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Role of hypoxia and EGF on expression, activity, localization and phosphorylation ofcarbonic anhydrase IX in MDA-MB-231 breast cancer cells
Ying Li a, Hai Wang a, Chingkuang Tu b, Kathleen T. Shiverick b, David N. Silverman b, Susan C. Frost a,⁎a Department of Biochemistry and Molecular Biology, University of Florida, Gainesville, FL 32610-0267, USAb Department of Pharmacology and Therapeutics, University of Florida, Gainesville, FL 32610-0267, USA
⁎ Corresponding author. 1600 SW Archer Road,Biochemistry and Molecular Biology, University of FloriTel.: +1 352 392 3207; fax: +1 352 392 2953.
Article history:Received 17 May 2010Received in revised form 24 September 2010Accepted 27 September 2010Available online 12 October 2010
Keywords:Carbonic anydrase IXMDA-MB-231 breast cancer cellHypoxiaEGFLipid raftDimerization
Carbonic anhydrase IX (CAIX) is a zinc metalloenzyme that catalyzes the reversible hydration of CO2. CAIX isoverexpressed in many types of cancer, including breast cancer, but is most frequently absent incorresponding normal tissues. CAIX expression is strongly induced by hypoxia and is significantly associatedwith tumor grade and poor survival. Herein, we show that hypoxia induces a significant increase in CAIXprotein in MDA-MB-231 breast cancer cells. Using a unique mass spectrophotometric assay, we demonstratethat CAIX activity in plasma membranes isolated fromMDA-MB-231 is correlated with CAIX content. We alsoshow that CAIX exists predominantly as a dimeric, high-mannose N-linked glycoprotein. While there is someevidence that the dimeric form resides specifically in lipid rafts, our data do not support this hypothesis. EGF,alone, did not affect the distribution of CAIX into lipid rafts. However, acute EGF treatment in the context ofhypoxia increased the amount of CAIX in lipid rafts by about 5-fold. EGF did not stimulate tyrosinephosphorylation of CAIX, although EGFR and down-stream signaling pathways were activated by EGF.Interestingly, hypoxia activated Akt independent of EGF action. Together, these data demonstrate that theactive form of CAIX in theMDA-MB-231 breast cancer cell line is dimeric but that neither lipid raft localizationnor phosphorylation are likely required for its dimerization or activity.
Box 100245, Department ofda, Gainesville, FL 32610, USA.
Carbonic anhydrase (CA) is a family of zinc metalloenzymes thatcatalyze the reversible hydration of CO2: CO2+H2O↔HCO3
−+H+. Todate, 16 isoforms have been identified inmammals, of which three arenot catalytically active (CA-RP VIII, X and XI) [1]. The 13 catalyticallyactive isoforms are further divided into 4 groups based on tissuedistribution and subcellular localization. CAI, II, III, VII, and XIII areexpressed in the cytosol. CAVA and CAVB are expressed inmitochondria, but show unique tissue distribution. CAVI is the onlysecreted CA and found in salivary gland of a number of mammalianspecies. There are five membrane-associated CA isoforms: CAIV, CAIX,CAXII, CAXIV and CAXV. CAXV is not found in humans.
Two CA isoforms (CAIX and CAXII) are associated with andoverexpressed in many solid tumors and cancer cell lines [2,3]. CAIXwas first sequenced and characterized as MN/CA9 in HeLa cells byPastorek et al. [4]. CAIX is a transmembrane N-linked glycoprotein thatcontains 422 amino acids and four distinct domains: an N-terminalproteoglycan-like domain, a conserved extracellular catalytic domain, atransmembrane region, and an intracellular C-terminal, cytoplasmic
domain. The activity of the catalytic domain of CAIX is nearly identical tothat of the well studied and very active cytoplasmic isoform of thisenzyme, CAII [5]. CAIX expression is mostly restricted to pre-malignantor malignant cells, including breast cancer, and rarely in normal tissuesor benign lesions cells [6–10]. Gastric tumors are unique in that differentparts of the normal mucosa express CAIX [11,12]. CAIX expression,which is regulated by the HIF-1 transcription factor complex, is stronglyinduced by hypoxia and is significantly associated with tumor grade,reduced survival, and poor prognosis in breast cancer [13]. We andothers have shown that CAIX activity is associated with extracellularacidosis [14–17]. Increased acidity is not permissive to normal cellgrowth and leads in vivo to apoptosis [18]. Yet cancer cells appear toestablish a new “set-point”which allows them to tolerate an interstitialpH of around 6.8 [19]. Work by Gatenby and Gillies suggest that thischange in set point is critical to tumor biology because acid will flowalong concentration gradients from the tumor to adjacent normal tissuecausing normal cell death, disruption of the extracellular matrix,promotion of angiogenesis, loss of immune response to tumor antigens,and resistance to therapeutic drugs [20,21]. Because of their increasedtolerance to extracellular pH, tumor cells are readily able to colonize thisadjacent damaged normal tissue providing a mechanism for continuedinvasion and growth.
Triple negative breast cancer (TNBC) is a descriptor for a subtype ofbreast cancer that does not express the estrogen receptor, progesteronereceptor, or HER2 receptor [22]. However, many of these cancers
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overexpress the EGF receptor (HER1), including the MDA-MB-231 cells[23]. Recent evidence has shown that the cytoplasmic domain of CAIXpossesses a tyrosine target for the EGF receptor (EGFR) kinase andparticipates in the PI3 kinase signaling pathway in renal clear cellcarcinoma(RCC) [24]. The authorsproposed that CAIX translocates fromthe bulk phase plasma membrane to lipid rafts to form dimers inresponse to EGF stimulation and directly mediate down-streamsignaling. Lipid rafts are unique, cholesterol-rich microdomains withinthe plasma membrane that sequester signaling proteins to amplifyintracellular signaling events.
In the current study, we took advantage of the constitutiveexpression of EGFR and hypoxia-inducible expression of CAIX in theMDA-MB-231 breast cancer cell line to examine the role of hypoxia andEGF in CAIX regulation. Our studies revealed that hypoxia-induced CAIXexists primarily in a dimeric form in the membrane and is responsiblefor most of the CA activity in isolated plasma membranes. Little CAIXresides in lipid rafts in either control or hypoxia-exposed cells indicatingits localization to lipid rafts is not required for activity. In addition, CAIXis not phosphorylated in response to EGF, although EGF induced a 5-foldincrease in CAIX translocation to lipid rafts. Hypoxia increased Aktphosphorylation independent of EGF action suggesting that hypoxiaengages unique mechanisms to initiate similar signaling paths. Takentogether, our data suggest that CAIX in the MDA-MB-231 cells is activeas a dimer and does not require lipid rafts or phosphorylation for eitherits activity or dimerization.
2. Materials and methods
2.1. Cell culture
The MDA-MB-231 cell line was a gift from Dr. Kevin Brown(University of Florida) and plated at a density of 8×104 cells/per10 cm dish in 8 mL of DMEM (Gibco) containing 10% FBS (AtlantaBiological). Cells were grown in a humidified atmosphere containing 5%CO2 for 3 days before treatment with 100 μM desferoxamine mesylate(DFO) or exposure to hypoxia (1% O2, 5% CO2, and balance N2) in ahumidified Modulator Incubator Chamber (MIC-101, Billups-Rothen-berg, Inc) for 16 h. Parallel normoxic cells were incubated in ahumidified atmosphere at 37 °C in air with 5% CO2.
2.2. Plasma membrane isolation
To isolate plasma membranes, we have used a modification of themethod published by Sennoune et al. [25]. Cells were washed threetimes with Buffer A (Tris, 10 mM; EDTA, 1 mM; NaCl, 150 mM; PMSF,1 mM; pH 7.4) and then scraped into the same buffer. Aftercentrifugation at 1000 ×g for 7 min, the supernatant was removedand the pellet was resuspended in 3 mL of Buffer B (Tris, 10 mM;EDTA, 1 mM; NaCl, 5 mM; pH 7.4) and incubated on ice for 10 min.The cell suspension was then homogenized in a Potter Elvehjemhomogenizer with 15 up and down strokes. Cell debris was collectedby centrifugation at 500 ×g for 5 min. The supernatant was removedand kept on ice. TwomL of Buffer B was added to the pellet which wasthen re-homogenized. After centrifugation at 250 ×g for 5 min, thissecond supernatant was combined with the first supernatant. Five mLof Buffer C (Tris, 160 mM; EDTA, 20 mM; NaI, 2 M; MgCl2, 5 mM; pH7.4) was added to the above solution and stirred on ice for 10 min.Twenty mL of Buffer D (Tris, 10 mM; EDTA, 1 mM; pH 7.4) was addedto the above solution. After mixing, the solution underwentultracentrifugation at 105,000 ×g for 45 min at 4 °C. The pellet waswashed 3 times with Buffer D. After resuspending the pellet withhomogenizing buffer (Tris, 50 mM; EGTA, 1 mM; sucrose, 250 mM;pH 7.4), the solution was loaded on a step gradient of 20 and 40%sucrose (20% or 40% sucrose, Tris,10 mM; EDTA, 1 mM; pH 7.4) andcentrifuged at 200 k×g for 1 h at 4 °C. The interface between the steps(plasma membranes) was collected and resuspended in Buffer D.
After centrifugation at 100 k×g for 30 min at 4 °C, the pellet wasresuspended in homogenizing buffer and kept at −80 °C. Proteinrecovery was determined by a modification of the Lowrymethod [26].
2.3. Membrane inlet mass spectrometry (MIMS) to determine CAIXactivity
An Extrell EXM-200mass spectrophotometer was used tomeasuredirectly the content of CO2 through a membrane which is permeableto CO2 but not to bicarbonate. This allows an estimation of the rate ofexchange of 18O from CO2 and bicarbonate to water at chemicalequilibrium as previously described [27]. This process is catalyzed byand is a measure of carbonic anhydrase activity. In our plasmamembrane samples, this activity is exclusively CAIX.
2.4. CAIX oligomerization
MDA-MB-231 cells were exposed to hypoxia for 16 h. Totalmembranes were isolated as previously described [28] with minormodifications. Briefly, cellswerewashed3 timeswith 5 mLKrebsRingerPhosphate buffer (NaCl, 128 mM; KCl, 4.7 mM;MgSO4, 1.25 mM; CaCl2,1.25 mM; sodium phosphate, 5 mM; pH, 7.4) and incubated for 10 min.Cells were then homogenized in a Potter Elvehjem homogenizer in aTris–based buffer (TES1p) containing 20 mM Tris-HCl, 25 mM sucrose,1 mM EDTA and protease inhibitor (Sigma). A total membrane fractionwas collected at 212,000 ×g. The pellet waswashed oncewith TES1 andresuspended in TES1p. Fifty μg of total membrane protein from hypoxiccells was denatured in 0.5% SDS in a final volume of 30 μL for 10 min at100 °C. The samples were brought to 60 μL with 50 mM sodiumphosphate buffer, pH 7.5, 1% NP-40 and incubated at 37 °C for2 h. Proteins were separated on 10% polyacrylamide gels in thepresence or absence of 1% β-mecaptoethanol. Proteins were thentransferred to nitrocellulose and probed for CAIX using the M75monoclonal antibody developed by Pastorekova et al. [29]. Forsome experiments, we also used CAIX antibodies purchased fromNovus Biologicals (NB100-417). Densitometry of individual bandsin these experiments, and all other immunoblot analyses, wasperformed using UnScanIt (Silk Scientific).
2.5. CAIX glycosylation
To determine CAIX glycosylation, 50 μg of total membrane protein(or cell lysates) from hypoxic MDA MB-231 cells was denatured in40 mMDTT/ 0.5% SDS in a final volume of 30 μL for 10 min at 100 °C. ForN-glycosidase F digestion, the samples were brought to 60 μL in 50 mMsodium phosphate, pH 7.5, 1% NP-40, containing 2 μL N-glycosidaseF (1000 U) ( Biolabs) and incubated at 37 °C for 2 h. For endoglycosidaseH digestion, denatured protein sampleswere brought to 60 μl in 50 mMsodium citrate, pH 5.5, containing 2 μL endoglycosidase (1000 U)( Biolabs) and incubated at 37 °C for 2 h. Twenty μL of 4× sampledilution buffer was added to each of the samples which were thenloaded onto a 10% SDS-PAGE gel for protein separation followed bywestern blot analysis for CAIX expression using the M75 monoclonalantibody.
2.6. Localization of CAIX in lipid rafts
Total membranes from MDA-MB-231 cells were isolated asdescribed above. Five mg of total membrane protein was extracted in1.0 mL ice-cold MBS buffer (25 mM MES and 150 mM NaCl, pH 6.5)containing 1% Triton X-100 (TX-100) and supplemented with proteaseinhibitor. The samples weremixed end-over-end for 20 min at 4 °C andthen homogenized with 10 strokes in a Dounce homogenizer. Thehomogenizing flask was rinsed with 0.5 mL MBS/TX-100 and added tothe extracted sample. The samples were thenmixedwith 1.5 mL of 80%sucrose. The samples, now at 40% sucrose, were placed at the bottom of
Fig. 1. Hypoxia increases CAIX expression and activity inMDA-MB-231 cells. Panel A: Totalmembranes were collected from control cells or cells exposed to DFO or hypoxia for 16 h.Western blot analysis of CAIX expression was performed using the NB-100 antibody. Thisblot is typical of at least four independent experiments. C: Control; D:DFO (desferoxaminemesylate), H: Hypoxia. Panel B: Plasmamembraneswere isolated from control or hypoxiccells and CAIX activity was measured by 18O exchange. These data represent duplicateexperiments.
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centrifuge tubes and overlaid with 6 mL of 30% sucrose in MBS and3.5 mL of 5% sucrose in MBS. After centrifugation at 240,000 ×g in aBeckman SW41 rotor for 18 h, 1.0 mL fractionswere collected into tubescontaining protease inhibitor at 4 °C by upward displacement using 60%sucrose. The fractions were mixed and 500 μL of ice-cold, 30%tricholoroacetic acid was added to each fraction collected. The samplesweremixed and incubatedon ice for 30 min. Theproteinprecipitatewascollected at 16,000 ×g for 10 min after which it was washed 2 times in1 mL of ice-cold acetone. The precipitate was then dissolved in 65 μL of0.1% SDS. Total membrane protein (100 μg) and precipitated proteinfrom fractions 2–13 from the sucrose gradients were resolved on a 10%SDS-PAGE gel under reducing conditions. The proteinswere transferredto nitrocellulosemembrane and probed for CAIX, GLUT1 (characterizedpreviously [30]), caveolin (Transduction Laboratories #C13630/L5), andtransferrin receptor (Alpha Diagnostics, #TFR12-M).
2.7. Phosphorylation of EGFR and Akt
MDA-MB-231 cells, grown to 50% confluence in 10 cm culturedishes, were exposed to serum-free medium overnight undernormoxic or hypoxic conditions. Recombinant EGF (rEGF, SantaCruz) was dissolved in 10 mM acetic acid containing 0.1% BSA at astock concentration of 10 μg/mL. EGF (16 nM, final concentration) wasadded to the serum-starved, control and hypoxic cells for specifictimes as indicated in the figure legends. Immediately after treatment,cells were placed on ice, washed with ice-cold PBS (10 mM sodiumphosphate salts, 120 mM NaCl, pH 7.4), and lysed in RIPA buffer [1%NP-40, 10 mM phosphate buffer, 0.1% SDS, 150 mMNaCl, 0.5% sodiumdeoxycholate, 1 mM sodium orthovanadate, 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and protease inhibitor (Roche Diagnostics),pH, 7.4]. Cell lysates were clarified by centrifugation at 16,000 ×g for15 min at 4 °C. Protein concentration of the clarified supernatants wasdetermined using a modification of the Lowry assay. Proteins wereseparated on an 8% PAGE gel and transferred to a nitrocellulosemembrane for western blot analysis. The following antibodies wereused in the analysis: EGFR (Cell Signaling Technology #2232); pEGFR(Y1173: Santa Cruz Biotechnology #sc-101668); Akt1 (Sigma#p1601); pAkt (S473) (Cell Signaling Technology #D9E); Mapkinase/Erk2 (Calbiochem #442700); pErk1/2 (Biolabs #9106).
2.8. Phosphorylation of CAIX
Cells were treated as described above from which total mem-branes were prepared. Total membrane proteins were lysed in RIPAbuffer. Then, lysates were processed for immunoprecipitation. Briefly,lysed samples (1 mg protein) were treated with 50 μL of a 50%suspension of protein A/G plus-agarose (Santa Cruz) for 1 h at 4 °C toeliminate non-specific protein binding. The beads were removed bycentrifugation at 3,000 ×g for 1 min. To the precleared supernatants,50 μL of a 50% solution of protein A/G plus-agarose beads and 3 μg of a(goat) polyclonal antibody against CAIX (R&D Systems #AF2188)were added and subjected to gentle end-over-endmixing overnight at4 °C. The immune complexes were collected by centrifugation at3,000 ×g for 5 min. Immunoprecipitates were subjected to gelelectrophoresis and blotted onto nitrocellulose membranes. Phos-phorylated CAIX was detected with antibody against phosphotyrosine(Santa Cruz Biotechnology, #sc-7020). Total CAIX was detected withthe M75 monoclonal antibody.
3. Results
3.1. Hypoxia increases CAIX expression and activity in MDA-MB-231cells
Hypoxic-dependent expressionof CAIXwas investigatedbyWesternimmunoblot. By densitometric analysis, CAIX expression was increased
by 6-fold in total membranes by desferoxamine mesylate (D), an ironchelator which mimics the effect of hypoxia, or hypoxia (H) comparedto controls (C) (Fig. 1A).Wehavepreviously shown that CAIX is theonlymembrane-associated CA inMDA-MB-231 cells [14] which allows us tounequivocally assay CAIX activity in plasma membranes. Using 18Oisotopic exchange between CO2 and water detected by mass spectrom-etry, we demonstrate that there is a 7-fold increase in CA activity inplasma membranes isolated from hypoxic cells relative to controls(Fig. 1B). This demonstrates that there is a strong correlation betweenCAIX expression and activity.
3.2. Membrane-bound CAIX is a dimer
It has been suggested that CAIX in renal carcinoma cells istranslocated to lipid rafts where it forms a dimeric structure, a processthat is controlled by EGF action [24]. To investigate the oligomerizationstatus of CAIX in hypoxic MDA-MB-231 cells, total membranes wereanalyzed by SDS-PAGE under reducing and non-reducing conditions(Fig. 2A). Under reducing conditions, CAIX migrated as a 54/58 kDadoublet which has been observed by many investigators [31]. Undernon-reducing conditions, CAIX migrated as a single, higher molecularweight band which represents 90% of the CAIX pool. Taking intoconsideration the activity data (Fig. 1B), we conclude that the dimericform of CAIX represents most of the activity in MDA-MB-231 cells. Wealso investigated the glycosylation state of CAIX through endoglycosi-dase digestion. N-glycosidase F (PNGF) releases the entire N-linkedglycanwhile endoglycosidase H (endo H) releases the glycan only if thestructure is highmannose or a hybrid form, but not a complex structure.The 54/58 doublets were both sensitive to PNGF showing more rapidmigration of these species in SDS-PAGE gels (Fig. 2B). This indicates thatthe 54 kDa protein is not a deglycosylated formof the 58 kDa protein. Inother words, the 54 kDa species may be a truncated isoform of CAIX orthe58 kDa formmaybepost-translationallymodifiedbymechanisms inaddition to glycosylation. Further, both formswere completely sensitiveto endo H indicating that the glycans were of high mannose structure.
3.3. CAIX activity does not require its localization to lipid rafts
Lipid rafts are microdomains within the plasma membrane thatserve as signaling platforms. These are enriched in specific lipids and
Fig. 2. Oligomerization and glycosylation of CAIX in MDA-MB-231 cells. Panel A: Total membranes were isolated fromMDA-MB-231 cells exposed to hypoxia for 16 h. Fifty μg of totalmembrane protein were separated on an SDS-PAGE gel in the presence or absence of 1% β-mercaptoethanol (β-ME). CAIX expression was detected by western blotting using theM75 monoclonal antibody. Panel B: Total membranes were isolated from MDA-MB-231 cells exposed to hypoxia for 16 h. Fifty μg of protein was digested with 2 μL N-glycosidase F(PNGF) in the presence of absence of protease inhibitor (PI) for 2 h at 37 °C. Panel C: Cell lysates were isolated fromMDA-MB-231 cells exposed to hypoxia for 16 h. Fifty μg of proteinwas treated with 2 μL endoglycosidase H (endo H) in the presence or absence of protease inhibitor (PI) for 2 h at 37 °C. CAIX expression was detected by western blotting using theM75 monoclonal antibody. These blots represent duplicate experiments.
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are comprised of a select set of proteins that reside in, or transientlyassociate with, these domains. We have shown that GLUT1, in 3T3-L1adipocytes, is associated with lipid rafts [28] and show that the sameis true for GLUT1 inMDA-MB-231 cells. In Fig. 3, fractions 3 and 4 fromsucrose density gradients represent plasma membrane vesiclesderived from lipid rafts. These vesicles are resistant to disruption byTriton X-100 (TX-100) because of the high cholesterol and sphingo-
Fig. 3. Association of CAIX and GLUT1 with lipid rafts. MDA-MB-231 cells were exposed tocollected and extracted with 1% TX-100. Detergent-resistant membranes (lipid rafts) were seas numbered at the bottom of each panel). Expression of CAIX, GLUT1, caveolin and transferrtotal pool that migrates in lipid rafts. TM = total membrane fraction; Rafts = lipid raft-condata are representative of at least triplicate experiments.
lipid content of the lipid rafts. The fractions that are labeled as “TX-100 Soluble” represent membrane proteins that were not protectedfrom detergent extraction. Thus, these proteins were not in lipid raftsto begin with and were dissolved by detergent treatment. We haveused the identification of caveolin as a marker of lipid rafts and thetransferrin receptor as a protein excluded from lipid rafts and thussolubilized by TX-100 exposure. Neither caveolin nor transferrin
normoxic (Panel A) or hypoxia (Panel B) conditions for 16 h. Total membranes wereparated from extracted protein by flotation on sucrose gradients (fractions 2 through 13in receptor is indicated. The values to the right of the figures represent the percent of thetaining membranes; TX-100 Soluble = membrane proteins extracted by TX-100. These
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receptor expression or localization was influenced by hypoxia(compare Fig. 3B to A). The localization of GLUT1 to lipid rafts inboth control and hypoxic cells represents about 25% of the total pooldespite a significant 6-fold increase in expression in response tohypoxia. We would interpret this to mean that hypoxia does not havea specific influence on GLUT1 localization within the plasmamembrane. CAIX was not detected in lipid rafts in control cells(Fig. 3A), perhaps a result of the low total CAIX expression. Hypoxiaincreased the total expression of CAIX by 5-fold in this experiment(Fig. 3B) with only a small increase in the amount of CAIX associatedwith lipid rafts, about 1.1% of the total CAIX pool. With a 7-foldincrease in CAIX activity in response to hypoxia (Fig. 1B), it is unlikelythat the small shift of CAIX to lipid rafts accounts for this change inactivity. Thus, lipid raft association does not to appear to play a majorrole in regulating CAIX activity under hypoxic conditions.
3.4. EGF induces CAIX translocation to lipid rafts but not itsphosphorylation
We next determined the effect of EGF on CAIX distribution withinthe plasma membrane. In Fig. 4A, we show only CAIX expression.Consistent with data in Fig. 3A, little CAIX was detected in lipid raftsfrom control cells. EGF, under normoxic conditions, did not cause anytranslocation of CAIX to lipid rafts. Hypoxia increased the amount ofCAIX, again by only a small amount, while the combination of hypoxiaand EGF stimulation increased the amount of CAIX by 5-fold relativeto hypoxia alone. In this particular experiment, it appeared that therewas a little more CAIX in the TM fraction isolated from hypoxic cells
Fig. 4. EGF-dependent localization of CAIX. Panel A: MDA-MB-231 cells were serum-starved overnight under normoxic or hypoxic conditions and then stimulated with EGF(16 nM) for 30 min. Total membranes were collected and then lysed with TX-100.Detergent resistant proteins (lipid rafts) were separated from extracted proteins. CAIXexpression in each sample was detected by western blotting using the M75monoclonalantibody. TM= total membrane fraction; Rafts= lipid raft-containingmembranes; TX-100 Soluble = membrane proteins extracted by TX-100. Panel B: MDA-MB-231 cellswere exposed to hypoxia for 16 h and then treated with EGF (16 nM) for 30 min. Totalmembranes were prepared from which lipid rafts were isolated. CAIX and caveolinexpression were detected by western blotting. TM = total membrane proteins; LR =lipid raft containing membranes.
treated with EGF. This could of course influence the amount of CAIX inthe lipid raft fraction. We repeated this experiment and confirm inFig. 4B that CAIX expression did not change in the presence of short-term exposure to EGF. Together these data suggest that EGF inducesCAIX translocation to lipid raft only in the context of hypoxia,although the pool associated with lipid rafts is relatively small (about5%) compared to the total.
Recent data suggest that EGF stimulates CAIX tyrosine phosphor-ylation in the cytoplasmic domain in renal carcinoma cells whichleads to down-stream activation of the PI3-kinase pathway [24]. Toinvestigate this possibility in MDA-MB-231 cells, we first examinedEGF-dependent autophosphorylation of the EGF receptor (EGFR).Cells were serum-starved under hypoxic or normoxic conditions for16 h and then stimulated with EGF (16 nM) for specific times rangingfrom 10 min to 40 min. Under these conditions, we observed a time-dependent phosphorylation of EGFR on Y1173 (Fig. 5A). The kineticsof phosphorylation were similar for both the normoxic and hypoxicconditions peaking between 10 and 30 min of EGF exposure.Activation of EGFR led to down-stream phosphorylation of both Akt(Fig. 5B) and ERK1/2 (data not shown). Interestingly, Akt phosphor-ylation was relatively strong in hypoxic cells even in the absence ofEGF. Under the conditions of the experiment, EGF did not influenceCAIX expression (Fig. 5C).
To examine EGF-dependent phosphorylation of CAIX, cells wereexposed to hypoxia for 16 h and then 30 min with EGF. CAIX was thenimmunoprecipitated with a CAIX-specific polyclonal antibody. Whilethere appeared to be several phosphorylated proteins in the cellextracts (input) including those that were EGF-dependent, there wereno phosphorylation signals in the CAIX-immunoprecipitated samples(Fig. 6). Note the non-specific detection of the heavy chain IgG(arrow). The presence of CAIX protein in the immunoprecipitates wasverified by Western blot using the M75 monoclonal antibody. Theseresults indicate that CAIX is not phosphorylated on tyrosine underhypoxic conditions or in an EGF-dependent manner in MDA-MB-231breast cancer cells.
4. Discussion
CAIX expression is upregulated in a wide variety of human tumors[1] under the control of the HIF cascade [32–35]. In breast cancer, CAIXis a marker for hypoxic regions of tumors [36], is associated with poorprognosis [37–39], and is linked to acidification of the tumormicroenvironment [17] which favors cancer cell survival andresistance to chemotherapeutic agents [40]. CAIX expression hasalso been linked to the basal B, triple-negative phenotype [41]. Wehave shown that the MDA-MB-231 cell line is a model for thisphenotype [14] and confirm here that CAIX expression is induced byan hypoxic mimic and by hypoxia, itself. We have also shownpreviously that CAIX is the only expressed membrane-bound CAisoform in this cell line [14] which allows us to unambiguously assayfor CAIX activity. We have measured endogenous CAIX activity,directly, using a novel mass spectrometric technique. Plasmamembranes, isolated from MDA-MB-231 cells by a method specifi-cally developed for breast cancer cells [25], exhibited a 7-fold increasein CAIX activity in response to hypoxia which correlated well with theincreased expression of CAIX in total membranes.
The selective expression of CAIX thatwe have observed inMDA-MB-231 cells is clearly different from the expression pattern described byHsieh et al., also inMDA-MB-231 cells [42]. In their study, the expressionof CAXII appeared to be significantly higher than CAIX. Further, theauthors showed that knocking down expression of CAXII decreased theinvasion andmigration capability of the cells. So the question arises as tohow the same cell line showsdifferent expression for specific proteins. Itis appreciated that cancer arises from a stepwise accumulation ofgenetic changes that afford an incipient cancer cell the properties ofunlimited, self-sufficient growth and resistance to normal homeostatic
Fig. 5. EGF and hypoxia dependent activation of Akt. MDA-MB-231 cells were exposedto normoxic (C) or hypoxic (H) conditions for 16 h in the absence of serum and thenstimulated with 16nM EGF for specific times. Proteins (100 μg) from cell lysates wereseparated on SDS-PAGE gels and analyzed by western blotting. Panel A represents totaland phosphorylated pools of EGFR; Panel B represents total and phosphorylated poolsof Akt; Panel C represents the total pool of CAIX. This experiment is representative oftwo independent experiments.
Fig. 6. CAIX phosphorylation in response to EGF stimulation. MDA-MB-231 cells wereexposed to hypoxia or not for 16 h in the absence of serum. EGF (16nM) was added for30 min after which total membranes were isolated. CAIX was immunoprecipitated withan antibody generated in goat (R&D Systems, #AF2188) followed by western blottingwith an anti-phosphotyrosine antibody or the M75 mouse monoclonal antibody. C =control, H = hypoxia. These data represent triplicate experiments.
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regulatory mechanisms [43]. This genetic instability is considered toplay a key role in the generation of genetic and phenotypicheterogeneity in cancer cells. Recently, Masramon et al. have demon-strated genetic drift in clonal lines originating from isolated (colon)
cancer cells [44]. This indicates that genetic instability is not lost incultured cells and can continue to contribute to genetic and phenotypicdifferences. It is logical to assume that this genetic drift is responsible forthe protein expression differences in theMDA-MB-231 cells used in ourstudies and those used by Hsieh et al. That CAXII has similar propertiesto CAIX isnot unexpected based on studies from thePoussegeur lab [16].In these studies, Chiche et al. demonstrated that overexpression of CAIXand CAXII were equally able to regulate intracellular pH in severaldifferent cell lines. Thus our data do not detract from the finding byHsieh et al. that CAXII is involved in regulating migration and invasion.
It was originally proposed that CAIX could form trimers [29]. Morerecent characterization reveals that CAIX can also exist as a dimer [45].These later biochemical studies were done with soluble recombinantforms of CAIX (containing exofacial domains) which were expressedin insect cell expression systems which allows protein glycosylationprocess. Hilvo et al. showed that approximately 50% of the CAIXcatalytic domain constructs form dimers while in constructs contain-ing the proteoglycan-like combined with the catalytic domain, thedimers comprised about 60% of the pool [45]. While the catalyticdomain construct could adapt both trimeric and dimeric structures,the proteoglycan domain construct preferred the dimer structure.This suggests that the presence of the proteoglycan domain favorsdimerization. These investigators also identified two sets of specificsulfhydryl groups that potentially participate in forming intermolec-ular disulfide bridges. The recently published crystal structure of thesoluble form of CAIX has confirmed that the two catalytic domainsassociate to form a dimer, stabilized by the formation of a singleintermolecular disulfide bond [46,47]. While the TM domain does notappear to be required for dimerization, we hypothesized that theextent of dimerization may be affected by its presence. Indeed, in theMDA-MB-231 cells, dimers comprise 90% of the CAIX pool whichinfers that dimers provide the majority of the CAIX activity. The equalintensity of the 54/58 kDa doublet, which we observed on reducinggels, might suggest that the doublet pair is linked by a disulfide bondin the dimer as the dimeric species that we observed migrated as asingle band at about 119 kDa.
It was previously shown in HeLa cells that each of the proteins thatcomprise the 54/58 kDa doublet of CAIX are N-linked glycosylated [4].We show here that the same is true for CAIX in theMDA-MB-231 cells.The oligosaccharides on both of these doublets are of high mannosecharacter, based on endo H sensitivity, which is atypical for plasmamembrane proteins from normal cells. However, this is not uncom-mon in cancer cells [48] and specifically in breast cancer cells as wasshown recently [49]. In the later study, membrane proteins showed a
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significant increase in GlcNAc2Man5 structures although the complexglycan structures with multiantennary arrangements were notsignificantly different. Like CAIX, GLUT1 has a single N-linkedglycosylation site but migrates as a broad band suggesting heteroge-neous, complex glycosylation which is resistant to endo H digestion[50]. Both GLUT1 and CAIX are up-regulated by hypoxia but appear tobe differentially processed in the same cell. This preference for highmannose glycans may be an intrinsic feature of the CAIX structure asrecombinant CAIX (constructs containing either the catalytic domain,alone, or in combination with the proteoglycan domain) expressed ina baculovirus-insect cells or in murine cells also exhibit high mannoseglycan structures [45].
Dorai et al. demonstrated that EGF stimulates CAIX phosphor-ylation on tyrosine 412 in the cytoplasmic domain in a renalcarcinoma cell line, SKRC-01 [24]. This phosphorylation led to theability of CAIX to interact with PI3-kinase. In a similar line, SKRC-17, which does not express CAIX, the authors demonstrated thatEGF action leading to phosphorylation of Akt was more robustwhen CAIX was ecoptically expressed. This infers that EGFstimulates CAIX phosphorylation which independently activatesthe EGF signaling path. However, we were unable to demonstrateEGF-mediated CAIX phosphorylation in the MDA-MB-231 cells.Clearly, the EGFR is expressed in MDA-MB-231 cells, is phosphor-ylated in the presence of EGF, and initiates down-stream signalingevents. There are several reasons why we may not have observedphosphorylation of CAIX. The EGFR is known to reside in lipid raftsin both normal cells and cancer cells [51,52] and to mediate theactivation of down-stream signaling pathways in cancer cells whenrecruited to lipid rafts [53]. In addition, cholesterol levels changeEGFR function, trafficking, and activation [54,55]. Dorai et al.demonstrated, indirectly, that phosphorylated CAIX was present inlipid rafts which implies that EGF stimulation causes the recruit-ment of CAIX to lipid rafts. We were unable to detect any CAIX inlipid rafts in control cells with or without EGF but the levels ofCAIX are quite low in normoxic cells. On the other hand, if EGFR islocalized to lipid rafts, then the interaction between EGFR and CAIXmight not occur. We did observe an EGF-dependent increase in thecontent of CAIX in lipid rafts under hypoxic conditions whichrepresented about 5% of the total CAIX pool. If this particular poolwas phosphorylated, admittedly, it might go undetected. There isalso the issue of CAIX function in renal cell carcinoma versus breastcancer cells. CAIX in RCC is not upregulated by the condition ofhypoxia, as it is in triple negative breast cancers and MDA-MB-231cells. Rather, mutations in the prolyl hydroxylase which normallymediates the degradation of HIF1α drives CAIX expression. Thusthe environment surrounding RCC cells and breast cancer cells isquite different. Further, CAIX expression in RCC is a positivepredictor of survival [6] while CAIX expression in breast cancer isan indicator of poor prognosis [13]. How these differences play outwith respect to EGF action is not known at this point.
PTEN mutation or loss, which leads to sustained activation ofPI3-kinase, is associated with many forms of cancer, includingbreast cancer [56]. However, MDA-MB-231 cells do not lack PTEN[57] which means that the PI3-kinase pathway is not constitutivelyactivated. This allowed us to examine both the effect of hypoxiaand EGF on this signaling path. EGF, as expected, stimulated Aktphosphorylation by activating EGFR. This is consistent with earlierstudies [57]. Our data also showed that hypoxia, independent ofEGFR activation, enhanced Akt phosphorylation. Others haveshown that this activation is mediated through PI3-kinase [58,59]and leads to protection from apoptosis [58]. Further, hypoxia-induced activation of Akt can be blocked by inhibitors of transcrip-tion or protein synthesis, although the levels of Akt are not affectedby these treatments [58]. While preparing the present manuscript,Mardilovich and Shaw showed that IRS2 is transcriptionallyupregulated by hypoxia in MDA-MB-231 cells [60]. Thus, the cells
are poised for activation by the IGF1 signaling system. Yet in ourexperiments, serum was absent for the duration of hypoxictreatment. Thus, IGF-mediated phosphorylation of IRS2 is notresponsible for the hypoxic activation of Akt in our setting.
Akt activation is also involved in localizing GLUT1 to the cellsurface andmaintaining hexokinase activity [61] which favors aerobicglycolysis in tumor cells [62]. While GLUT1 is transcriptionallyupregulated by HIF1α, hypoxia-activated Akt may also help to recruitGLUT1 to the cell surface and maintain the distribution betweenintracellular membrane stores and the plasma membrane. Whilethere are data suggesting that lipid rafts influence GLUT1 activity[28,63], the increase in localization of GLUT1 to lipid rafts inMDA-MB-231 cells is equivalent to the increased expression of GLUT1 inresponse to hypoxia. As well, the increase in glucose uptake inresponse to hypoxia (data not shown) is equivalent to GLUT1expression. It would seem logical to conclude that GLUT1 activity isa reflection of the total pool.
In conclusion, we have shown that CAIX activity and expressionare well correlated in hypoxia-treated MDA-MB-231 cells. EGFrecruits CAIX to lipid rafts, in the context of hypoxia, but it is unlikelythat this association regulates its oligomerization status or its activity.Further, we did not detect any EGF-dependent CAIX phosphorylation,despite an intact EGF signaling system. Rather, hypoxia activates Aktindependently from EGF action to induce what Dorai et al. called thevicious cycle [24]. Thus, hypoxia induces the stabilization of HIF1αwhich mediates the expression of CAIX through transcriptionalactivation of its gene, and increases Akt activation which enhancescap-dependent translation of HIF1α to further induce expression oftarget genes.
Funding Source
Thework in this manuscript was supported in part by grants to SCFfrom the Department of Defense (BC073020) and NIH (DK45035) andDNS from NIH (GM25154).
Acknowledgements
The authors would like to thank Dr. Egbert Oosterwijk for the M75antibody and Ms Xiao Wei Gu for her excellent assistance in cellculture.
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