Inhibition of carbonic anhydrase IX (CA9) sensitizes renal cell carcinoma to ionizing radiation (2024)

Introduction

Partial or radical nephrectomy for primary renalcell carcinoma (RCC) achieves excellent rates of cure (1), yet the procedure is invasive and oftenresults in loss of normal renal parenchyma leading to thedevelopment of renal insufficiency with its associated long-termmorbidity and even mortality (1).Radiation treatment of primary RCC is rarely employed for curativeintent as RCC is generally believed to be radiation-resistant. Ofnote, in clinical practice, particular care is taken to keep kidneyradiation doses within acceptable tolerance limits as normal renalparenchyma is considered relatively sensitive to radiation(2). One possible explanation forthis conundrum may be the differential expression of proteins, suchas carbonic anhydrase IX (CA9), involved in radiation resistance inRCC cells compared to normal renal cells. CA9 is not expressed innormal kidney cells (3), yet itsexpression is ubiquitous in clear cell RCC (ccRCC), most likely dueto the fact that expression of CA9 is transcriptionally regulatedby hypoxia-inducible factor-1α (4),which accumulates in ccRCC cells as a result of frequentinactivating mutations in the von Hippel-Lindau tumor-suppressorgene (5).

The family of carbonic anhydrase enzymes catalyzesthe dissolution of CO2 in water as carbonic acid andprotons (6). CA9 contributes to theacidification of the local tumor environment and guards tumor cellsagainst acidosis. We hypothesized that upregulation of CA9 in RCCcells may account, at least in part, for the radiation resistanceof RCC, and thus targeting CA9 expression or enzymatic activity maysensitize RCC to ionizing radiation.

Materials and methods

Cell culture and transfection

Mycoplasma-free human ccRCC 786-O and human prostateadenocarcinoma LNCaP cells (ATCC, Manassas, VA, USA) werepropagated in RPMI-1640 medium supplemented with 10% FBS(Invitrogen, Burlington ON, Canada). The 786-O cell identity wasverified by STR analysis (ATCC). Murine RCC RAG cells and humanglioblastoma LN-18 cells (ATCC) were maintained in Eagle's MEM andDulbecco's MEM, respectively, supplemented with 10% FBS.

The shRNA vector for human CA9 (CA9 shRNA) and thenon-effective negative scrambled control were purchased fromOrigene (Rockville, MD, USA). Transfection of human ccRCC 786-Ocells was performed using 12% Fugene (Promega, Madison, WI, USA).Cells stably transfected with shCA9 or the scrambled control wereselected with 1.0 µg/ml puromycin (Sigma-Aldrich, Oakville,ON, Canada).

Clonogenic survival experiments

RAG, 786-O, and LN-18 cells (250 per well) wereseeded onto 6-well plates in 3 ml of medium and allowed to adhereby incubation at 37°C and 5% CO2 for 4 h. For LNCaP,1000 cells were seeded per well and allowed to adhere for 24 h.

For the AEBS treatment, a stock solution of4-(2-aminoethyl)benzene sulfonamide (AEBS, 33 mM, Sigma-Aldrich)was prepared fresh with H2O and filter-sterilized usinga 0.2 µm syringe filter. AEBS at concentrations ranging from3.3 µM to 3300 µM was added to 100 µl ofH2O to 3 ml media. Solvent controls were also included.The medium was aspirated and replaced with 3 ml of the appropriatedrug solutions in media in duplicate wells. After 24 h ofincubation, the media were aspirated, and 3 ml of fresh medium wasadded to each well.

Ionizing radiation (IR) of cells was performed usinga Varian Linear Accelerator (LINAC) generating six MV X-rays(Varian Medical Systems, Inc., Palo Alto, CA, USA). Cells plated induplicate 6-well plates were irradiated at a distance of 100 cm ina 16 cm by 20 cm field. A 19-mm-thick acrylamide sheet was placedon the plates as a build-up region. Thermoluminescent dosimeterswere used to measure and calibrate the dose. The cells receivedfrom 1 to 8 Gy of 6 MV X-ray radiation.

Following treatment, the cells were incubated for anadditional 6 days (786-O), 7 days (RAG), or 12 days (LNCaP andLN-18), after which the medium was aspirated and the colonies werestained with crystal violet (0.25% in 95% ethanol) for 10 min.Colonies of 50 cells or more were counted. Survival was expressedas a percentage of the corresponding untreated controls, andIC50 values were calculated using CalcuSyn softwareversion 1.2 (Biosoft, Cambridge, UK). Experiments were repeatedthree times.

CA9 activity assay

Confluent RCC cells in 6-well plates were washedtwice with 3 ml of PBS. One ml of 0.9% saline (adjusted to pH 8.0with NaOH) containing 0.15 mg/ml phenol red was added to each well.Deionized water (100 µl) with or without AEBS at variousconcentrations was added to the wells. Starting 5 sec after theaddition of saline to the cells, the absorbance at 565 nm wasmeasured using a Powerwave HT spectrophotometer (BioTek, Winooski,VT, USA) at 1 sec intervals for 20 sec. The relative absorbance ofa particular well throughout the 20 sec was determined as a percentof the no cell control average at that time interval (A/A nocells).

Western blot analysis

Cultured cells were lysed using RIPA lysis buffer.Lysate (40 µg) was resolved on a 10% SDS-PAGE gel andtransferred onto nitrocellulose membrane. Primary antibodies usedwere CA9 (1:1,000 Epitomics, Burlingame, CA, USA) and β-actin(1:2,000, Sigma-Aldrich). Secondary HRP-conjugated antibody (1:200,Dako, Carpinteria, CA, USA) was used in conjunction withchemiluminescence detection.

Animal studies

All protocols for animal studies were reviewed andapproved by the institutional Animal Research Ethics Board (AUP#12-09-37). Per group, 7–10 female inbred nude (Balb/c nu/nu) mice(Charles River, St. Constant, QC, Canada) 5 weeks of age were used.786-O parental, shCA9 or scrambled control 786-O cells[1–3×106 in 50% (v/v) Matrigel] were injectedsubcutaneously into the right flank of each mouse. Tumor size wasmeasured every three days using Vernier calipers, and the tumorvolume was determined using the formula π/6(length x width xheight) until the largest tumor reached 400 mm3. Themice were sacrificed 7–12 weeks after tumor cell injection, whenthe tumors were dissected, weighed, fixed in formalin and embeddedin paraffin.

For mice in the IR group, 21–27 days post injection,when tumors were palpable, the animals were anaesthetized usingisoflurane and positioned in sterile, acrylamide cylindersconnected to a portable anaesthetic machine. Cylinders weretransported to the treatment area, where they were positioned at adistance of 100 cm to the source and irradiated with 6 Gy in a 2 cmby 2 cm field using a Varian Linear Accelerator generating 6 MVX-rays. A 5-mm-thick sheet of superflab bolus material served as abuild-up region. Animals in non-irradiated control groups wereanaesthetized for a similar time period.

Animals in the AEBS-treatment groups received 50 or200 µg/ml AEBS in the drinking water supplied fresh everytwo days starting two days before IR. No adverse effects of thetreatment were observed.

The serum levels of VEGF were determined using amouse VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA)according to the manufacturer's instructions on a BMG LabtechSpectroStar Nano multi-well plate reader. Immunostaining of 4µm-thick tumor xenograft sections for CD31 and subsequentimage analysis was performed as previously described (7) resulting in the microvessel densityexpressed as endothelial length (in µm) permm2.

AEBS mass spectrometry

Trifluoroacetic acid (2 µl) and methanol (500µl) were added to 100 µl mouse serum, vortexed for 10sec and centrifuged at 5000 rpm for 10 min. Resulting supernatant(300 µl) was removed and blown down to dryness withnitrogen. The sample was reconstituted in 200 µl ofmethanol/water (1:1) containing 25 µg/ml phenylalanine(internal standard) and filtered using a 13-mm syringe filter (0.2µm GHP membrane). The sample (5 µl) was run on theLC-MS (Agilent 6340 Ion Trap coupled to an Agilent 1200 HPLC,Agilent Technologies Inc, Mississauga, ON, Canada) at the McMasterregional centre for mass spectrometry. Analysis was performed usingmultiple reaction monitoring on AEBS and phenylalanine with thetransition at 201-184 (m/z) and 166-120 (m/z), respectively.Control serum was spiked with AEBS at 1–16 µg/ml.

Statistical analysis

Values are expressed as the mean ± the standarderror of the mean. Where appropriate, results are presented with95% confidence intervals (CI). Dependent on whether the data werenormally distributed or not, parametric (Student's t-test) ornonparametric methods (Mann-Whitney U-test) were used with ap-value <0.05 indicative of statistical significance.

Results

CA9 is present in the radiation-resistant786-O and RAG RCC cells

Protein expression of CA9 was determined in thelysates of human prostate adenocarcinoma LNCaP, human ccRCC 786-O,murine RCC RAG and immortalized human embryonic kidney HEK-293cells by western blot analysis (Fig.1A). CA9 was detectable in the 786-O and RAG cells, but not inthe LNCaP or HEK-293 cells. Clonogenic survival experimentsdemonstrated that RAG cells were more sensitive to IR than 786-Ocells (p<0.05), whereas LNCaP cells were the most sensitiveamong the cell lines tested (Fig.1B). Human glioblastoma LN-18 cells exhibited similar toleranceto IR as the RAG cells. The 786-O cells displayed significantlydecreased survival at a dose of 2 Gy and above (p<0.001),whereas LNCaP cells displayed significantly decreased survival atall radiation doses (p<0.001). The calculated IC50values for each cell line are presented in Table I.

TableI Sensitivity of the different celllines to ionizing radiation in vitro as determined by theIC50 value measured by clonogenic survival.

Knockdown of CA9 expression by shRNA in786-O cells leads to radiosensitivity

To investigate the significance of CA9 expression onRCC radiosensitivity, human ccRCC 786-O subclones stably expressingshRNA specific for CA9 were generated. These shCA9 cells showed 92%knockdown of CA9 protein expression compared to the respectivecontrol cells (scrambled shRNA) (Fig.2A). Clonogenic survival of the 786-O scrambled control andshCA9 cells was determined after IR with increasing doses of 1–8 Gyand clearly demonstrated that knockdown of CA9 confers sensitivityto IR (p<0.001). The IC50 value decreased by >50%,from 3.64 Gy (95% CI: 3.27–4.05) in the scrambled control cells to1.81 Gy (95% CI: 1.44–2.27) in the shCA9 cells (Fig. 2B). To further demonstrate the effectof CA9 knockdown in vitro, the activity of the CA9 enzymewas measured using phenol red as an indicator. In comparison to thescrambled control cells, shCA9 cells had significantly decreasedacidification capacity (p<0.001) and experienced 51% of theabsorbance change observed in the scrambled control cells (Fig. 2C).

The effect of CA9 knockdown on the in vivogrowth after subcutaneous injection of shCA9 or scrambled controlcells (1×106) into nude mice (n=7/group) was determined.A tumor take rate of 64% was achieved. The tumor volume from miceinjected with the shCA9 cells was not significantly different frommice injected with cells transfected with the scrambled controlshRNA (Fig. 3A and B). However,when the subcutaneous tumors were also irradiated 21 days aftercell injection, we observed that IR of the shCA9-transfected 786-Ocells led to decreased tumor growth (p<0.001) and a 78.7%decrease in tumor volume at sacrifice (Fig. 3B). IR of the scrambledcontrol-injected mice led to a 20.7% reduction in tumor volumeafter 12 weeks (Fig. 3A). Westernblot analysis of the tumor hom*ogenates showed that CA9 remainedreduced in the shCA9-injected mice (53%) at sacrifice, 12 weeksafter tumor cell injection (Fig.3C).

AEBS inhibits CA9 activity in vitro andleads to radio-sensitivity

AEBS is a known inhibitor of CA9's enzymaticreaction with a Ki of 33 nM (8).However, to the best of our knowledge, AEBS has not been usedpreviously to inhibit CA9 in vivo, in contrast toacetazolamide, a similar sulfonamide. In the presence of 33µM AEBS, the human ccRCC 786-O cells exhibited a significantdecrease in clonogenic survival after IR when compared to theuntreated control (Fig. 4A,p<0.001). Similarly, the more radiation-sensitive murine RAGcells also exhibited a decrease in clonogenic survival after IRwhen treated with the same concentration of AEBS for 24 h (Fig. 4B, p=0.018). To further demonstratethe effectiveness of AEBS in vitro, the acidification of theextracellular environment of 786-O cells was determined (Fig. 4C). In comparison to the untreatedcontrol 786-O cells, incubation with either 33 µM or 33 nMAEBS caused significantly less acidification (p<0.001), leadingto a decrease of 70 and 36%, respectively. AEBS was not cytotoxic;by clonogenic survival the IC50 values for AEBS amountedto 1,360 and >5,000 µM in the 786-O and RAG cells,respectively (data not shown).

Radiosensitization is more pronouncedwhen the radiation is hypofractionated

To investigate whether a hypofractionated regimen ofradiation delivery could increase the survival difference betweenCA9-inhibited and non-inhibited RCC cells, 786-O cells expressingscrambled control shRNA or shCA9 or with and without the additionof AEBS to the medium were subjected to either one treatment of 6Gy or three treatments of 2 Gy for three consecutive days. Cellsreceiving AEBS or expressing shCA9 had significantly reducedsurvival (p<0.001) after receiving a single dose treatment of 6Gy compared to the fractionated radiation treatment of three dosesof 2 Gy (Fig. 5).

AEBS administration increasesradiosensitivity in vivo

We determined the effect of CA9 inhibition on thein vivo growth after subcutaneous injection of 786-O cells(3×106) into nude mice (n=20/group). A tumor take rateof 85% was achieved. Mice were treated with either AEBS starting 25days after cell injections or received IR on day 27 and weresacrificed 24 days later. One untreated control group was includedand one group of mice received both IR and AEBS. The tumor volumein mice treated with AEBS was significantly smaller than that inthe control mice (Fig. 6A; p=0.03ANOVA). When the subcutaneous tumors were also irradiated 27 daysafter cell injection, we observed that the combination treatmentled to decreased tumor growth compared to either AEBS or IR alone(p<0.0005 and p=0.04, respectively). At sacrifice, this led toan average increase of a mere 12% in tumor volume from the time ofIR on day 27 to sacrifice on day 51 in the combination group(Fig. 6B). In comparison, the tumorvolumes of the untreated control mice had increased in size byalmost 3-fold. Protein levels of CA9 in the mouse tumors did notdiffer between treatment groups (Fig.6C). Mass spectrometry showed that measurable andCA9-inhibitory levels of AEBS were achieved in mouse serum(Fig. 6D). Levels reached 26.5µM 18 h after starting the treatment and remained similarthroughout the experiment (data not shown).

Using immunohistochemical staining of thesubcutaneous tumors for CD31, an endothelial marker, we alsodemonstrated a decrease in the microvessel density in theirradiation-treated mice, with or without AEBS, with a linearmicrovessel length decrease of 55.6 and 64.5% compared to thecontrol (Fig. 7A and B, p=0.04 andp=0.03). Serum VEGF levels at endpoint were significantly decreasedin the AEBS-treated and irradiated mice compared to the controlmice (Fig. 7C).

Discussion

This study presents proof-of-concept for CA9 being atarget for inhibition to increase the sensitivity of RCC cells toradiation. We elected CA9 as a target as it regulates intracellularpH which has been suggested to play a key protective role inirradiated cells (9). Moreover, CA9is highly expressed on the majority of ccRCC cells, but absent innormal kidney (3). In the presentstudy, we employed two different methods to show that CA9 confersradiation resistance in RCC; we used knockdown of CA9 expressionvia transfection with specific shRNA and we inhibited the enzymaticactivity of CA9 using AEBS, a sulfonamide that has previously beenshown to efficiently inhibit CA9 by competition for the active site(8). We demonstrated, for the firsttime, that by adding AEBS to the drinking water (50–200µg/ml), we achieved serum concentrations that were severalmagnitudes (26.5 µM) higher than its Ki (Fig. 6D). While AEBS is relatively specificfor CA9, it can also efficiently inhibit carbonic anhydrase XII(CA12) with a 10-fold lower Ki (8).Employing an agent that efficiently blocks the enzymatic activityof both CA9 and CA12 could be of therapeutic advantage, as CA12functions similarly to CA9, and is also significantly expressed inRCC (10). Similar sulfonamideshave been used as anti-bacterial agents before the discovery ofantibiotics and are currently used as diuretics and anti-glaucomaagents due to their ability to mediate water transport and pressurein various tissues. Sulfonamides are well tolerated and are usuallyassociated with few side effects other than potential allergicreactions (11).

The treatment of primary RCC is currently limited tosurgical removal of the tumor via partial or radical nephrectomy orthermal ablation. Radiation therapy has typically been dismissed asa curative therapeutic option since RCC is generally regarded as aradiation-resistant tumor, even though normal kidney is consideredradiation-sensitive. For this reason, nephropathy is a complicationobserved in gastrointestinal and retroperitoneal non-Hodgkin'slymphoma patients receiving abdominal radiotherapy as their primarytreatment (12). Moreover, thehistorical landmark trial by the Copenhagen Renal Cell Cancer Studygroup which randomized RCC patients at high risk for recurrencepost-nephrectomy to receive 50 Gy vs. no radiation found nosurvival benefit and closed prematurely due to a toxicity-relatedmortality rate of 20% (13). Withthe utilization of more accurate CT-based image-guided delivery ofradiation, a retrospective study demonstrated more acceptablecomplication rates (14).Nevertheless, radiation treatment of RCC is currently limited tothe treatment of oligometastases or inoperable disease (15).

Comparing two CA9-positive RCC cell lines (human786-0 and murine RAG) with tumor cell lines that are consideredradiation-sensitive (LNCaP) (16)and radiation-resistant (LN-18) (17), we confirmed by clonogenic survivalthat these RCC cells are indeed radiation resistant (Fig. 1B). Both 786-O and RAG RCC cells weresignificantly more resistant to radiation than glioblastoma LN-18cells with IC50 values of 3.52, 2.29 and 1.94 Gy,respectively.

We performed both in vitro and in vivoinvestigations to test the potential radiation sensitizationeffects of CA9 inhibition at the level of RCC cells in vitroand using human RCC tumor xenografts where the effect on tumorvascularization can be evaluated. Our study found, for the firsttime, that in ccRCC both the pharmacological inhibition of CA9activity and knockdown of the expression of CA9 sensitized RCCcells to radiation in vitro (Figs. 2B, 4Aand B) and in vivo (Figs.3B, 6A and B). Inhibition ofCA9 activity by treatment of mice with AEBS in combination with IRhad an inhibitory effect on the growth rate of the RCC xenograftsand had a significantly larger effect compared with radiation alone(p=0.04, Fig. 6A and B). Similarly,using a xenograft model of human colorectal adenocarcinoma cells,Mclntyre et al demonstrated that knockdown of CA9 reducedthe growth rate of xenografts (18). Doyen et al showed thatsilencing of CA9 significantly increased radiation-induced celldeath in another human colorectal adenocarcinoma cell line, whileectopic expression of CA9 in fibroblasts lacking CA9 expressionimproved survival following radiation in an acidic environment(9). Treatment of mice bearinghuman colon carcinoma xenografts with acetazolamide, anothersulfonamide similar to AEBS, which has been demonstrated to inhibitCA9 in 786-O cells via induced apoptosis (19), also led to sensitization toradiation (20). This lendscredibility to the concept that CA9 is associated with radiationtolerance by limiting a cell's ability to avoid apoptosis afterirradiation.

Cells treated with AEBS or expressing shCA9exhibited significantly reduced survival (p<0.001) afterreceiving a single dose treatment of 6 Gy compared to thefractionated radiation treatment of three doses of 2 Gy. Thisreduction in survival underscores the importance of CA9 in theradiation resistance of 786-O cells and is important in the contextof RCC. Whereas conventional 1.8–3.0 Gy fractions do not causesufficient endothelial apoptosis, high-dose radiotherapyefficiently induces endothelial apoptosis via increased ceramideproduction and is expected to be detrimental in RCC, which istypically highly vascularized (21). While clinical evidence for theefficacy of stereotactic body radiotherapy of primary RCC iscurrently sparse (15), a renewedinterest in this treatment option using novel CT-based image-guidedradiation delivery for primary RCC has recently been expressedwhich awaits confirmation in the setting of prospective randomizedtrials (22). In our mice, IRdelivered as one radiotherapy dose of 6 Gy significantly reducedthe microvessel density within the tumors, alone and in combinationwith AEBS (p=0.03 and p=0.04, respectively), whereas the mouseserum VEGF levels were decreased after IR and after inhibition ofCA9 by AEBS compared with the control untreated animals (p=0.04,Fig. 7).

In conclusion, our study presents experimentalproof-of-concept for the potential role of CA9 inhibition as ameans to sensitize RCC to radiation. Specifically, it offers theconcept of pharmacological inhibition of CA9 by sulfonamides, suchas AEBS and acetazolamide, which have already been in clinical usefor decades (11). While furthermechanistic studies are underway, our data warrant consideration toperform phase I clinical trials.

Acknowledgments

This research was financially supported by McMasterSurgical Associates (W.C.M.D. and J.H.P.). We are grateful for theassistance by Dr Kirk Green and Sujan Fernando of the McMasterRegional Centre for Mass Spectrometry.

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References