the pkc/nf-kb signaling pathway induces ... · link between a major signal transduction pathway and...

Molecular and Cellular Pathobiology

The PKC/NF-kB Signaling Pathway InducesAPOBEC3BExpression inMultiple HumanCancersBrandon Leonard1,2, Jennifer L. McCann1,2, Gabriel J. Starrett1,2, Leah Kosyakovsky1,2,3,Elizabeth M. Luengas1,2, Amy M. Molan1,2, Michael B. Burns1,2,4, Rebecca M. McDougle1,2,5,Peter J. Parker6,7,William L. Brown1,2, and Reuben S. Harris1,2,8

Abstract

Overexpression of the antiviral DNA cytosine deaminaseAPOBEC3B has been linked to somatic mutagenesis inmany cancers. Human papillomavirus infection accounts forAPOBEC3B upregulation in cervical and head/neck cancers,but the mechanisms underlying nonviral malignancies areunclear. In this study, we investigated the signal transductionpathways responsible for APOBEC3B upregulation. Activationof protein kinase C (PKC) by the diacylglycerol mimic phorbol-myristic acid resulted in specific and dose-responsive increasesin APOBEC3B expression and activity, which could then

be strongly suppressed by PKC or NF-kB inhibition. PKCactivation caused the recruitment of RELB, but not RELA, tothe APOBEC3B promoter, implicating noncanonical NF-kBsignaling. Notably, PKC was required for APOBEC3B upregula-tion in cancer cell lines derived from multiple tumor types. Byrevealing how APOBEC3B is upregulated in many cancers, ourfindings suggest that PKC and NF-kB inhibitors may be reposi-tioned to suppress cancer mutagenesis, dampen tumor evolution,and decrease the probability of adverse outcomes, such as drugresistance and metastasis. Cancer Res; 75(21); 4538–47. �2015 AACR.

IntroductionSomatic mutations are essential for nearly every hallmark of

cancer (1). Mutations occur when DNA damage escapes repair.Cancer genome deep sequencing studies are confirming previ-ously known sources of mutation as well as helping to discovernew ones (2–4). Established sources ofmutation includeUV lightin skin cancer, tobacco carcinogens in lung cancer, and hydrolyticdeamination of methyl-cytosine as a function of age in nearly allcancers. One newly discovered source is the plant-derived dietarysupplement aristolochic acid, which causes A-to-T transversionmutations in liver and bladder cancers (5). A second and largersource of mutation is the APOBEC family of DNA cytosinedeaminases, which cause signature C-to-T transition and C-to-G transversion mutations in breast, head/neck, bladder, cervical,lung, and ovarian cancers (2–4, 6–11). The majority of these

mutational events are dispersed throughout the genome, but aninteresting minority is found in strand-coordinated clusterstermed kataegis (2, 12).

Expression profiling and functional studies independentlydiscovered APOBEC as a major source of mutation in cancer(6, 8). Human cells have the potential to express up to sevendistinct antiviral APOBEC3 enzymes (13). APOBEC3B is the onlyfamily member clearly upregulated in breast and ovarian cancercell lines andprimary tumors (6, 8). APOBEC3B is predominantlynuclear, and knockdown experiments demonstrated that itaccounts for all measurable DNA cytosine deaminase activity incancer cell line extracts and, likewise, is also responsible forelevated levels of genomic uracil and higher mutation rates(6, 8). In addition, APOBEC3B levels correlated with higher C-to-T and overall base substitution mutation loads. Importantly,the biochemical preference of recombinant APOBEC3B deducedin vitro closely resembles the actual cytosine mutation bias inbreast cancer as well as in several of the other tumor types listedabove (i.e., strong bias toward 50-TC dinucleotides; refs. 6–10).

Human papillomavirus (HPV) infection was recently shown toinduce APOBEC3B expression in cell culture experiments, whichhelps explain APOBEC3B upregulation and mutation biases invirus-positive cervical and head/neck tumors (14, 15). However,the mechanism responsible for APOBEC3B upregulation in othertumor types (i.e., non-HPV cancers) is presently unknown, butnot due to obvious processes such as chromosomal translocation,gene amplification, or promoter demethylation (6). Here, weshow that the protein kinase C (PKC)/NF-kB pathway specificallyinduces APOBEC3B expression, providing the first mechanisticlink between a major signal transduction pathway and cancermutagenesis. Multiple experimental approaches combined todemonstrate direct transcriptional upregulation of APOBEC3Bby a signal transduction pathway involving the classical PKCisoform PKCa and the noncanonical NF-kB transcription factor

1Department of Biochemistry, Molecular Biology, and Biophysics, Uni-versityofMinnesota,Minneapolis,Minnesota. 2MasonicCancerCenter,UniversityofMinnesota,Minneapolis,Minnesota. 3FacultyofMedicine,University of British Columbia, Vancouver, Canada. 4Department ofGenetics, Cell Biology, and Development, University of Minnesota,Minneapolis, Minnesota. 5Medical School, University of Minnesota,Minneapolis,Minnesota. 6ProteinPhosphorylationLaboratory, FrancisCrick Institute, London, United Kingdom. 7Division of Cancer Studies,King's College London, London, United Kingdom. 8Howard HughesMedical Institute, University of Minnesota, Minneapolis, Minnesota.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Author: Reuben S. Harris, Department of Biochemistry, Molec-ular Biology and Biophysics, University of Minnesota, 321 Church Street South-east, 6-155 Jackson Hall, Minneapolis, MN55455. Phone: 612-624-0457; Fax: 612-625-2163; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-15-2171-T

�2015 American Association for Cancer Research.

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RELB. PKC inhibition also leads to APOBEC3Bdownregulation inseveral cancer cell lines, suggesting that existing compounds maybe repurposed to control mutagenesis in cancer.

Materials and MethodsCell lines

Cell line information is given in Supplementary Table S1.

qRT-PCRRNA was extracted from healthy cells using the High Pure

RNA Isolation Kit (Roche), and triplicate cDNA reactions weremade using Transcriptor RT (Roche). qPCR was performedusing primer-probe combinations for each APOBEC (16).The primers for PKCa were 50-TGGTTTTGGTTCCCATTTCT and50-CATCCGGGTTTCCTGATTC and the probe was Roche UPL 1.The primers for TNFa were 50-CAGCCTCTTCTCCTTCCTGATand 50-GCCAGAGGGCTGATTAGAGA, and the probe was RocheUPL 29.

ImmunoblottingThe development of the rabbit mAb against APOBEC3Bwill be

described elsewhere (W.L. Brown and colleagues; unpublisheddata). The mAb used here is 10-87-13, and it recognizes endog-enous APOBEC3B in a variety of assays, including immunoblot-ting, as demonstrated in several experiments. This mAb cross-reacts with APOBEC3A and APOBEC3G but these proteins can beeasily distinguished by faster and slower SDS-PAGE migrationdifferences, respectively (e.g., Fig. 4). The anti-tubulin (Covance,MMS-407R) and anti-PKCa (Cell Signaling Technology, 2056P)antibodies were used according to company specifications.

Deaminase activity assaysssDNA cytosine deaminase activity assays were performed as

reported (14). A total of 4 pmol 50-ATTATTATTATTCAAATG-GATTTATTTATTTATTTATTTATTT-fluorescein was treated withcell extract containing 0.025U/rxn UDG (New England BioLabs),UDGbuffer, and 1.75U/rxn RNase A (Qiagen) for 2 hours. Abasicsites were cleaved by treatment with 100 mmol/L NaOH at 95�Cfor 10 minutes. Substrate was separated from the product using15% TBE-urea gel electrophoresis. Gels were analyzed using aFujiFilm Image Reader FLA-7000.

Phorbol-myristic acid induction and PKC/NF-kB inhibitorsA total of 2.5 � 105 cells were plated in a 6-well plate 1 day

prior to drug treatment. Phorbol-myristic acid (PMA) was thenadded to the media and incubated at 37�C with 5% CO2 for 6hours unless otherwise indicated. For experiments utilizinginhibitors, cells were pretreated with inhibitors 30 minutesprior to PMA induction (25 ng/mL). PMA (Fisher Scientific),cyclohexamide (Acros Organics), G€o6983 (Cayman Chemical),LY294002 (EMD Chemicals), UO126 (EMD Chemicals), bisin-dolylmaleimide-1 (BIM-1; Cayman Chemical), G€o6976 (EnzoLife Sciences), AEB071 (Medchem Express), BAY 11-7082 (R&DSystems), MG132 (Fisher Scientific), and TPCA-1 (CaymanChemical) were stored as recommended.

PKC knockdownspLKO.1-based lentiviruses were produced in 293T cells as

reported (6). MCF10A cells were transduced with PKCa#1 (Open Biosystems, TRCN0000001691), PKCa #2 (Open

Biosystems, TRCN0000001692), PKCa #3 (Open Biosystems,TRCN0000001690), or a control lentivirus. Ninety-six hourslater the transduced pools were treated with 25 ng/mL PMA for3 (RNA) or 6 (protein) hours and then harvested and analyzedas described above.

RNA sequencingParallel sets of MCF10A cells were treated every 8 hours with

media supplemented with PMA or DMSO for 48 hours. RNA wasextracted using an RNeasy Mini Kit (Qiagen). Total RNA wassubmitted to the University of Minnesota Genomics Center forsequencing on the IlluminaHiSeq 2000 platform. Raw reads wereanalyzed using both DESeq2 (17) and the Tuxedo suite (18) toidentify changes in mRNA expression in PMA treated versusuntreated cells.

Chromatin immunoprecipitationsMCF10A cellswere treatedwith eitherDMSOor 25ng/mLPMA

for 2 hours. Cross-linking was performed with 1% formaldehydefor 10 minutes at room temperature and quenched with 150mmol/L glycine. Cells were then lysed in Farnham Lysis Buffer at4�C for 30 minutes. Nuclei were pelleted, resuspended in RIPABuffer, and sonicated (Diagenode Pico Sonicator) to generateapproximately 600-bp DNA fragments. Immunoprecipitationswere done using Protein G Dynabeads (Invitrogen) and 2 mgantibody per sample. Samples were washed in 1mL low salt washbuffer, 1 mL high salt wash buffer, 1 mL LiCl wash buffer, andeluted at 65�C for 30 minutes. Samples were reverse cross-linkedusing 200 mmol/L NaCl and treated with Proteinase K for 12hours at 65�C. DNA was purified using a ChIP DNA Clean andConcentrator Kit (Zymo Research) and qPCRwas performedwithSYBR Green master mix (Roche) on a Roche LightCycler 480.Values represent the percentage of input DNA immunoprecipi-tated (IP DNA) and are the average of three independent biologicreplicates. All chromatin immunoprecipitation (ChIP) reagentsare listed in Supplementary Table S2.

ResultsSpecific upregulation of APOBEC3B by PMA

The first cDNAs representing APOBEC3A and/or APOBEC3Bwere cloned from PMA-treated primary human keratinocytes(19). PMA is a diacylglycerol (DAG) analogue known to triggerPKC signaling as well as activate a number of other cellularprocesses (20–23). Due to high levels of homology betweenAPOBEC3A and APOBEC3B (92%), including stretches of perfectidentity, it is not clear which gene may have been represented bythese original cDNAs. Moreover, the primary tissues used in thisstudy consisted of multiple epithelial cell types and most likelyalso infiltrating immune cells making it unclear where the cDNAsmay have originated. These distinctions are important given thefact that APOBEC3A (not APOBEC3B) is upregulated >100-foldby IFNa treatment of myeloid cell types (24, 25), and thatAPOBEC3B (not APOBEC3A) is upregulated by HPV infectionof keratinocytes (14, 15).

To resolve these issues and get a molecular handle on APO-BEC3B transcriptional regulation, a panel of cell lines was treatedwith PMA or equal amounts of DMSO as a negative control, andthe mRNA levels of all 11 human APOBEC family members werequantified by qRT-PCR (16). APOBEC3B mRNA was induced atleast 2-fold in all lines by PMA treatment (except 293T), with the

APOBEC3B Upregulation by PKC/NF-kB

www.aacrjournals.org Cancer Res; 75(21) November 1, 2015 4539




greatest magnitude occurring in the immortalized normal breastepithelial cell line MCF10A (Supplementary Fig. S1). Understandard cell culture conditions MCF10A expresses low levels ofAPOBEC3B and APOBEC3F, even lower levels of APOBEC3G andAPOBEC3H, high levels ofAPOBEC3C, and undetectable levels ofall other APOBEC family members. PMA treatment caused aspecific 100-fold upregulation of APOBEC3B mRNA, with nodetectable changes in the expression levels of any other APOBECfamily members (Fig. 1A and Supplementary Fig. S2).

APOBEC3B was induced with as little as 1 ng/mL PMA, and itsinduction was dose responsive and near maximal at 25 ng/mLPMA (Fig. 1B, histogram). APOBEC3B mRNA levels correlated

with a rise in steady-state protein levels as measured by immu-noblotting (Fig. 1B, immunoblot) and enzymatic activity asmeasured by a gel-based ssDNA cytosine deamination assay(Fig. 1B, polyacrylamide gel). Moreover, significant APOBEC3BmRNA induction was detected 30 minutes after PMA treatmentand maximal levels were observed 3 hours after treatment(Fig. 1C, histogram). APOBEC3Bprotein andactivity levels laggedshortly behind mRNA levels and persisted throughout the dura-tion of the 6-hour time course (Fig. 1C, immunoblot andpolyacrylamide gel). An extended time course revealed thatAPOBEC3B mRNA levels begin to decrease by 12 hours andreturn to near basal levels by 24 hours after PMA treatment

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Figure 1.APOBEC3B upregulation by PMA. A, a histogramshowing the specific upregulation ofAPOBEC3BmRNAbyPMA.MCF10Acellswere treatedwith PMA (25 ng/mL) orvehicle control for 6 hours, and mRNA levels were measured by qRT-PCR (mean and SD are shown for triplicate qRT-PCR reactions normalized to TBP).The same data points are shown in the context of a larger PMA dose–response experiment in Supplementary Fig. S1. B, a histogram demonstrating the doseresponsiveness of APOBEC3B upregulation by PMA. Normalization and quantification were calculated as in A. The middle images show immunoblots forcorresponding APOBEC3B and TUBULIN proteins levels, and the lower image shows DNA cytosine deaminase activity for the corresponding whole cell extracts(S, substrate; P, product; percent deamination quantified below each lane). C, a histogram depicting the rapid kinetics of APOBEC3B upregulation followingPMA treatment. MCF10A cellswere treatedwith a single concentration of PMA (25 ng/mL), andmRNA, protein, and activity levels are reported as in B. D, newproteinsynthesis is dispensable for APOBEC3B mRNA upregulation by PMA. Representative dose–response experiment for MCF10A cells treated with the indicatedconcentrations of PMA following a 30-minute pretreatment with 10 mg/mL cyclohexamide. mRNA, protein, and activity levels are reported as in B.

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Cancer Res; 75(21) November 1, 2015 Cancer Research4540




(Supplementary Fig. S3). Importantly, APOBEC3B upregula-tion is likely to be a direct result of signal transduction as thekinetics of mRNA upregulation were not affected by simulta-neously treating cells with the protein translation inhibitorcyclohexamide (Fig. 1D). Cycloheximide treatment was effec-tive as evidenced by disrupted APOBEC3B protein accumula-tion. Altogether, these data demonstrate that APOBEC3B isstrongly and specifically upregulated by a PMA-induced signaltransduction mechanism in multiple cell lines and most strong-ly in the immortalized normal breast epithelial cell lineMCF10A. Notably, APOBEC3B upregulation can be as high as100-fold and this mRNA level is on par with those observed inmany different cancer cell lines and tumor types, including alarge fraction of breast and ovarian cancers (i.e., mRNA levels 2-to 5-fold higher than those of the housekeeping gene TBP;refs. 6–8, 14).

PKC is required for APOBEC3B induction by PMAPMA is a known agonist of PKC signaling, but is also capable of

affecting other cellular processes (20–23). To determine whetherAPOBEC3B induction by PMA occurs through PKC signal trans-duction or an alternative mechanism, MCF10A cells were pre-treated for 30 minutes with varying concentrations of the pan-PKC inhibitor G€o6983 (26) and then incubated for 6 hours withan optimal amount of PMA (25 ng/mL). In comparison to strongAPOBEC3B upregulation observed with PMA treatment alone,pretreatment with G€o6983 caused a dose-responsive suppressionof APOBEC3B induction (Fig. 2A). APOBEC3Bwas suppressed tobackground levels by as little as 5 mmol/L G€o6983 (Fig. 2A).Moreover, no morphologic defects or viability issues wereobserved at these concentrations of G€o6983 (Supplementary Fig.S4). As additional controls, MCF10A cells were pretreated inparallel with the PI3K inhibitor LY294002 and theMEK inhibitorUO126prior to PMA induction (Fig. 2B andC). In both instances,APOBEC3B was still induced fully by PMA.

Human cells can express up to nine different PKC genes(20, 21, 23). The resulting PKC proteins (conventionally calledisoforms) are divisible into three classes based primarily onactivation mechanism: classical PKC (cPKC) isoforms requireboth DAG and increased levels of intracellular calcium, novelPKC (nPKC) isoforms require only DAG, and atypical PKC(aPKC) isoforms are activated by other signals. To test whichclass of PKC isoforms is responsible for APOBEC3B upregula-tion, we utilized two additional inhibitors known to havepotency similar to G€o6983, but greater selectivity for certainPKC classes. First, we pretreated MCF10A cells with BIM-1,which is known to inhibit both the cPKC and nPKC classes(27), and then induced with optimal PMA concentrations. Anearly identical dose-dependent suppression of APOBEC3Binduction was observed (Fig. 2D). This result was expected, asDAG mimics do not generally activate aPKCs. Second, wepretreated MCF10A cells with G€o6976, which is an inhibitorof cPKC proteins (28). The dose responsiveness of APOBEC3Brepression was again similar to G€o6983 (Fig. 2E). Takentogether, these chemical inhibition data strongly implicateda cPKC isoform in APOBEC3B induction by PMA.

One of themost potent and clinically advanced PKC inhibitorsis AEB071, which selectively inhibits cPKC and nPKC isoforms(29, 30). AEB071 has shown results in preclinical studies andphase I clinical trials for treatment of uveal melanoma (31–34).To fortify the pharmacologic approaches elaborated above, we

askedwhether pretreatment ofMCF10A cells with AEB071wouldproduce a similar reductive effect on PMA-induced APOBEC3Bexpression as the above PKC inhibitors. Indeed, a clear dose-dependent response was observed and, importantly, AEB071caused a complete suppression of APOBEC3B expression at500 nmol/L, which is approximately 10-fold more potent thanG€o6983, BIM-1, or G€o6976, consistent with reported lower IC50values for this molecule (Fig. 2F; refs. 26–29, 35).

RNAseq data revealed that PKCa (PRKCA) is the only cPKCisoform expressed in MCF10A cells (Fig. 2G). PKCamRNA levelswere unchanged by PMA treatment consistent with a mechanismby which PMA signals through preexisting PKCa to ultimatelystimulate APOBEC3B transcription (Fig. 2G). To further test theinvolvement of PKCa in this regulatory pathway, we depletedPKCa expression using three independent shRNA-encoding len-tiviral constructs. In each case, PKCa knockdown resulted in acorresponding reduction in the level of APOBEC3B mRNAinducedbyPMA(Fig. 2H). Immunoblots confirmedPKCa knock-down and proportional reductions in APOBEC3B (Fig. 2I). Alto-gether, the pharmacologic and genetic approaches provide astrong case for PKCa as the predominant PKC isoform drivingPMA-mediated upregulation of APOBEC3B.

NF-kB is required for APOBEC3B induction by PMAWe next asked which downstream transcription factor is

responsible for driving APOBEC3B upregulation in response toPMA. PKC is known to signal through several different transcrip-tion factors, including ERK, JNK, NF-kB, and others (20, 21, 23).We therefore started at the DNA level and examined the APO-BEC3Bpromoter region for binding sites of knownPKC-regulatedtranscription factors. Interestingly, these in silico analyses revealedseveral NF-kB binding sites within 2.5 kb of the APOBEC3Btranscriptional start site (50-GGRRNNYYCC). NF-kB is known tohave multiple roles in immunity and inflammation (36), and adirect NF-kB-mediated relay to APOBEC3B expression could bephysiologically beneficial, given APOBEC3B's known roles ininnate immunity.

To test for a mechanistic link between NF-kB and APOBEC3Btranscription, we used two compounds known to block NF-kBsignaling through independent mechanisms. First, we treatedMCF10A cells with varying amounts of BAY 11-7082, which isan NF-kB inhibitor that acts by inhibiting upstream ubiquitinassembly (37). This small molecule caused strong dose-respon-sive drops in APOBEC3B induction by PMA treatment (Fig. 3A).Second, we pretreated the MCF10A cells with a titration of theproteasome inhibitor,MG132, prior to PMA stimulation. It iswellknown that both the canonical andnoncanonicalNF-kB signalingpathways require proteasome-mediated degradation of IkB andprocessing of p100, respectively, for efficient signal transductionand that MG132 blocks these events (36). Consistent with thismechanismof action,MG132 treatment caused adose-dependentdecrease in APOBEC3B expression (Fig. 3B). As above, neitherBAY 11-7082 nor MG132 caused cell cycle or morphologicchanges throughout the durations of these experiments (Supple-mentary Fig. S4).

RNAseq data revealed that MCF10A expresses both thecanonical NF-kB components, RELA and NFKB1, and the non-canonical NF-kB components, RELB and NFKB2, and levels ofthese mRNAs are unaffected by PMA treatment (Fig. 3C).Canonical signaling is known to require IKKb, whereas nonca-nonical NF-kB signaling is strictly dependent on IKKa-catalyzed






phosphorylation of p100 (36). To distinguish between thesepathways, we used TPCA-1, which is known to have a 22-foldselectivity for IKKb (canonical) over IKKa (noncanonical;ref. 38). MCF10A cells were pretreated with a titration ofTPCA-1 concentrations spanning the IC50 values of both pro-teins, and then PMA was used to induce APOBEC3B upregula-tion. APOBEC3B expression was inhibited closer to the reported

IC50 of IKKa, consistent with involvement of the noncanonicalNF-kB pathway (Fig. 3D). As an additional control, we alsoanalyzed TNFa, which is regulated by the canonical pathway(39, 40). As expected, TNFa expression was inhibited by muchlower concentrations of TCPA-1 confirming the differential selec-tively of this compound and further implicating the noncanonicalNF-kB pathway (Fig. 3D).

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Figure 2.APOBEC3B upregulation by PMA is dependent on PKC. A–F, histograms reporting the impact of the indicated small molecules on PMA-induced APOBEC3Bupregulation.APOBEC3B inductionwas inhibitedbyG€o6983 (pan-PKC inhibitor), BIM-1 (classical andnovel PKC inhibitor), G€o6976 (classical PKC selective inhibitor),and AEB071 (preclinical PKC inhibitor) but not by LY294002 (PI3K inhibitor) or UO126 (MEK inhibitor). MCF10A cells were treated with PMA following a30-minute pretreatment with the indicated concentrations of each inhibitor. mRNA expression is reported as the mean of three independent qRT-PCR reactionsnormalized to TBP (error bars, SD from triplicate assays). G, histogram depicting PKC isoforms expressed in MCF10A cells treated with PMA or vehiclecontrol. mRNA expression was determined by RNA-seq and is reported as fragments per kilobase of exon per million fragments mapped (FKPM) and normalized toTBP. H, histogram showing that PKCa knockdown inhibits APOBEC3B induction by PMA. MCF10A cells were treated with PMA following PKCa knockdownusing three independent PKCa-specific shRNA encoding lentiviruses and a control. mRNA levels for both PKCa (blue) and APOBEC3B (red) are reported.I, immunoblots confirming PKCa knockdowns and proportional reductions in APOBEC3B protein levels.

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Figure 3.NoncanonicalNF-kBsignaling is responsible forAPOBEC3BupregulationbyPMA.AandB, histogramsdepicting thedose-responsive inhibitionofPMA-inducedAPOBEC3Bupregulation by BAY 11-7082 (ubiquitination inhibitor) and MG132 (proteasome inhibitor). MCF10A cells were treated with PMA following a 30-minute pretreatmentwith the indicated concentrationsof each inhibitor.APOBEC3BmRNAexpression is reported as themeanof three independent qRT-PCR reactions normalized toTBP (errorbars reportSD fromtriplicateassays).C, histogramdepictingNF-kB subunitmRNA levels inMCF10Acells treatedwithPMAorvehicle control. ExpressionwasdeterminedbyRNA-seq and is reported as per million fragments mapped (FKPM) and normalized to TBP. D, plot depicting inhibition of PMA-induced APOBEC3B expression by the IkBkinase (IKK) inhibitor, TPCA-1, near the IC50 for IKKa, not IKKb. The MCF10A cells were treatedwith PMA following treatment with varying concentrations of TPCA-1. TNFa(blue) andAPOBEC3B (red)mRNA levels are reported as themeanof three independent qRT-PCR reactions normalized to TBP (error bars report SD from triplicate assays).Thedotted linesdenote previously reported in vitro IC50 values for IKKa and IKKb inhibition by TPCA-1 (38). E, histogram showing the kinetics ofNFKBIAupregulationPMA.MCF10Acellswere treatedwithPMA for the indicated times andmRNAvalueswere quantified as inA. F,APOBEC3BandNFKBIApromoter regions contain several putativeNF-kB binding sites (TSS, transcriptional start site). G, RELB and p100/p52 are specifically and robustly recruited to the APOBEC3B promoter region by PMA.ChIPwas performed after a treatmentwith PMAor vehicle control for 2 hours.APOBEC3B sites 4 and 5 and the twoNFKBIA sites are reported together because theyweretoo close to each other to be distinguished by this procedure. qPCR results are reported as a percentage of the total chromatin input.






RELB and p100/p52 are recruited to the APOBEC3B promoterregion in response to PMA

We next performed ChIP experiments to further test whetherthe noncanonical NF-kB pathway is responsible for upregulatingAPOBEC3B. Primer sets were designed for each of the predictedNF-kB binding sites near the APOBEC3B transcriptional start site(Fig. 3F). As a control, an additional primer set was made for thepromoter region of NFKBIA, which contains NF-kB binding sitesand is also upregulated by the PMA with similar kinetics asAPOBEC3B (NFKBIA encodes IkB; Fig. 3E and F). ChIP wasperformed for RELA, RELB, p100/p52, RNA POL II (positivecontrol), and isotype-matched IgG (negative control). Asexpected, RELA, RELB, p100/p52, and RNA POL II were all boundto the NFKBIA promoter following PMA treatment (Fig. 3G). Wealso found RNA POL II bound to the APOBEC3B gene near thetranscriptional start site and throughout the gene body inresponse to PMA (Fig. 3G). Interestingly, both RELB and p100/

p52were recruited to the same sites as RNA POL II following PMAtreatment, indicating that these factors are also involved in drivingAPOBEC3B expression in response to PMA (Fig. 3G). An expand-ed ChIP experiment replicated these data and showed that RNAPOL II, RELB, and p100/p52 binding are dependent on PKCsignaling as treatment with AEB071 completely ablated all bind-ing to the APOBEC3B promoter (Supplementary Fig. S5). TheseChIP data strongly implicate the noncanonical NF-kB pathway,specifically the RELB and p100/p52 heterodimer (and not RELAand p105/p50), in directly inducing APOBEC3B transcription inresponse to PMA activation of PKC.

Endogenous APOBEC3B expression requires PKC in multiplecancer cell lines

We next asked whether the constitutively high levels of endog-enous APOBEC3B observed in many human cancer cell linesoccurs through the PKC pathway (6, 8, 14). For this series of

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Figure 4.The PKC pathway drives endogenous APOBEC3B expression in cancer cells. A, APOBEC3BmRNA levels in representative breast, ovarian, bladder, and head/neckcancer cell lines. mRNA expression is reported as the mean of three independent qRT-PCR reactions normalized to TBP (error bars report SD from triplicateassays). B, AEB071 downregulates APOBEC3B inmultiple cancer cell lines. The histogram reportsAPOBEC3BmRNA levels normalized to the vehicle-treated controlfor each line. The dotted line represents a 50% decrease of APOBEC3B expression due to AEB071. The corresponding immunoblots show APOBEC3B andTUBULIN levels. Each line was treated with AEB071 (10 mmol/L) or vehicle control for 48 hours prior to mRNA and protein analysis.

Leonard et al.





experiments, we selected 4 breast, 4 ovarian, 4 bladder, and 4head/neck cancer cell lines expressing a 10-fold range of endog-enousAPOBEC3BmRNA levels (Fig. 4A). Each linewas treated for48 hours with 10 mmol/L AEB071, the most potent PKC inhibitoridentified above, and then APOBEC3B mRNA and protein levelswere quantified by qRT-PCR and immunoblotting. As above, noeffects on the cell cycle or cell viability were observed (Supple-mentary Fig. S6). This is important because higher concentrationsof AEB071 are known to cause cell-cycle perturbations andapoptosis in certain cell types (31–33). APOBEC3B mRNA levelswere reduced by more than half in 7/16 cell lines, including thebreast cancer cell lines MDA-MB-468, MDA-MB-453, andHCC1806, the ovarian cancer cell line OVCAR5, and the head/neck lines SQ-20B, JSQ3, and TR146 (Fig. 4B, histogram).Changes of protein levels largely mirrored the mRNA results (Fig.4B, immunoblot). Interestingly, several cell lines, including all ofthe bladder cancer cell lines, showed little decrease in APOBEC3Bexpression upon treatment with AEB071, suggesting that at leastone additional induction mechanism exists. Altogether, thesedata demonstrate that the PKC axis is responsible for the consti-tutive upregulation of endogenous APOBEC3B in a variety ofcancer cell lines representing multiple distinct cancer types.

DiscussionThese studies are the first to establish mechanistic linkages

between the PKC/NF-kB signal transduction pathway and upre-gulation of the DNAmutating enzyme APOBEC3B in cancer. Ourstudies suggest amodel in which PKCa activation signals throughthe noncanonicalNF-kBpathway and results in the recruitment ofRELB to the APOBEC3B gene and its transcriptional activation(Fig. 5). This mechanism is remarkably specific to APOBEC3B, asexpression of the relatedAPOBEC familymembers is not affected.This specificity is concordantwith our prior studies indicating thatAPOBEC3B is the only DNA deaminase family member upregu-lated in these and other cancer types in comparison to normaltissues (6–8, 14). Moreover, PKC inhibitor studies with breast,head/neck, bladder, andovarian cancer cell lines demonstrate thatthe PKC/NF-kB pathway contributes to the constitutively high

levels of endogenous APOBEC3B associated with cancer muta-genesis. Additional studieswill beneeded to determine the preciseproportions of each tumor type affected by this APOBEC3Bupregulation mechanism that, based on prior studies from ourlaboratory and others, is expected to endow cancer cells withmutational fuel for accelerated tumor evolution.

A recent publication implicated both the interferon responseand the canonical and noncanonical NF-kB pathways in APO-BEC3A and APOBEC3B upregulation and clearance of HBV epi-somes from infected cells (41). Activation of the lymphotoxin-breceptor through treatment of infected hepatocytes with bivalentor tetravalent antibodies led to the nuclear translocation of bothRELA and RELB and the activation of known NF-kB pathwaygenes. These antibody treatments also led to the upregulation ofAPOBEC3A and/orAPOBEC3B and to the gratuitous deaminationof HBV cccDNA cytosines, viral DNA degradation, and long-termvirus suppression. Taken together with our results presented here,it is tempting to speculate that both thePKCand the lymphotoxin-b receptor signaling mechanisms converge upon the noncanon-ical RELB-dependent NF-kB pathway in order to activate APO-BEC3B expression. Thus, our work suggests additional strategiessuch as PMA treatment to induce APOBEC3B upregulation andclearance of HBV from infected hepatocytes. However, thesestrategies may induce collateral damage through genomic DNAmutagenesis and should be approached carefully.

Clear evidence for APOBEC3B overexpression and mutationsignatures in cervical and head/neck cancers suggested that HPVinfection might trigger an innate immune response that includesDNA deaminase upregulation (7, 9). Subsequent work demon-strated that infection by high-risk (not low-risk) HPV types causesthe specific upregulation of APOBEC3B, suggesting that this is notsimply a gratuitous response to viral infection (14, 15).Moreover,the E6 oncoprotein alone from high-risk types was sufficient totrigger APOBEC3B upregulation (14). It is notable that the overallfold induction by HPV is lower than that described here, duepartly to higher background and partly to a smaller magnitude ofinduction. The mutator phenotype induced by HPV infection islikely fueling tumor evolution as the pattern of PI3K-activatingmutations in HPV-positive tumors is biased toward cytosine

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Figure 5.Model for APOBEC3B upreglation by thePKC/NF-kB pathway. PKCa activation by DAGor PMA leads to IKKa phosphorylation andproteasome-dependent cleavage of NF-kBsubunit p100 into the transcriptionally active p52form. The noncanonical NF-kB heterodimercontaining p52 and RELB is then recruited to theAPOBEC3B promoter to drive transcription. Redlabels represent the small molecules andapproaches used to interrogate this signaltransduction pathway.






mutations in APOBEC-like motifs in the helical domain of thekinase, whereas the pattern in HPV-negative tumors is splitbetween the helical and kinase domains of the enzyme(10, 11). Obviously, HPV-mediated upregulation of APOBEC3Bonly impacts cervical cancers and a proportion of head/neck andbladder carcinomas. In contrast, a larger number of tumor types islikely to be using the mechanism described here.

It will also be interesting to determine the relationship betweenAPOBEC3B upregulation and immunotherapy responsiveness, asrecent reports have suggested that increased tumor mutationloads correlate with stronger anticancer immune responses(42, 43). It may therefore be useful to induce APOBEC3B, asdescribed here, to increase tumor neoantigens in order to boostefficacies of current immunotherapies.

Although PKC mutations are rare in cancer, altered expressionof several PKC isoforms is observed and associated with poorclinical outcomes (20, 21). In addition, mutations in GNAQ andGNA11 occur in approximately half of all uveal melanomasamples [(44, 45); illustrated as Gq in Fig. 5]. Inhibition of PKCin these uveal tumors leads to clinical benefits attributed to cell-cycle arrest and apoptosis (31–34). It is possible that down-regulation of APOBEC3B and a subsequent decrease in tumorevolution through loweredmutation rates may also contribute tothese encouraging clinical responses. Based on substantive priorwork from our lab and others demonstrating a major role forAPOBEC3B in cancer mutagenesis and correlating high levels ofAPOBEC3B with poor prognoses for ER-positive breast cancers(46, 47), togetherwith the studies presented here, we propose thatexisting inhibitors of the PKC/NF-kB axis such as AEB071may berepurposed to treat primary tumors in combination with existingtherapies and help prevent detrimental outcomes such as drugresistance and metastases.

Disclosure of Potential Conflicts of InterestR.S.Harris has ownership interest (includingpatents) inApoGenBiotechnol-

ogies Inc. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: B. Leonard, G.J. Starrett, L. Kosyakovsky, P.J. Parker,R.S. HarrisDevelopment of methodology: B. Leonard, R.M. McDougleAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): B. Leonard, J.L. McCann, L. Kosyakovsky, E.M.Luengas, A.M. Molan, W.L. BrownAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis):B. Leonard, J.L.McCann,G.J. Starrett, L. Kosyakovsky,R.S. HarrisWriting, review, and/or revision of the manuscript: B. Leonard, J.L. McCann,G.J. Starrett, A.M. Molan, R.S. HarrisAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): B. LeonardStudy supervision: B. Leonard, R.S. HarrisOther (performed initial experiments and research): M.B. Burns

AcknowledgmentsThe authors thank lab members for critical discussions and P. Howley, P.

Lambert, S. Kaufmann, D. Yee, and M. Herzberg for providing cancer cell lines,C. McDonald-Hyman and B. Blazar for PKC inhibitors, and C. Deip and C.Lange for PI3K and MEK inhibitors.

Grant SupportB. Leonard is supported in part by a Cancer Biology Training Grant (NIH T32

CA009138) and G.J. Starrett by a National Science Foundation GraduateResearch Fellowship (DGE 13488264). Cancer research in the Harris laboratoryis supported by grants from the Department of Defense Breast Cancer ResearchProgram (BC121347), Jimmy V Foundation for Cancer Research, NorwegianCentennial Chair Program, Minnesota Ovarian Cancer Alliance, MinnesotaPartnership for Biotechnology and Medical Genomics, and Randy ShaverCancer Research and Community Fund. R.S. Harris is an Investigator of theHoward Hughes Medical Institute.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received August 7, 2015; accepted August 12, 2015; published OnlineFirstSeptember 29, 2015.

References1. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell

2011;144:646–74.2. Nik-Zainal S, Alexandrov LB, Wedge DC, Van Loo P, Greenman CD, Raine

K, et al. Mutational processes molding the genomes of 21 breast cancers.Cell 2012;149:979–93.

3. Alexandrov LB, Nik-Zainal S, Wedge DC, Aparicio SAJR, Behjati S, BiankinAV, et al. Signatures of mutational processes in human cancer. Nature2013;500:415–21.

4. LawrenceMS, Stojanov P, Polak P, Kryukov GV, Cibulskis K, Sivachenko A,et al. Mutational heterogeneity in cancer and the search for new cancer-associated genes. Nature 2013;499:214–8.

5. Poon SL, Pang S-T,McPherson JR, YuW,Huang KK,Guan P, et al. Genome-wide mutational signatures of aristolochic acid and its application as ascreening tool. Sci Transl Med 2013;5:197ra101.

6. Burns MB, Lackey L, Carpenter MA, Rathore A, Land AM, Leonard B, et al.APOBEC3B is an enzymatic source of mutation in breast cancer. Nature2013;494:366–70.

7. Burns MB, Temiz NA, Harris RS. Evidence for APOBEC3B mutagenesis inmultiple human cancers. Nat Genet 2013;45:977–83.

8. Leonard B, Hart SN, Burns MB, Carpenter MA, Temiz NA, Rathore A, et al.APOBEC3Bupregulation and genomicmutationpatterns in serous ovariancarcinoma. Cancer Res 2013;73:7222–31.

9. Roberts SA, Lawrence MS, Klimczak LJ, Grimm SA, Fargo D, Stojanov P,et al. An APOBEC cytidine deaminasemutagenesis pattern is widespread inhuman cancers. Nat Genet 2013;45:970–6.

10. Henderson S, Chakravarthy A, Su X, Boshoff C, Fenton TR. APOBEC-mediated cytosine deamination links PIK3CAhelical domainmutations tohuman papillomavirus-driven tumor development. Cell Rep 2014;7:1833–41.

11. Cancer Genome Atlas Network. Comprehensive genomic characteriza-tion of head and neck squamous cell carcinomas. Nature 2015;517:576–82.

12. Roberts SA, Sterling J, ThompsonC,Harris S,MavD, ShahR, et al. Clusteredmutations in yeast and in human cancers can arise from damaged longsingle-strand DNA regions. Mol Cell 2012;46:424–35.

13. Refsland EW, Harris RS. The APOBEC3 family of retroelement restrictionfactors. Curr Top Microbiol Immunol 2013;371:1–27.

14. Vieira VC, Leonard B, White EA, Starrett GJ, Temiz NA, Lorenz LD,et al. Human papillomavirus E6 triggers upregulation of the antiviraland cancer genomic DNA deaminase APOBEC3B. MBio 2014;5:e02234–14.

15. Warren CJ, Xu T, Guo K, Griffin LM, Westrich JA, Lee D, et al. APOBEC3Afunctions as a restriction factor of human papillomavirus. J Virol 2015;89:688–702.

16. Refsland EW, Stenglein MD, Shindo K, Albin JS, Brown WL, Harris RS.Quantitative profiling of the full APOBEC3 mRNA repertoire in lympho-cytes and tissues: implications for HIV-1 restriction. Nucleic Acids Res2010;38:4274–84.

17. Love MI, Huber W, Anders S. Moderated estimation of fold change anddispersion for RNA-seq data with DESeq2. Genome Biol 2014;15:550.


Leonard et al.




18. Trapnell C, Roberts A, Goff L, Pertea G, KimD, Kelley DR, et al. Differentialgene and transcript expression analysis of RNA-seq experiments withTopHat and Cufflinks. Nat Protoc 2012;7:562–78.

19. Madsen P, Anant S, Rasmussen HH, Gromov P, Vorum H, Dumanski JP,et al. Psoriasis upregulated phorbolin-1 shares structural but not functionalsimilarity to the mRNA-editing protein apobec-1. J Invest Dermatol 1999;113:162–9.

20. RosseC, LinchM,Kermorgant S,CameronAJM,Boeckeler K, Parker PJ. PKCand the control of localized signal dynamics. Nat Rev Mol Cell Biol2010;11:103–12.

21. Griner EM, Kazanietz MG. Protein kinase C and other diacylglyceroleffectors in cancer. Nat Rev Cancer 2007;7:281–94.

22. Spitaler M, Cantrell DA. Protein kinase C and beyond. Nat Immunol2004;5:785–90.

23. Mackay HJ, Twelves CJ. Targeting the protein kinase C family: are we thereyet? Nat Rev Cancer 2007;7:554–62.

24. Stenglein MD, Burns MB, Li M, Lengyel J, Harris RS. APOBEC3 proteinsmediate the clearance of foreign DNA from human cells. Nat Struct MolBiol 2010;17:222–9.

25. Thielen BK, McNevin JP, McElrath MJ, Hunt BVS, Klein KC, Lingappa JR.Innate immune signaling induces high levels of TC-specific deaminaseactivity in primary monocyte-derived cells through expression of APO-BEC3A isoforms. J Biol Chem 2010;285:27753–66.

26. Gschwendt M, Dieterich S, Rennecke J, Kittstein W, Mueller HJ, JohannesFJ. Inhibition of protein kinase Cmu by various inhibitors. Differentiationfrom protein kinase c isoenzymes. FEBS Lett 1996;392:77–80.

27. Toullec D, Pianetti P, Coste H, Bellevergue P, Grand-Perret T, Ajakane M,et al. The bisindolylmaleimide GF 109203X is a potent and selectiveinhibitor of protein kinase C. J Biol Chem 1991;266:15771–81.

28. Martiny-Baron G, Kazanietz MG, Mischak H, Blumberg PM, Kochs G, HugH, et al. Selective inhibition of protein kinase C isozymes by the indolo-carbazole G€o 6976. J Biol Chem 1993;268:9194–7.

29. Evenou J-P,Wagner J, ZenkeG, BrinkmannV,Wagner K,Kovarik J, et al. Thepotent protein kinase C-selective inhibitor AEB071 (sotrastaurin) repre-sents a new class of immunosuppressive agents affecting early T-cellactivation. J Pharmacol Exp Ther 2009;330:792–801.

30. Wagner J, Matt von P, Sedrani R, Albert R, Cooke N, Ehrhardt C, et al.Discovery of 3-(1H-indol-3-yl)-4-[2-(4-methylpiperazin-1-yl)quinazolin-4-yl]pyrrole-2,5-dione (AEB071), a potent and selective inhibitor of pro-tein kinase C isotypes. J Med Chem 2009;52:6193–6.

31. Wu X, Li J, Zhu M, Fletcher JA, Hodi FS. Protein kinase C inhibitor AEB071targetsocularmelanomaharboringGNAQmutationsviaeffectson thePKC/Erk1/2 and PKC/NF-kB pathways. Mol Cancer Ther 2012;11:1905–14.

32. ChenX,WuQ, Tan L, PorterD, JagerMJ, EmeryC, et al. CombinedPKCandMEK inhibition in uveal melanoma with GNAQ and GNA11 mutations.Oncogene 2014;33:4724–34.

33. Musi E, Ambrosini G, de Stanchina E, Schwartz GK. The phosphoinositide3-kinase a selective inhibitor BYL719 enhances the effect of the proteinkinase C inhibitor AEB071 in GNAQ/GNA11-mutant uveal melanomacells. Mol Cancer Ther 2014;13:1044–53.

34. Piperno-Neumann S, Kapiteijn E, Larkin JMG, Carvajal RD, Luke JJ, SeifertH, et al. Phase I dose-escalation study of the protein kinase C (PKC)inhibitorAEB071 inpatientswithmetastatic uvealmelanoma. J ClinOncol2014;32:9030.

35. Wagner J, Matt von P, Faller B, Cooke NG, Albert R, Sedrani R, et al.Structure-activity relationship and pharmacokinetic studies of sotrastaurin(AEB071), a promising novelmedicine for preventionof graft rejection andtreatment of psoriasis. J Med Chem 2011;54:6028–39.

36. Vallabhapurapu S, Karin M. Regulation and function of NF-kappaBtranscription factors in the immune system. Annu Rev Immunol 2009;27:693–733.

37. Strickson S, Campbell DG, Emmerich CH, Knebel A, Plater L, Ritorto MS,et al. The anti-inflammatory drug BAY 11-7082 suppresses the MyD88-dependent signalling network by targeting the ubiquitin system. Biochem J2013;451:427–37.

38. Podolin PL, Callahan JF, Bolognese BJ, Li YH, Carlson K, Davis TG, et al.Attenuation of murine collagen-induced arthritis by a novel, potent,selective small molecule inhibitor of IkappaB Kinase 2, TPCA-1 (2-[(ami-nocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide),occurs via reduction of proinflammatory cytokines and antigen-induced Tcell Proliferation. J Pharmacol Exp Ther 2005;312:373–81.

39. Foxwell B, Browne K, Bondeson J, Clarke C, de Martin R, Brennan F, et al.Efficient adenoviral infection with IkappaB alpha reveals that macrophagetumor necrosis factor alpha production in rheumatoid arthritis is NF-kappaB dependent. Proc Natl Acad Sci 1998;95:8211–5.

40. Liu H, Sidiropoulos P, Song G, Pagliari LJ, Birrer MJ, Stein B, et al. TNF-alpha gene expression in macrophages: regulation by NF-kappa B isindependent of c-Jun or C/EBP beta. J Immunol 2000;164:4277–85.

41. Lucifora J, Xia Y, Reisinger F, Zhang K, Stadler D, Cheng X, et al. Specific andnonhepatotoxic degradation of nuclear hepatitis B virus cccDNA. Science2014;343:1221–8.

42. Snyder A, Makarov V, Merghoub T, Yuan J, Zaretsky JM, Desrichard A, et al.Genetic basis for clinical response to CTLA-4 blockade in melanoma.N Engl J Med 2014;371:2189–99.

43. Lo JA, Fisher DE. The melanoma revolution: from UV carcinogenesis to anew era in therapeutics. Science 2014;346:945–9.

44. Van Raamsdonk CD, Bezrookove V, Green G, Bauer J, Gaugler L, O'BrienJM, et al. Frequent somatic mutations of GNAQ in uveal melanoma andblue naevi. Nature 2009;457:599–602.

45. Van Raamsdonk CD, Griewank KG, Crosby MB, Garrido MC, Vemula S,Wiesner T, et al. Mutations in GNA11 in uveal melanoma. N Engl J Med2010;363:2191–9.

46. Sieuwerts AM, Willis S, Burns MB, Look MP, Meijer-Van Gelder ME,Schlicker A, et al. Elevated APOBEC3B correlates with poor outcomesfor estrogen-receptor-positive breast cancers. Horm Cancer 2014;5:405–13.

47. Cescon DW, Haibe-Kains B, Mak TW. APOBEC3B expression in breastcancer reflects cellular proliferation, while a deletion polymorphism isassociated with immune activation. Proc Natl Acad Sci U S A 2015;112:2841–6.






2015;75:4538-4547. Published OnlineFirst September 29, 2015.Cancer Res Brandon Leonard, Jennifer L. McCann, Gabriel J. Starrett, et al. in Multiple Human Cancers

B Signaling Pathway Induces APOBEC3B ExpressionκThe PKC/NF-

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