JAK kinases promote invasiveness in VHL-mediated renal cell carcinoma bya suppressor of cytokine signaling-regulated, HIF-independent mechanism
Karen L. Wu, Hui Miao, and Shenaz KhanDepartment of Medicine, Case Western Reserve University, School of Medicine, MetroHealth Campus, Rammelkamp Centerfor Research, Cleveland, Ohio
Submitted 26 February 2007; accepted in final form 24 September 2007
Wu KL, Miao H, Khan S. JAK kinases promote invasivenessin VHL-mediated renal cell carcinoma by a suppressor of cytokinesignaling-regulated, HIF-independent mechanism. Am J PhysiolRenal Physiol 293: F1836–F1846, 2007. First published September26, 2007; doi:10.1152/ajprenal.00096.2007.—von Hippel-Lindau(VHL) disease is a cancer syndrome, which includes renal cellcarcinoma (RCC), and is caused by VHL mutations. Most, but not allVHL phenotypes are due to failure of mutant VHL to regulateconstitutive proteolysis of hypoxia-inducible factors (HIFs). Januskinases (JAK1, 2, 3, and TYK2) promote cell survival and prolifer-ation, processes tightly controlled by SOCS proteins, which havesequence and structural homology to VHL. We hypothesized that inVHL disease, RCC pathogenesis results from enhanced SOCS1 deg-radation, leading to upregulated JAK activity. We find that baselineJAK2, JAK3, and TYK2 activities are increased in RCC cell lines,even after serum deprivation or coincubation with cytokine inhibitors.Furthermore, JAK activity is sustained in RCC stably expressingHIF2� shRNA. Invasion through Matrigel and migration in wound-healing assays, in vitro correlates of metastasis, are significantlygreater in VHL mutant RCC compared with wild-type cells, andblocked by dominant-negative JAK expression or JAK inhibitors.Finally, we observe enhanced SOCS2/SOCS1 coprecipitation andreduced SOCS1 expression due to proteasomal degradation in VHL-null RCC compared with wild-type cells. The data support a newHIF-independent mechanism of RCC metastasis, whereby SOCS2recruits SOCS1 for ubiquitination and proteasome degradation, whichlead to unrestricted JAK-dependent RCC invasion. In addition tocommonly proposed RCC treatment strategies that target HIFs, ourdata suggest that JAK inhibition represents an alternative therapeuticapproach.
Janus kinase; suppressor of cytokine signaling; von Hippel-Lindausyndrome
VON HIPPEL-LINDAU (VHL) disease is a heritable multisystemcancer syndrome caused by germline mutations in VHL, whichis located on chromosome 3p25 (30). The main causes of deathare complications linked to highly angiogenic renal carcinomasand hemangioblastomas within the central nervous system (45,59). The VHL gene is ubiquitously expressed (60) and the roleof the VHL gene product (pVHL) in renal cell carcinoma(RCC) has been extensively studied (31, 49). There are cur-rently no established, successful therapies, e.g., radiation orchemotherapy, for RCC other than nephrectomy in situationswith no evidence of metastases.
pVHL contains an �100-residue NH2 terminus (�-domain)and a smaller COOH-terminal �-helix (�-domain). Undernormoxic conditions, the pVHL �-domain interacts with
�-subunits of hypoxia-inducible factors (HIF)-1 and -2 tran-scription factors (48, 60), while the VHL �-domain and a smallportion of the �-domain interact with Elongin C, a componentof an E3 ubiquitin ligase complex that targets HIF-1� subunitsfor proteasomal degradation (31, 43, 64) and thereby preventsHIF transcription factor binding to target genes. Mutations ineither VHL �- or �-domains disrupt HIF degradation, whichcontributes to tumorigenesis by permitting transactivation ofHIF target genes, including vascular endothelial growth factor(VEGF), platelet-derived growth factor (PDGF), transforminggrowth factor (TGF)-�, erythropoietin, epidermal growth fac-tor (EGF) receptor, Glut1 glucose transporter, and carbonicanhydrase IX (CAIX), with the net effect of upregulatedangiogenesis and/or cell proliferation (22, 23, 44, 62). Inaddition to HIFs, pVHL also targets other proteins for degra-dation (3, 6, 20, 53–55, 57, 58), suggesting that the RCCphenotype may not be regulated entirely by HIF-dependentpathways.
The Janus kinase (JAK)-signal transducer and activator oftranscription (STAT) pathway contributes to tumorigenesisthrough regulation of growth factor activity, apoptosis, andangiogenesis. Constitutive activation of STAT1, STAT3, andSTAT5 has been implicated in many primary cancers, includ-ing RCC (8). Stimulation of cytokine and growth factor recep-tors leads to direct receptor-JAK interaction, and receptortyrosine kinase phosphorylation of JAKs. Phosphorylated/ac-tivated JAKs then recruit and phosphorylate STAT proteins,which promote dimerization. STAT homo- or heterodimers arerapidly transported from the cytoplasm to the nucleus and arecompetent for DNA binding (1).
JAK activity is modulated by a negative feedback loop,wherein STATs transcriptionally upregulate suppressor of cy-tokine signaling (SOCS) expression (11). SOCS then modu-lates JAK activity by multiple mechanisms. All SOCS iso-forms (SOCS 1-7 and CIS) contain a central SH2 domain,which binds and inhibits the tyrosine-phosphorylated JAKcatalytic domain (11). In addition, SOCS1 has an NH2-terminal JAK pseudosubstrate domain, which inhibits JAKactivity by blocking JAK access to true substrates (50).Finally, SOCS proteins bind the Elongin C component of theubiquitin ligase complex, which stabilizes SOCS, and leadsto proteasomal degradation of SOCS-bound proteins, in-cluding JAKs. A recent report from Tannahill et al. (67)demonstrates that SOCS3 and SOCS2 heterodimerize, andbinding of the complex to Elongin C led to SOCS2 stabili-zation and SOCS3 degradation.
Address for reprint requests and other correspondence: K. L. Wu, CaseWestern Reserve Univ., School of Medicine, Dept. of Nutrition, ResearchTower, RT600, 2109 Adelbert Rd., Cleveland, OH 44106 (e-mail: [email protected]).
The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Renal Physiol 293: F1836–F1846, 2007.First published September 26, 2007; doi:10.1152/ajprenal.00096.2007.
In this study, we show that VHL mutant RCC cell linesexhibit constitutive, HIF-independent JAK-STAT pathway ac-tivation, which regulates RCC invasion. We also find that JAKactivity is increased by a mechanism involving SOCS2/SOCS1association, SOCS1 ubiquitination, and proteasomal degrada-tion, resulting in diminished steady-state SOCS1 expression.The data suggest that RCC therapy directed toward JAK-STATabrogation may synergize with HIF inhibition strategies.
MATERIALS AND METHODS
Cell lines. 786-0 RCC cells were transfected with either emptyvector (RC3) or wild-type VHL (WT8) (gifts from Dr. B. Kaelin).RCC4 cells (VHLSer65Trp) (32) were transfected with wild-typeVHL (WTVHL) or an inactivating point mutant (L188V) (gifts fromDr. E. Maher). Cells were cultured in DMEM medium (Invitrogen)supplemented with 10% FBS and 0.5 g/l G418 in humidified incubatorwith 5% CO2 at 37°C. Stably transfected 786-0 cells that overexpressshort-hairpin siRNA (shRNA) targeting HIF2� or empty vector weregifts from Dr. O. Iliopoulos. MCF7 cells were from ATCC andcultured in 10% FBS containing DMEM in humidified incubator with5% CO2 at 37°C.
Reagents. Anti-phospho-JAK2, anti-phospho-TYK2, and anti-phosphotyrosine-STAT3 antibodies were obtained from Cell Signal-ing (Danvers, MA). Anti-JAK2, anti-JAK3, anti-TYK2, anti-SOCS1,anti-ubiquitin, and anti-phosphotyrosine were from Santa Cruz Bio-technology (Santa Cruz, CA). Anti-SOCS2 antibody was purchasedfrom Abcam (Cambridge, MA). Horseradish peroxidase-conjugatedanti-rabbit and anti-mouse antibodies were products from AmershamBiosciences (Piscataway, NJ). Anti-�-tubulin, anti-FLAG M2 IgG,cycloheximide, crystal violet, and IGF-1 were from Sigma (St. Louis,MO). Anti-HIF1� was from NeoMarkers (Fremont, CA). AG490,JAK inhibitor I, JAK3 inhibitor V, AG9, anti-TGF-�, Tranilast,AG1296, PD153035, lactacystin, MG132, and PSI were purchasedfrom Calbiochem (San Diego, CA). TUNEL Assay Kit was fromChemicon. [35S]Methionine was from ICN. Matrigel was purchasedfrom BD Biosciences.
Plasmids and transient transfection. Tyrosine kinase domain-de-leted dominant-negative TYK2 construct, �TK-TYK2, was a kind giftfrom Dr. S. Pellegrini (69). JAK2 inactivating point mutants, K882Eand Y1007F, were kind gifts from Dr. O. Silvennoinen (68). Plasmidswere transformed into Top 10 competent bacteria cells according tothe manufacturer’s protocol (Invitrogen, Carlsbad, CA), extractedusing a Maxiprep kit (Qiagen, Valencia, CA), and amplified by culturein Luria-Bertani-ampicillin broth. cDNAs were transiently transfectedinto cells according to previously described methods (73). Briefly,cells were plated into six-well plates (0.25 � 106 cells/well) andcultured overnight in complete medium. The transient mixture, whichcontained 1.0 �g of plasmid DNA and 6 �l of Fugene 6 transfectionreagent (Roche Diagnostics, Indianapolis, IN) in 100 �l of serum-freeDMEM medium (Invitrogen), was mixed for 20 min at room temper-ature and then added to each well with complete medium for 48 h.
Scratch-wound assays. Wounding assays were performed as de-scribed previously (15). Briefly, confluent WT8 and RC3 cells platedin 24-well gridded plates were incubated in serum-free DMEMmedium 1 h before wounding. Cells’ monolayer was scratch-woundedwith a P200 pipette tip. Cells were then incubated with or withoutJAK inhibitors AG490 (100 �M), P131 (300 �M), JAK3I-V (50 �M),AG9 (100 �M), or JAKI-1 (500 �M). Migration was determined atidentical locations by phase-contrast microscopy (�10 magnification)5 and 15 h after incubation. Representative images were photo-graphed.
Immunoprecipitation and immunoblot analyses. The methods havepreviously been described in detail (73). Cells were lysed in RIPAbuffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 0.5 mMdeoxycholate, 0.1% SDS, 1% Triton X-100) containing protease
inhibitor cocktail (Roche Diagnostics, Mannheim, Germany) for 1 h at4°C. The lysates were centrifuged at 12,000 rpm for 30 min at 4°C.Proteins in the supernatant were assayed for protein content (Bio-Rad)and 500 �g of protein were immunoprecipitated with appropriateantibodies (1 �g/each, 4°C, overnight). The samples were incubatedwith �-bind protein G-Sepharose beads (Amersham; 4°C, 1 h) andthen washed in RIPA buffer. Immunoprecipitates were dissolved in 40�l of 2� SDS sample buffer (125 mM Tris, pH 6.8, 2% SDS, 5%glycerol, 1% �-mercaptoethanol, and 0.003% bromophenol blue) andthen evaluated by immunoblotting according to the following meth-ods. Whole cell lysates or immunoprecipitates in 2� SDS samplebuffer were denatured by boiling for 5 min, and then samples werefractionated by 6 or 4–20% SDS-PAGE (Invitrogen). The proteinswere transferred to polyvinylidene difluoride membranes, blockedwith 5% nonfat milk (5% BSA for anti-phosphotyrosine, -phospho-JAK2, and -phospho-TYK2 antibodies), and incubated with appropri-ate antibodies (4°C, overnight), followed by horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies (1:10,000, 1 h, roomtemperature). In some experiments, the blots were stripped with BlotRestoration Solutions (Chemicon, Temecula, CA) and reprobed withappropriate antibodies. Protein bands were detected by enhancedchemiluminescence (Amersham Biosciences).
Invasion assay. Matrigel (50 �l at 1:8 dilution in DMEM medium)was coated on the top of Transwell filters (6.5-mm diameter, 8.0-�mpore size, Costar, Corning, NY) for 2 h at 37°C. Chemoattractant (0.5�M IGF-1) was added to bottom chambers of transwells. Cells(6.25 � 104) were plated on Matrigel in DMEM and allowed tomigrate overnight at 37°C through Matrigel. Migrated cells were fixedin methanol (100%, 20 min, room temperature) and stained withcrystal violet (0.5 in 20% methanol, 30 min, room temperature).Matrigel and cells, which did not migrate, were gently removed fromthe upper chamber with Q-tips. Cells migrating through Matrigel tothe lower chamber were counted under a dissecting microscope fromthree to six random fields.
TUNEL assay. TUNEL assay was performed according to themanufacturer’s instruction, as described previously (74). WT8 andRC3 cells were cotransfected with GFP and dominant-negative TYK2and JAK2 constructs. TUNEL-positive cells within the GFP-express-ing population were counted to assess apoptosis.
Protein degradation assay [35S]-labeled pulse chase. Methodshave previously been described in detail (74). Cells were cultured tosubconfluence, washed in PBS, and incubated with [35S]methionine inmethionine-free DMEM (0.1 mCi/ml, 2 h, 37°C). Cells were washedin PBS and chased in methionine-containing DMEM for up to 16 h.Protein lysates (400 �g per sample) were immunoprecipitated withanti-SOCS1 (1 �g, overnight) antibody and resolved by SDS-PAGE.Autoradiograms were developed from dried gels. Individual bandswere digitized by phosphoimager (Molecular Dynamics), quantifiedwith Image Quant 5 software (Molecular Dynamics), and normalizedto control values.
As an alternative to [35S]-labeled pulse chase, protein degraded wasalso assessed by cycloheximide-based assay. Cells were plated insix-well dishes (0.25 � 106 cells/well) and cultured overnight. Thefollowing day, cells were treated with protein synthesis inhibitor,cycloheximide (20 �g/ml in serum-free medium for up to 16 h).Whole cell lysates were resolved by SDS-PAGE and probed withanti-SOCS1 antibodies.
Statistics. Data are representative of three to five experiments percondition, and unless otherwise noted in figure legends, expressed asmeans � SE. Statistical significance between two groups was assessedby Student’s t-test and by one-way ANOVA for all other comparisons.
RESULTS
Increased JAK activities in RCC. JAK-STAT pathways havebeen implicated in carcinogenesis, including in RCC (8). Initialimmunoblots demonstrated JAK2, JAK3, and TYK2 expres-
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sion in RC3 and RCC4 cell lines, whereas JAK1 expressionwas not detected in either RCC cell line (data not shown).JAK2 activity was investigated in RCC4, wild-type, andL188V cell lines by immunoblot analysis with anti-phospho-JAK2 antibodies. Figure 1A demonstrates increased JAK2activity in RCC4 or L188V compared with wild-type cells. Toassess JAK3 activity in RCC, cell lysates were immunopre-cipitated with anti-phosphotyrosine antibodies and then probedwith anti-JAK3 antibodies. The data show robust JAK3 ty-rosine phosphorylation in VHL mutant (RCC4, L188V, andRC3) cells compared with wild-type controls (Fig. 1B). Fur-thermore, TYK2 activation was also assessed in both sets ofVHL cell lines by immunoprecipitation and immunoblottingwith anti-phosphotyrosine antibodies. As shown in Fig. 1C,significant TYK2 tyrosine phosphorylation was observed inVHL mutant RCC cells compared with wild-type cells.
To confirm these results, WT8 and RC3 cells were treatedwith the specific JAK2 inhibitor AG490 (50 �M, 72 h) andlysed whole cell lysates were immunoblotted for expression ofphospho-STAT3, the downstream effector for JAK2. Figure1D shows that RC3 cells contained increased tyrosine phos-
phorylated STAT3 levels, which was abolished by AG490,indicating that JAK2 is functionally active in RCC cells. Basalphospho-STAT3 was unaffected by AG490 in wild-type cells,suggesting that STAT3 is phosphorylated by a kinase otherthan JAK2 in the basal state. Taken together, JAK2, JAK3, andTYK2 are activated in two different RCC cell lines.
Increased JAK activity is independent of HIF�. JAK acti-vation in the cell line expressing the L188V VHL mutation wassurprising because L188V is associated with VHL syndromesthat primarily include pheochromocytoma, rather than RCC. Insome instances, L188V overexpression has been shown tosuppress RCC growth (27), suggesting that JAKs mediate RCCphenotypes other than proliferation. Since the L188V VHLmutant retains the ability to bind HIF� subunits and Elongin C,and downregulate HIF activity (12, 27), the data suggest thatJAK activation may be HIF-independent in RCC. To test theeffect of HIF on JAK activity, we examined RC3 cells thatstably express short-hairpin siRNA (shRNA) targeting HIF2�.Figure 1E demonstrates that HIF2� shRNA expression did notinhibit JAK2 activation, suggesting that JAK activity in RCCcells is indeed independent of HIF� regulation. To confirm that
Fig. 1. Renal cell carcinoma (RCC) JAK activity is increased by a hypoxia-inducible factor (HIF)-independent mechanism. A: RCC4, wild-type, and L188V vonHippel-Lindau (VHL) mutant cell lines were maintained in serum-free media overnight, to avoid cytokine stimulation. Top: whole cell lysates were then probedwith anti-phospho-JAK2 antibodies by immunoblot analysis. Bottom: blot was stripped and reprobed for JAK2 expression and protein loading with anti-JAK2antibodies. B: depicted cells were maintained in serum-free media overnight. Cell lysates were immunoprecipitated with anti-phosphotyrosine antibody (�-PY)and probed with anti-JAK3 antibodies. Bottom: parallel lysates were immunoblotted with anti-JAK3 antibodies. C: cell lines were maintained in serum-free mediaovernight. Top: whole cell lysates were then immunoprecipitated with anti-TYK2 antibodies and probed with �-PY. Bottom: stripped blot was reprobedanti-TYK2 antibody as a loading control. D: WT8 and RC3 cells were incubated with JAK2 inhibitor AG490 (50 �M, 72 h). Top: cell lysates were immunoblottedwith anti-phospho-STAT3 antibodies. Middle: parallel lysates were immunoblotted with anti-STAT3 antibodies. Bottom: blot was stripped and reprobed withanti-�-tubulin antibodies as a protein loading control. E: RC3 cells were stably transfected with either mock vector or shRNA construct targeted to HIF-2�. Bothgroups were maintained in serum-free media overnight. Top: whole cell lysates were immunoblotted with anti-phospho-JAK2 antibodies. Panel 2: blots werestripped and reprobed for JAK2 expression. Panel 3: to verify efficacy of shRNA knockdown, lysates were probed for HIF-2� expression by immunoblotanalysis. Bottom: blot was stripped and reprobed with anti-�-tubulin antibodies as a loading control. F, top: RC3 and MCF7 cell lysates were immunoblottedwith anti-HIF1� antibodies. Bottom: blot was stripped and reprobed with anti-tubulin antibodies.
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HIF1� isoform is not expressed RC3 cells (48, 77), we exam-ined HIF1� protein expression levels in RC3 cells and acontrol cell line, MCF7 cells. RC3 cell lysates did not displayHIF1� protein expression, whereas MCF7 cells did (Fig. 1F,top). The stripped blot was reprobed with anti-tubulin antibod-ies as a loading control (Fig. 1F, bottom).
JAK activities are constitutive and cytokine-independent inRCC. In most circumstances, JAKs are activated followingligation with cytokine receptors. However, recent studies dem-onstrate that in some cell lines, JAKs can be constitutivelyactivated (40). To determine cytokine dependence for RCCJAK activities, cytokines were depleted by incubating WT8and RC3 cells in serum-free media for up to 24 h. WT8 cellsdisplayed progressively decreased phospho-JAK2 levels after6- and 24-h incubation in serum-free media. RC3 cells dem-onstrated relatively sustained phospho-JAK2 levels after 6- and24-h depletion of serum, although some decreased activity hasbeen shown (Fig. 2A). Cytokine depletion resulted in markedlydecreased phospho-TYK2 content in WT8 cells after 6 h,which was almost undetectable by 24 h (Fig. 2A). However,RC3 cells displayed sustained TYK2 activity even after 24 h ofserum starvation. These results suggest that JAK2 and TYK2activation in RCC cells is cytokine-independent.
To more directly address this issue, cells were cultured in thepresence and absence of PD153035 (EGFR inhibitor), AG1296(PDGF inhibitor), or Tranilast (VEGF inhibitor) for 48 h, andJAK2 and TYK2 activities were determined by immunoblot-ting with anti-phospho-JAK2 antibodies. Exposure to cytokineinhibitors diminished phospho-JAK2 and phospho-TYK2 lev-els in WT8 cells, whereas RC3 cells showed sustained JAK2and TYK2 phosphorylation (Fig. 2B), indicating that JAK2 andTYK2 are constitutively activated in RCC cells in a cytokine-independent fashion.
Increased JAK activity promotes RCC invasion. To test JAKregulation of pathophysiologically relevant phenotypes, wescreened RC3 and RCC4 cells for altered proliferation, apo-ptosis, and fibronectin secretion. Of these assays, only fi-bronectin secretion was abnormal, consistent with prior reports(53). However, JAK inhibition had no effect on secretion offibronectin by RCC or wild-type cell lines (data not shown).
Metastatic potential was measured by assaying RC3 andWT8 invasion through Matrigel (7, 14). To test JAK regulationof invasion, cells were preincubated with pan-JAK inhibitor(JAKI-1), JAK2-specific inhibitor (AG490), JAK3 inhibitor(JAK3I-V), and TYK 2 inhibitor (AG9). Figure 3A demon-strates three- to fourfold increases in invasion of RC3 com-pared with WT8 cells, which were diminished by JAKinhibitors. The greatest inhibition of RC3 invasion wasobserved with JAKI-1 and AG490, indicating that JAK2-dependent signaling pathways may be important in RCCmetastasis in vivo. Suggestive, but statistically insignificant,effects were observed with JAK3I-V and AG9. None of theJAK inhibitors significantly altered invasion in WT8 cells (datanot shown). The activity of downstream effector of JAKs,STAT3, was evaluated by testing tyrosine phosphorylation ofparallel cell lysates and was diminished by all JAK inhibitors.The greatest inhibition of STAT3 phosphorylation was foundwith JAKI-1 and AG490, which is consistent with invasionassay data (Fig. 3A, bottom).
Invasion through Matrigel was also assessed in RCC ex-pressing dominant-negative JAK constructs: TYK2 (1-895),also referred to as TYK2 (�TK), which has a tyrosine kinasedomain deletion, as well as two JAK2-inactivating point mu-tants (K882E and Y1007F). Figure 3B shows that enhancedRC3 cell invasion was abrogated by expression of dominant-negative TYK2. The parallel cell lysates were immunoblotted
Fig. 2. JAK activities are constitutive and cytokine independent in RCC. A: WT8 and RC3 cells were cultured in serum-free DMEM media overnight, and thenfor additional time periods, as shown. Lysates were immunoblotted with either anti-phospho-JAK2 (panel 1) or anti-phospho-TYK2 (panel 3) antibodies. Parallellysates were probed with anti-tubulin antibodies (panel 2). The anti-phospho-TYK2 blot was stripped and reprobed with anti-TYK2 antibodies as a proteinloading control. B: WT8 and RC3 cells were pretreated for 48 h in serum-free media with no additions (control), epidermal growth factor (EGF) receptor inhibitorPD153035 (10 �M), platelet-derived growth factor (PDGF) inhibitor AG1296 (50 �M), or vascular endothelial growth factor (VEGF) inhibitor Tranilast (80�M). Top: whole cell lysates were then immunoblotted with anti-phospho-JAK2 antibodies. To verify equal protein loading, parallel lysates were probed forJAK2 expression by immunoblot analysis (panel 2) and the stripped membrane was reprobed with anti-�-tubulin antibodies (panel 3). Panel 4: similarly,phospho-TYK levels were tested by probing the whole cell lysates with anti-phospho-TYK2 antibodies. Panel 5: blot was stripped and reprobed with anti-tubulinantibodies.
F1839JAK KINASES IN RCC
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for TYK2 tyrosine phosphorylation as an indicator of TYK2activity. The data show abolished TYK2 activity with TYK2dominant-negative construct transfection (Fig. 3B, bottom).Figure 3C demonstrates that expression of catalytically inac-tive JAK2 point mutants also inhibited invasion of RC3, butnot WT8 cells. Inhibition was slightly greater in RC3 cellscotransfected with both JAK2 and TYK2 dominant-negativevectors (Fig. 3C), suggesting an additive effect. As expected,JAK2 activity was reduced dramatically in cells overexpress-ing JAK2 dominant-negative constructs (Fig. 3C, bottom).
To determine whether RC3 invasion could be confounded bycytotoxicity from dominant-negative TYK2 and JAK2 expres-sion, transfected cells were assessed for apoptosis by TUNELassay. These experiments revealed no difference in apoptosisbetween RC3 cells transfected with dominant-negative JAKsor empty vector (data not shown). In summary, RC3, but notWT8, cells demonstrate an invasive phenotype, which is reg-ulated by JAK2 and TYK2.
As an alternative approach to assessing effects of JAKs onRCC metastatic potential, scratch-wound assays were con-ducted in confluent WT8 and RC3 cells in the presence andabsence of JAK inhibitors. In the absence of JAK inhibitors,WT8 cells failed to fill the wound gap after 15 h (Fig. 4, top).However, RC3 cells closed the gap completely in the sametime period, thereby confirming invasion assay results bydemonstrating that VHL mutant RCC cell migrate more exten-sively than wild-type VHL-expressing cells. Moreover, inhibi-tion of JAK2 by AG490 and JAK3 by P131 and JAK3I-V
significantly attenuated RC3 cell migration, whereas inhibitionof TYK2 by AG9 had a more modest effect on RC3 migration(Fig. 4). Taken together, the data from Figs. 3 and 4 indicateJAKs regulate RCC invasion and migration.
SOCS1 undergoes enhanced proteasomal degradation inRCC. A major mechanism of JAK regulation is through down-regulation by SOCS proteins. To initially investigate the pos-sibility of JAK activation through evasion of SOCS-dependentdegradation in RCC, SOCS protein expression was assessed byimmunoblot analysis in RCC and wild-type cells. VHL mutantRCC cells (RCC4, RC3, and L188V) demonstrated signifi-cantly less steady-state SOCS1 expression compared withwild-type, VHL-transfected control cell lines (Fig. 5A, top).
To determine whether decreased SOCS1 expression is due toprotein degradation, SOCS1 half-life was measured in WT8and RC3 cells by [35S]pulse chase methods. Figure 5, B and C,demonstrates that SOCS1 decay is enhanced in RCC cells,consistent with enhanced SOCS1 degradation. As an alterna-tive strategy to verify SOCS1 degradation, WT8 and RC3 cellswere treated with the protein synthesis inhibitor cyclohexi-mide. The rate of decline of SOCS1 expression followingcycloheximide removal was then measured as an index ofSOCS1 degradation. Similar to [35S]pulse chase results, thesestudies demonstrated accelerated degradation of SOCS1 inRC3 cells compared with WT8 cells (Fig. 5D).
It has been suggested that SOCS1 and SOCS3 are degradedthrough the proteasome pathway (50, 76). To determinewhether increased SOCS1 decay is due to proteasomal degra-
Fig. 3. Increased JAK activity promotes RCC invasion through Matrigel matrix. Pretreated WT8 or RC3 cells were plated on 8-�m-pore transwell filtersprecoated with Matrigel (0.5-mm thickness, 1:8 dilution), and allowed to migrate from the lower chamber, through the Matrigel and transwell filter in responseto chemoattractant stimulation (IGF-1, 0.5 �M, 16 h, 37°C) presented in the bottom chamber. Cells were fixed in 100% methanol, stained with crystal violet,and counted under a dissecting microscope. A: cells were treated with chemical inhibitors of JAKs: JAKI-1 (500 nM), AG490 (100 �M), JAK3I-V (50 �M),and AG9 (100 �M). The parallel lysates were immunoblotted with anti-phospho-STAT3 and anti-�-tubulin antibodies. B: cells were transfected with emptyvector or construct encoding TYK2 with tyrosine kinase domain deletion (TYK2-�TK). The parallel lysates were probed with anti-phospho-TYK2 andanti-�-tubulin antibodies. C: cells were transfected with empty vector, inactivating JAK2 point mutations (K882E or Y1007F), TYK2-�TK or a combinationof JAK mutants. The parallel lysates were immunoblotted with anti-phospho-JAK2 and anti-�-tubulin antibodies. *P 0.05 compared with WT8 cell invasion.
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dation, SOCS1 protein expression was examined in RC3 andWT8 cells preincubated with the proteasome inhibitors lacta-cystin, MG132, and PSI. Figure 5E demonstrated once againthat SOCS1 expression is relatively decreased in VHL mutantRC3 cells. However, proteasome inhibition restored SOCS1levels in RC3 (Fig. 5E), consistent with a model, in whichSOCS1 undergoes enhanced proteasomal degradation in VHLmutant RCC cells.
SOCS1/SOCS2 interaction leads to SOCS1 degradation inRCC. To investigate whether SOCS1 degradation is driven byElongin B/C E3 ligase complex-directed ubiquitination, WT8and RC3 cell lysates were immunoprecipitated with anti-SOCS1 antibodies and then immunoblotted with anti-ubiquitinantibodies. In Fig. 6A, increased SOCS1 ubiquitination wasobserved in RCC cells compared with wild-type, VHL-ex-pressing cells (top), as demonstrated by the upper molecularweight polyubiquitin smear. Although the data are consistentwith SOCS1 degradation by direct binding to the Elongin B/Ccomplex, we were unable to coprecipitate SOCS1 with ElonginC (data not shown), suggesting that ubiquitinated SOCS1 isbound to the Elongin B/C through an intermediary protein.Based on a recent report demonstrating that SOCS2 can accel-erate SOCS3 proteasomal degradation in Ba/F3 cells (67), wequeried whether SOCS2 could play a similar role in RCC cells,
by interacting with SOCS1. Figure 6B shows enhancedcoprecipitation of SOCS1 with SOCS2 in RC3 vs. WT8cells. However, no difference in SOCS2/Elongin C interactionwas observed between RCC and WT8 cells (data not shown).
DISCUSSION
Our findings provide clear evidence that JAK2, JAK3, andTYK2 tyrosine kinase activities were enhanced in VHL muta-tion-induced RCC. The JAK/STAT signaling pathway hasbeen extensively characterized in many systems, includinghematopoietic, gastrointestinal, prostate, and vascular endothe-lial cells (2, 4, 5, 17). In the context of cancers, JAK kinasescan activate STATs, as well as many other signals, to promotecell survival and proliferation. Constitutive activation ofSTATs, particularly STAT3, has been reported in a number oftumors, including RCC (70). Several recent reports indicatethat gain-of-function JAK2 mutations may account for themajority of cases of polycythemia vera (37, 39, 65). To the bestof our knowledge, this is the first report regarding the role ofJAK/STAT signaling in VHL mutation-induced RCC patho-genesis.
Most previous studies implicated cytokine- and growth fac-tor- induced JAK/STAT activation in disease pathogenesis (4,
Fig. 4. Increased JAK activity promotes RCC migration in scratch-wound assays. Confluent WT8 and RC3 cells in gridded 24-well plates were wounded witha P200 pipette tip and incubated in serum-free DMEM medium with no addition (control), JAK2-specific inhibitor AG490 (100 �M), JAK3 inhibitors P131 (300�M), JAK3I-V (50 �M), or TYK2 inhibitor AG9 (100 �M) for 5 and 15 h. Photographs were taken from 3 different sites observed under phase-contrastmicroscopy. Three different experiments were performed and representative images are shown.
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19, 63, 72). However, recent evidence in transformed pre-Bcells demonstrates that cytokine-independent phosphorylationof SOCS1 on Ser/Thr residues by v-Abl allowed JAKs tobypass SOCS regulation, resulting in constitutive JAK activa-tion (40). Consistent with these results, our data illustrate thatincreased JAK activity in VHL mutation-induced RCC isindependent of cytokines and growth factors. The mechanismof constitutive JAK activation was not established, but wespeculate it may be related to posttranscriptional SOCS1 mod-ification, which promotes both SOCS1 degradation and JAKrelease.
It has been repeatedly reported that enhanced JAK activ-ity promotes cell proliferation and survival in both malig-nant and benign tumors (16, 18, 29, 38, 42, 56). NeitherRCC cell line demonstrated significant differences in pro-liferatiion or apoptosis, so the effect of JAK inhibition couldnot be determined for these phenotypes. JAKs were notrequired for diminished extracellular fibronectin secretion,which is a pathologic feature unique to RCC. However, JAKactivity was linked to cell migration and invasion throughMatrigel, suggesting that JAKs may regulate RCC cellmetastasis in vivo.
Fig. 5. SOCS1 undergoes enhanced proteasomal degradation in RCC. A, top: whole cell lysates from VHL-deficient RCC cells (RCC4), RCC4 cells stablytransfected with wild-type VHL (WTVHL) or L188V VHL point mutant (L188V), VHL-deficient RCC stably transfected cells transfected with wild-type VHL(WT8), or empty vector (RC3) were immunoblotted with anti-SOCS1 antibodies. Bottom: blot was stripped and reprobed with anti-�-tubulin antibodies asloading control. B: WT8 and RC3 cells were pulsed with [35S]-Met in Met-free media for 2 h, and then chased with Met-replete medium for 16 h. [35S]-SOCS1content in cell lysates was determined by immunoprecipitation, separation by SDS-PAGE, and visualization by autoradiography. C: mean value of band densitieswas obtained from 3 experiments by STORM phosphoimager analysis and the rate of SOCS1 decline was shown. D: WT8 (top) and RC3 (bottom) cells weretreated with the protein synthesis inhibitor cycloheximide (CHX; 20 �g/ml, 16 h). Residual SOCS1 levels (reflecting SOCS1 t1/2) were measured byimmunoblotting with anti-SOCS1 antibodies. Immunoblot data from 3 cycloheximide experiments were quantitated by Scion Image analysis. E: WT8 and RC3cells were preincubated with proteasome inhibitors lactacystin (Lac; 10 �M, 1.5 h), MG132 (20 �M, 2 h), or PSI (30 �M, 2 h). Top: whole cell lysates werethen probed with anti-SOCS1 antibodies. Bottom: blot was stripped and reblotted with anti-�-tubulin antibodies as a loading control. Immunoblot data from 3proteasome inhibition experiments were quantitated by Scion Image analysis.
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The mechanism of JAK regulation in RCC is complex,including interaction with SOCS proteins, which are endoge-nous inhibitors of JAKs and are upregulated by JAK/STATactivation (26). There are at least three families of proteins thatinhibit JAK/STAT signaling: SOCS, protein inhibitors of ac-tivated STATs (PIAS), and the SH2-containing phosphatase(SHP-1). SOCS proteins are of particular interest in this studydue to structural similarity with VHL. All SOCS gene productscontain a COOH-terminal SOCS box, which is homologous toBC box in the �-domain of VHL, which confers ability to bindthe Elongin B/Elongin C/Cullin 2 or Cullin 5 ubiquitin ligasecomplex, resulting in proteasome-mediated protein degrada-tion (21, 32, 33, 64). Furthermore, SOCS1 and SOCS3 possessan NH2-terminal kinase inhibitory region, which directly reg-ulates JAK activity (71).
Transcriptional regulation of SOCS has been investigated inmany diseases. For example, inactivation of the SOCS gene bymethylation has been previously described in hepatocellular,pancreatic, lung, ovarian, and breast carcinomas (25, 35, 51,66, 75). Since CpG island methylation generally results incomplete absence of transcription, and increased baselineSOCS1 synthesis was observed in RC3 relative to WT8 cells(see Fig. 5B), we reason that hypermethylation of SOCS1 is not
a mechanism in RCC. However, our results provide strongevidence that SOCS1 undergoes rapid proteasomal degradationin VHL mutant RCC compared with wild-type VHL-express-ing cells. Although a specific mechanism was not determined,we speculate that posttranslationally modified SOCS1 may bemore susceptible to degradation. Recent reports revealed thatphosphorylation of nontyrosine SOCS1 residues, possibly byPim1 kinase, disrupts SOCS1 interaction with the proteasome,thereby preventing SOCS1 from targeting activated JAKs fordegradation (10, 40, 41). Tyrosine-phosphorylated SOCS3 wasalso found to interact with and activate Nck and Crk-L (61).Finally, Haan et al. (24) reported that JAK-mediated tyrosinephosphorylation of SOCS3 disrupted interaction with ElonginC and accelerated SOCS3 proteasomal degradation. Tyrosinephosphorylation of SOCS1 in VHL mutation-induced RCCwas not observed (data not shown).
Among eight SOCS family molecules, SOCS1 and SOCS3have been most extensively investigated as regulators of kinaseactivities, particularly in JAK/STAT signaling pathways. Inaddition, recent studies demonstrated regulatory cross-talk be-tween SOCS family members. For example, Tannahill et al.(67) found that SOCS2 can accelerate SOCS3 proteasomaldegradation in Ba/F3 cells by binding with both Elongin C andSOCS3. Our data are consistent with a similar role for SOCS2in RCC in this study. Based on these data, we speculate thatSOCS2 constitutively binds Elongin B/C to form an E3 ligasecomplex in wild-type and RCC cells, but SOCS2 preferentiallybinds SOCS1 in RCC, leading to enhanced SOCS1 ubiquiti-nation and proteasomal degradation.
The net effect of diminished SOCS1 expression wasdecreased JAK inhibition, thereby promoting RCC migra-tion through activation of JAK-dependent pathways. Be-cause in vitro invasion and migration are surrogate assaysfor metastasis in vivo, we suggest that JAKs may regulateRCC metastasis through a SOCS-dependent mechanism. Toassess JAK regulation of RCC invasion and migration, we usedpharmacological inhibitors and dominant-negative JAK mu-tants in two different assay systems. In some cases, there wasslight discrepancy in magnitude of the effect on invasion/migration between different inhibitors of the same kinase,which could be due to variable intracellular access to JAKs oroverlapping specificities. However, when viewed in aggregate,we conclude that JAK2, JAK3, and TYK2 each contribute toregulation of RCC motility.
The VHL literature is replete with studies highlighting thesignificance of HIF in RCC pathogenesis (36, 46, 48, 52).HIF1� interaction with wild-type VHL is required for consti-tutive HIF1 ubiquitination, proteasomal degradation, and as aconsequence, inhibition of HIF1 substrates activities (13, 34).HIF1� activation due to impaired binding with mutant VHLresults in the angiogenic phenotype of VHL-associated tumorsin VHL kidneys (46, 48); reconstitution of VHL restoresHIF1� ubiquitination and reverses the angiogenesis phenotype(34). However, HIF1� activation may not be sufficient toexplain all aspects of VHL mutation-dependent RCC (47). Arecent study showed that VHL inactivation disrupts intercellu-lar junctions and cell shape through HIF-independent events inRCC cells, supporting the concept that VHL has additionalfunctions beside its role in the regulation of HIF (9). Hsu et al.(28) demonstrated that in VHL null RCC cell lines, deficientFGF receptor cycling, resulting in enhanced cell migration,
Fig. 6. SOCS1/SOCS2 interaction leads to SOCS1 degradation in RCC.A: WT8 and RC3 cells were incubated in serum-free medium overnight todeplete cytokines and growth factors. Top: cells were then immunoprecipitatedwith anti-SOCS1 antibodies and immunoblotted with anti-ubiquitin antibodies.Bottom: stripped blot, which was reprobed with anti-SOCS1 antibodies.B: WT8 and RCC cells were maintained in serum-free medium overnight.Top: cell lysates were immunoprecipitated with anti-SOCS2 antibodies, re-solved by SDS-PAGE, and then blotted with anti-SOCS1 antibodies. Bottom:blot was stripped and reblotted with anti-SOCS2 antibodies as a proteinloading control.
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was also independent of HIF. We now provide additionalevidence for HIF-independent regulation of RCC phenotype,namely JAK-dependent cell migration and invasiveness.
In summary, in VHL mutant RCC cells, SOCS1-SOCS2interaction led to enhanced SOCS1 ubiquitination, SOCS1instability and degradation, resulting in constitutive JAK ac-tivity and accelerated RCC invasiveness. These observationssuggest that impaired SOCS-dependent JAK degradation mayrepresent a biochemical mechanism of RCC metastasis in vivo.There are currently no established, successful therapies, e.g.,radiation or chemotherapy, for RCC other than nephrectomy,which applies only to patients with no evidence of metastases.We maintain that a better understanding of pathophysiologicalmechanisms involving JAK-STAT pathway activation mayprovide new therapeutic approaches for RCC.
ACKNOWLEDGMENTS
We thank Drs. J. Schelling and J. Sedor for assistance with study design,data interpretation, and editing of the manuscript. We appreciate Dr. M. Zhoufor anti-HIF-1� antibodies.
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