3067 56 M pRCC 1 18.38 1.056561 0.840031 1.2668 68 M pRCC 3 15.75 1.058377 0.379668 2.7969 76 F pRCC 2 135.00 0.516938 0.85798 0.6070 64 M pRCC 3 6.40 0.452739 0.035901 12.6171 54 M pRCC 3 29.16 0.533865 0.774859 0.6972 74 M pRCC 2 11.40 0.659463 1.050666 0.6373 66 M pRCC 2 9.00 0.348974 1.123551 0.3174 62 F pRCC 2 11.25 1.283757 0.78487 1.6475 59 M pRCC 3 3.89 1.008619 0.321475 3.1476 N/A F pRCC N/A N/A 0.462544 0.593796 0.7877 68 F pRCC N/A N/A 0.179510 0.063258 2.8478 72 M pRCC 2 108.00 1.351564 2.875968 0.4779 42 M pRCC 3 60.75 1.635751 0.229260 7.1380 74 M othere N/A 31.35 1.441819 1.13391 1.2781 81 M other 3 N/A 0.57296 0.665227 0.8682 55 M other 3 238.00 0.524639 0.495360 1.0683 71 F other 1 27.00 0.889320 0.846540 1.0584 54 F other N/A N/A 0.5994036 0.025449 23.5585 83 F other N/A N/A 3.623511 0.092719 39.08
aTumor size (cm) in maximal diametersb FADD expression was normalized against actinc Normal kidney versus tumord N/A: not availablee other RCC: chromophobe, Sarcomatoidf 1−f 17 Ratios of N/T for these ccRCCs < 0.9
of FADD staining were consistent throughout the entire sec-tion as well as in replicate experiments. Western blot analysisconfirmed these variations in nontumor tissues (Figure 2(a),comparing the FADD expression in the nontumor tissues be-tween patients 56 and 31). It is likely that these variations maybe associated with each patient’s individual clinical characteris-tics, such as age, previous medication, or the presence of otherkidney complications.
Reduction of FADD in ccRCC
To investigate FADD expression in RCC, we examined thelevels of the FADD protein in the same pair of tumor andmatched nontumor tissues by IHC staining and by Western blot.IHC staining of normal kidney tissue and the respective RCCtissue (both tissues were mounted on the same slide) detectedccRCCs and pRCCs that express either reduced or nonreducedFADD when compared to the respective normal kidney tissues(Figure 1). In both ccRCC and pRCC, FADD largely stays inthe cytosol (Figure 1). To quantify FADD expression in nor-mal kidney and in RCC tissues, we performed Western blotanalysis of FADD for 85 pairs of normal kidney tumor tis-sues (Figure 2). Since the cellular content or cellularity differsin RCC and in nontumor kidney tissue, we normalized FADDexpression against actin, an intracellular protein. In compari-
son to the respective normal kidney tissues, 67.7% (42/62) ofccRCCs express reduced FADD, as their ratios of FADD (nor-mal vs. tumor) are larger than onefold (Figure 3(a), Table 1),while 32.2% (20/62) of ccRCCs express FADD at levels thatare not lower than those of normal kidney tissues (Figure 3(a),Table 1), suggesting that FADD is downregulated in ccRCC (seethe “Discussion” section for details).
To confirm this suggestion, we determined whether the lowlevels of FADD observed in ccRCC are statistically significant.Paired t-test revealed that ccRCC expresses significantly lowerlevels of FADD than those expressed in normal kidney tissues(p = .001) (Table 2). This is not due to the two patients (patients26 and 30, Table 1) whose ratios (normal kidney vs. RCC) ofFADD expression are substantially higher than those of the
Table 2. Significant reduction of FADD in ccRCC
Tumor number Mean/ Mean/type of cases normala tumora p value
ccRCC 62 0.9866 0.7307 0.001pRCC 17 0.8926 0.8752 0.916
a FADD expression in normal and tumor tissues wasnormalized against the respective action expression.
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Figure 1. Expression of FADD in RCC. ccRCC and pRCC and the matched nontumor tissue were cut and mounted on the same slide followedby H&E staining (data not shown) and IHC detection of FADD. Scale bar represents 60 µm. Insets show enlarged cells in the marked regions.
Figure 2. Western blot analysis of FADD in RCC and matchednontumor tissues. Tissue lysates were prepared for all 85 pairsof normal kidney and RCC tissues as described in the “Materialsand Methods” section, followed by Western blot analysis for FADDand actin. Representative Western blot results for the indicatedpatients are shown. N: normal kidney tissue; C: cancer.
general population, as removing these two patients does notsignificantly change the level of significance (p = .002) (datanot shown). Taken together, the above observations demonstratethat ccRCC expresses reduced FADD compared to the respectivenormal kidney tissues.
No differences in FADD expression duringccRCC progression and in pRCC
We further examined whether reduction of FADD expressioncorrelates with the disease progression. Pearson’s ϕ coefficientwas used to test potential correlations between changes in FADDexpression, using the ratios of FADD (normal kidney vs. RCC)(Table 1), and Fuhrman grades or tumor sizes. We did not detectsignificant correlation between changes in FADD and Fuhrmangrades (p = .633) or tumor sizes (p = .592). This indicates thatreduction of FADD expression plays a role in ccRCC initiationbut not disease progression. Alternatively, our sample size maynot be sufficient to detect any potential correlation (see the“Discussion” section for details).
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Figure 3. FADD expression in ccRCC and pRCC. Levels of FADD expression in normal kidney and RCC were normalized against the levelsof respective actin. The ratios of FADD expression (Normal/RCC) were determined and their distribution was graphed. The onefold line showsan equal expression of FADD between RCC and the matched nontumor kidney tissue. Data points above or below the line represent RCCsthat express decreased or increased FADD, respectively. (a) All ccRCC data present in Table 1, excluding patients 26 and 30, are graphed; (b)pRCC.
We were also unable to detect whether FADD expressionchanges significantly in pRCC (Figure 3(b), Table 2). This maybe attributable to the small sample size. The very small samplesize also limited our examination of changes in FADD expres-sion in other RCCs (Table 1).
DISCUSSION
Apoptosis is a major mechanism of tumor suppression. Whileevasion of apoptosis promotes tumorigenesis (3), restoration ofapoptosis or, more specifically, induction of apoptosis is themain strategy of current cancer therapy. It is thus not sur-
prising that most therapeutic agents currently used in cancertherapy induce apoptosis through either DR-mediated (extrin-sic) or mitochondria-mediated (intrinsic) apoptotic pathways(19). Failure to undergo apoptosis may thus be attributable totreatment-resistant tumors. As RCC is highly resistant to a va-riety of therapies, RCC may contain defections in executingapoptosis. We provide evidence suggesting that one of these po-tential defections is attenuation of FADD-mediated apoptosis.
FADD is the central feature in extrinsic apoptosis (20) andalso plays a role in intrinsic apoptosis (24). TRAIL-initiatedextrinsic apoptosis has been heavily investigated as a ther-apeutic option to kill cancer cells (22). Interestingly, both
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apoptosis-inducing TRAIL receptors, DR4 and DR5, directlybind to FADD to initiate apoptosis (25). However, whetherchanges in FADD may impact on tumorigenesis may dependon the tissue or cancer type. FADD also plays important rolesin cell proliferation. FADD is required for T-cell developmentand activation (26). FADD is phosphorylated on Ser194 at G2/Mphase (27), which plays a role in activation of G2/M checkpoints(28–30). Accumulating evidence suggests that the distributionof FADD in different cellular compartments may contribute toits apoptotic and nonapoptotic functions. While cytosolic FADDis recruited to DR on the plasma membrane to induce apoptosis,phosphorylation of Ser194 results in FADD’s nuclear localiza-tion, which contributes to FADD’s nonapoptotic functions (31).Consistent with the concept that Ser194-phosphorylated FADD(p-FADD) is associated with nonapoptotic functions, p-FADDhas been reported to associate with poor outcome in lung ade-nocarcinomas (32).
In kidney and ccRCC, FADD distributes predominantly inthe cytosol and nuclear FADD was not convincingly observedin ccRCC (Figure 1), suggesting that FADD largely functions inDR-induced apoptosis in kidney and ccRCC. This is consistentwith our observed reduction of FADD in ccRCCs in compar-ison to the respective nontumor kidney tissues. Proteins maybe heterogeneously expressed in ccRCCs (Figure 2, comparingFADD expression in ccRCCs in patients 56 and 30–32) (see Ta-ble 1). In comparison to the matched nontumor kidney tissues,while 27.4% (17/62) ccRCCs showed enhanced FADD expres-sion (ratios of N/T < 0.9, Table 1), 67.7% (42/62) ccRCCsdisplayed reduced FADD (ratios of N/T > 1.1, Table 1). Fur-thermore, ccRCCs as a group (including the 27.4% and 67.7%subgroups) show statistically significant reduction of FADD ex-pression, when compared to the respective nontumor tissues.Taken together, we demonstrated that FADD expression wasreduced in the majority (67.7%) of ccRCCs. However, our ob-servation does not exclude the possibility that for the minorpopulation (27.4%) of ccRCCs, enhanced FADD may facili-tate ccRCC tumorigenesis. Alternatively, variations in FADDexpression in these ccRCCs may simply result from the hetero-geneity of protein expression. Evidence supporting this possi-bility is the heterogeneous expression of FADD in the nontumorkidney tissues of ccRCC patients (see the “Results”section fordetails). Additional studies will be required to distinguish thesetwo possibilities using a larger patient population with ccRCCdemonstrating elevated FADD expression.
Reduction of FADD in ccRCC might be an important mech-anism for ccRCC to evade apoptosis. This is consistent withour observations showing that FADD expresses in the epithelialcells of proximal and distal tubules (Figure 1), and that FADDexpression is reduced in ccRCC (Table 1). Interestingly, ccRCCoriginates from the proximal tubular epithelium (33), whichprovides further support for the notion that FADD may func-tion to suppress ccRCC tumorigenesis. Although we detectedpredominant cytosolic FADD in pRCC and other types of RCC,we are unable to detect significant changes in FADD expressionin these RCCs in comparison to their respective normal kidneytissues due to the very limited sample size.
While the potential mechanisms leading to reduction ofFADD in ccRCCs are currently unclear, reducing FADD genetranscription and enhancing FADD protein degradation are po-tential mechanisms governing the reduction of FADD expres-sion in ccRCC. Regardless of what exactly these mechanismsare, we provide the first evidence showing the reduction of theFADD protein in ccRCC.
ACKNOWLEDGMENTS
The authors would like to thank the Department of Pathologyof St. Joseph’s Hospital at Hamilton, Ontario, Canada, for theirassistance in diagnosis and preparation of slides. This researchis supported in part by The Kidney Foundation of Canada (Grantno. 2000HO3867 to D.T.), National Cancer Institute of Canada.
CONFLICT OF INTEREST/DISCLOSURE
None.
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