PRAK Suppresses Oncogenic ras-Induced Hematopoietic Cancer Development by Antagonizing ... ·...

Signaling and Regulation

PRAK Suppresses Oncogenic ras-Induced HematopoieticCancer Development by Antagonizing the JNK Pathway

Naoto Yoshizuka1, Maoyi Lai2, Rong Liao1, Ryan Cook2, Changchun Xiao2, Jiahuai Han3, and Peiqing Sun1

AbstractThe p38 mitogen-activated protein kinase (MAPK) pathway regulates multiple physiologic and pathologic

processes, including cancer development. PRAK, a p38 substrate protein kinase, has previously been implicated inthe suppression of skin carcinogenesis. In the current study, we show that PRAK deletion accelerates hematopoieticcancer development in a mouse model harboring an oncogenic ras allele, Em-N-RasG12D, specifically expressed inhematopoietic cells. Further investigation reveals that enhanced hematopoietic tumorigenesis by PRAK deficiencyis associated with hyperactivation of the c-jun-NH2-kinase (JNK) pathway both in vivo and in primaryhematopoietic cells isolated from spleens. In primary splenocytes, PRAK deficiency further enhanced oncogenicras-induced cell proliferation and promoted ras-mediated colony formation on semisolid medium in a JNK-dependent manner. In addition, deletion of PRAK leads to abrogation of ras-induced accumulation of senescencemarkers. These findings indicate that PRAK suppresses hematopoietic cancer formation in this mouse model byantagonizing oncogenic ras-induced activation of the JNKpathway.Our results suggest that PRAKmay function asa tumor suppressor in multiple types of cancers. Mol Cancer Res; 10(6); 810–20. �2012 AACR.

IntroductionThe p38 mitogen-activated protein kinase (MAPK) was

initially identified as a mediator of inflammation and stressresponses (1–4). Recent studies have revealed a novel func-tion of the p38 pathway in tumor-suppressing cellularresponses such as oncogene-induced senescence, cell contactinhibition, and DNA damage responses (5). These findingssuggest that p38 has a tumor-suppressing function. Indeed,tissue-specific deletion of p38a promotes the developmentof chemical-induced liver cancer and K-rasG12V–inducedlung cancer in mice (6, 7). Moreover, deletion of Wip1, ap38 phosphatase frequently amplified in human breasttumors, leads to p38 activation and reduced erbB2- andras-mediated mammary tumorigenesis in mice (8, 9).Like other MAPK pathways, the functions of p38 are

mediated by its downstream substrates. Numerous p38substrates, including serine/threonine protein kinases, tran-scription factors, and cell-cycle regulators, have been iden-tified that mediate various p38 functions (10). The p38downstream kinase substrates include MAPK-activated

kinases 2 (MK2) and 3 (MK3), MAPK-interacting proteinkinase 1 (MNK1), p38-regulated/activated kinase (PRAK),mitogen- and stress-activated protein kinases-1 (MSK1) and-2 (MSK2), and casein kinase 2 (CK2). Upon phosphory-lation by p38, these Ser/Thr protein kinases activate sub-strates such as heat shock proteins, transcription factors,translation initiation factors, and proteins that regulatemRNA stability. In a previous study, we showed that theability of p38 to mediate oncogene-induced senescence andtumor suppression relies, at least in part, on PRAK. Repli-cative senescence is a stable proliferative arrest associatedwith the exhaustion of replicative potential as a result oftelomere erosion during cell divisions (11). Telomerelength–independent, senescence-like proliferative arrest canalso be induced in young cells by activated oncogenes such asras (12). This second type of arrest state is thus operativelytermed as oncogene-induced premature senescence. Likeapoptosis, oncogene-induced senescence serves as an anti-tumorigenic defense mechanism (13–16). Our studiesrevealed that PRAK is essential for ras-induced senescenceand that PRAK deficiency disrupts oncogene-inducedsenescence and enhances 7,12-dimethylbenz(a)anthracene(DMBA)-induced skin carcinogenesis (17).While our previous results indicate that PRAK suppresses

skin carcinogenesis (17), it is unclear whether the tumor-suppressing activity of PRAK also operates in other types ofcancers. To this end, the consequence of PRAK inactivationwas analyzed in the current study using an N-rasG12D

transgenic mouse model previously shown to develophematopoietic cancer (15). Our data show that PRAKdeletion also accelerates tumor formation in this N-rasG12D

transgenic line and enhances cell proliferation and soft agar

Authors' Affiliations: Departments of 1Molecular Biology and 2Immunol-ogy and Microbial Science, The Scripps Research Institute, La Jolla,California; and 3State Key Laboratory of Cellular Stress Biology and Schoolof Life Sciences, Xiamen University, Xiamen, Fujian, China

Note: This Scripps manuscript number is 21440.

Corresponding Author: Peiqing Sun, Department of Molecular Biology,The Scripps Research Institute, 10550N. Torrey Pines Rd,MB-41, La Jolla,CA 92037. Phone: 858-784-9710; Fax: 858-784-9067; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-11-0576

�2012 American Association for Cancer Research.

MolecularCancer

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colony formation induced by activated ras in primarysplenocytes. Further studies indicate that enhancedhematopoietic tumorigenesis by PRAK deficiency isaccompanied by hyperinduction of the c-jun-NH2-kinase(JNK) pathway and downregulation of a subset of senes-cence markers, and that inhibition of JNK activity attenu-ates the hyperproliferation induced by oncogenic ras inhematopoietic cells isolated from PRAK-deficient mice.These findings suggest that PRAK may suppress thedevelopment of a broad range of cancers and that in thecase of ras-induced hematopoietic cancer, the tumor-suppressing function of PRAK may be attributed to itsability to antagonize the activation of tumor-promotingMAPK pathways by oncogenic ras.

Materials and MethodsMouse strainsTheEm-N-RasG12D transgenic strain (18)was intercrossed

with mice harboring a targeted deletion in the PRAK gene(17) to generate the Em-N-RasG12D;PRAKþ/� line (F1),which was then crossed with PRAKþ/� to obtain Em-N-RasG12D;PRAKþ/þ, Em-N-RasG12D;PRAKþ/�, and Em-N-RasG12D;PRAK�/� littermates (F2) for observation ofhematopoietic cancer development. The mice were in theBL6/129 background. All the mice carried only one copy ofthe ras transgene, as the F1 mice were heterozygous for thetransgene. Animals were genotyped by allele-specific PCR asdescribed previously. Time to death was defined as thelatency between birth and death or a terminal disease stageas indicated by symptoms of severe sickness (lethargy,distress, swollen neck, abdominal bulging). Statistical anal-ysis of Kaplan–Meier survival plots is based on the log-rank(Mantel–Cox) test. After euthanasia of mice with deepanesthesia byCO2, tissues were processed for histopathologyand subsequent staining with hematoxylin and eosin. Car-diac or tail vein blood was collected into Microvette tubes(Sarstedt) and analyzed by aHemavet 950 (Drew Scientific).

Immunogenotyping of hematopoietic tumor cellsOrgans such as spleen, thymus, and bone marrow were

isolated from mice and minced in PBS. The mixture wasthen filtered through a 70-mm nylon mesh (BD Falcon) toobtain single-cell suspensions. Isolated cells were stainedwith antibodies against CD11b (R-phycoerythrin–conju-gated) and GR-1 (Tricolor-conjugated), or CD3 (R-phyco-erythrin–conjugated) and B220 (Tricolor-conjugated;Caltag Laboratories), and analyzed by flow cytometry.

Isolation of primary splenocytes and transduction withrecombinant retroviruses ex vivoSpleen from 8- to 12-week-old nontransgenic mice served

as the source for primary splenocyte preparations. Isolatedspleens were minced in PBS and filtered through a 70-mmnylon mesh to obtain single-cell suspensions. Erythrocyteswere removed by incubating the cell suspension with Ackbuffer (0.15 mol/L NH4Cl, 1 m mol/L KHCO3, and 0.1 mmol/L EDTA). The primary splenocytes were cultivated in a

medium containing 45% Iscove minimum essential media,45% Dulbecco's Modified Eagle's Media (DMEM), 10%FBS, 4 mmol/L L-glutamine, 100 U/mL Pen-strep and 25mmol/L b-mercaptoethanol (cell culture grade; Life Tech-nologies). The medium was conditioned for 2 to 3 days ongamma-irradiated (20 Gy) NIH3T3 cells before addition tosplenocytes.Two retroviruses encoding short hairpin RNA (shRNA)

targeting both JNK1 and JNK2 (shJNK1/2-S1 and -S2)were based on a published article (19) and have the followingtargeting sequences, 50-AAA GAA UGU CCU ACC UUCUTT-30 and 50-AGA AGG UAG GAC AUU CUU UTT-30, respectively. Retroviruses encoding JNK1/2 shRNA,H-RasG12V (MSCV-puro-H-RasG12V), or N-RasG12D

(MSCV-puro-N-RasG12D) or corresponding vector controlswere packaged in an ecotropic packaging cell line LinX-E asdescribed previously (20, 21), except that the viruses wereproduced in the NIH3T3-conditioned splenocyte medium.A total of 1 � 106 freshly isolated splenocytes were mixedwith 2 mL of viral supernatant in the presence of 4 mg/mLpolybrene, plated onto 6-well plates, and centrifuged at2,700 rpm for 3 hours. After incubation at 32�C forovernight, cells were subjected to a second round of retroviralinfection and then collected by centrifugation and resus-pended in NIH3T3-conditioned splenocyte medium. Cellstransduced with the retroviruses were selected with 1 mg/mLpuromycin or 50 mg/mL hygromycin B.

Analysis of proliferation and colony-forming ability ofsplenocytes cultured ex vivoTo measure the rate of proliferation, primary spleno-

cytes transduced with oncogenic ras or vector control wereseeded onto 12-well plates in triplicates at a density of 1 �104 to 5 � 104 cells per well in NIH3T3-conditionedsplenocyte medium. Cells were harvested 4 to 8 days laterand their numbers counted in hemocytometer. To mea-sure colony formation on semisolid medium, 1 � 104 to 5� 104 of splenocytes were resuspended in the NIH3T3-conditioned splenocyte medium containing 0.3% lowmelting point agarose and plated onto a solidified bottomlayer medium containing 0.5% agarose in 6-well plates, intriplicates. Colonies were photographed after 2 to 3 weeks,stained with 0.02% Giemsa in PBS, and counted. Whennecessary, 2 mmol/L of SP600125, a JNK-specific inhib-itor, or dimethyl sulfoxide (DMSO) was included in themedium.

Immunohistochemical analysisFrozen tissue samples were sliced into 8-mm sections and

stored in�80�C until use. Frozen sections were fixed in 4%buffered paraformaldehyde at 4�C for 10 minutes andincubated with primary antibodies (anti-Ki-67, ThermoScientific or anti-phospho-JNK-T183/Y185, Cell Signal-ing) at 4�C for overnight. Signals were detected by Vectas-tain ABC kit (Vector Laboratories). Samples were counter-stained with hematoxylin (Vector Laboratories). Positivecells were quantified under microscope in 20 randomlychosen 40� fields.

PRAK Suppresses Hematopoietic Cancer

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Western blot analysisCells were lysed with protein lysis buffer (10 mmol/L Tris

HCl, pH 8.0, 140 mmol/L sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% SDS) supplemen-ted with 1 mmol/L sodium orthovanadate, 10 mmol/Lsodium fluoride, 1 mmol/L b-glycerophosphate, and com-plete protease inhibitor cocktail (Roche). Cleared cell lysateswere mixed with Laemmli sample buffer supplemented withb-mercaptoethanol and boiled for 5 minutes. Equal amountof protein from the cell lysates were fractionated on SDS-PAGE and transferred to the nitrocellulose membrane (Bio-Rad). The antibodies used in this study were purchased fromBDPharmingen (cyclinD1), Cell Signaling (phospho-JNK-T183/Y185, JNK, c-Jun, phospho-AKT-S473, AKT, phos-pho-ERK-T202/Y204, and ERK2), Santa Cruz (p53, RasC-20, p19ARF, and p16Ink4a-M-156), Sigma (actin), andStressgen (DcR2). The antibodies against PRAK wasdescribed previously (17). Antibody against mouse p53phosphorylated at S37 was a gift from Dr. Carol Prives(Columbia University, New York, NY).

Quantitative real-time PCRRNA was isolated from cells using TRIzol (Life Technol-

ogies). cDNA was synthesized with iScript RT Supermix(BioRad) and quantified by real-time PCR using SsoFastSYBR Green Supermix (BioRad) on a CFX96 Real-TimeSystem (BioRad). The primers used for Grap2 were 50-AAGGCCTCTCTCGACATCAA-30 and 50-AGATG-GACGTTGTCCGGTAG-30 (6).

ResultsPRAK deletion facilitates oncogenic ras-inducedhematopoietic tumor developmentOur previous study indicated that PRAK suppresses skin

carcinogenesis induced by an environmental carcinogenDMBA (17). To evaluate the role of PRAK in hematopoietictumor formation, we crossed the PRAK-targeted mice withthe Em-N-RasG12D transgenic line harboring an activatedN-RasG12D transgene under the control of the immunoglobulin(Ig) heavy chain (Em) promoter, which is expressed specif-ically in hematopoietic cells (18). Western blot analysisindicated that the ras transgene was expressed at 3- to 4-fold above the endogenous level (Fig. 1A, inset). These micedevelop hematopoietic tumors of myeloid and T lymphoidorigins. It was reported that targeted deletion of p53 orSuv39h1, a histone methyltransferase involved in ras-induced senescence, promotes tumor development in thesemice (15).We monitored cancer development among PRAKþ/þ,

PRAKþ/�, and PRAK�/� littermates carrying the Em-N-RasG12D transgene. The PRAKþ/þ mice developed hemato-poietic tumors in a time frame consistent with previousreports (15, 18). The median tumor-free survival of thesemice was 236 days. Tumor development was significantlyaccelerated in the PRAK�/� mice as compared with theirPRAKþ/þ littermates, with a median tumor-free survival of160 days (Fig. 1A). Tumor development was also enhanced

in the PRAKþ/� animals, although only to a moderate level(with amedian survival of 205 days).Western blot analysis ofthe spleens showed that these mice mostly expressedexpected levels of PRAK and N-Ras (Fig. 1B), indicatingthat PRAK suppresses oncogenic ras-induced hematopoietictumorigenesis in mice. It is of interest to note that in some ofthe wild-type tumors, PRAK expression was reduced tosimilar levels to that in the PRAKþ/� tumors. This findingsuggests that at least a subset of wild-type mice developedtumors as a result of spontaneous reduction in PRAKexpression. The other PRAKþ/þ tumors retained normal,wild-type PRAK expression, raising a possibility that muta-tions might have occurred in other components of thePRAK-mediated signaling pathway.

PRAK deletion accelerates formation of hematopoietictumors of both myeloid and T lymphoid originsIt has been reported that while the Em-N-RasG12D mice

develop hematopoietic tumors of either myeloid or T lym-phoid origin, deletion of the p53 or Suv39h1 gene mainlyenhances the development of T-cell lymphomas (15). Wethus analyzed the origin of the tumors from PRAK-deficientEm-N-RasG12D animals, by immunogenotyping the celltypes in hematopoietic compartments and analyzing the

Figure 1. PRAK deficiency accelerates hematopoietic cancerdevelopment in Em-N-RasG12D transgenic mice. A, Kaplan–Meier plotsfor tumor-free survival of Em-N-RasG12D;PRAKþ/þ, Em-N-RasG12D;PRAKþ/�, and Em-N-RasG12D;PRAK�/� littermates. Number of mice foreachgenotype (n),median survival (MS) and statistical significance in log-rank test (P) are shown. Inset, Western blot analysis of N-Ras expressionlevels in splenocytes of mice of indicated genotypes. Relative Rasexpression levels are indicated. B, Western blot analysis of spleens oftumor-bearing Em-N-RasG12D;PRAKþ/þ, Em-N-RasG12D;PRAKþ/�, andEm-N-RasG12D;PRAK�/� littermates.

Yoshizuka et al.

Mol Cancer Res; 10(6) June 2012 Molecular Cancer Research812




organs infiltrated by tumors. Consistent with previousreports, about 80% of the tumors developed in wild-typemicewere ofmyeloid origin and 20%of these tumors were ofT lymphoid origin. Although heterozygous deletion of p53increased the incidence of T-cell lymphoma to 45%, PRAKdeficiency did not significantly alter the ratio between the 2types of hematopoietic tumors (Table 1), despite the short-ened disease latency in PRAKþ/� and PRAK�/� animals(Fig. 1A).Detailed analysis revealed no difference in tumor char-

acteristics between wild-type and PRAK-deficient mice. TheT-cell lymphomas from both wild-type and PRAK-deficientanimals were usually associated with enlarged spleen con-taining increased percentage of T cells (CD3-positive),enlarged lymph nodes and thymus containing almost exclu-sively T cells, and increased percentage of T cells in bonemarrow (Fig. 2A and Table 2). The myeloid malignancies inPRAKþ/þ, PRAKþ/�, and PRAK�/� mice all infiltratedspleen and liver (an organ not involved in T-celllymphomas; Fig. 2A and B) and displayed increased per-centage of CD11bþGR-1� myeloid cells in bone marrowand spleen (Table 3). In addition, peripheral blood analysisrevealed signs of anemia in themyeloid tumor–bearingmice,whereas the white blood cell (including lymphocyte andmyeloid cell) counts appeared to be normal (Fig. 2C).Therefore, PRAK deficiency accelerates the onset of N-

RasG12D–induced hematopoietic cancer development with-out altering the spectrum or characteristics of the tumors.Our results thus suggest that PRAK functions as a tumorsuppressor in hematopoietic cells of either myeloid or Tlymphoid lineage.

PRAK deficiency confers proliferative advantage inhematopoietic cells transduced with oncogenic rasTo investigate the cellular mechanism underlying the

enhanced hematopoietic cancer development in PRAK-deficient mice, we isolated hematopoietic cells from thespleen of PRAKþ/þ, PRAKþ/�, and PRAK�/� littermates

that did not carry the N-rasG12D transgene and transducedthem with an oncogenic ras allele, H-rasG12V or N-rasG12D

(Fig. 3A, inset). While wild-type cells also attained a higherproliferation rate upon transduction of either of the activatedras alleles as compared with a vector control, ras-induced cellproliferation was much more robust in PRAK-deficient cellsthan in wild-type cells (Fig. 3A). We also examined theability of these cells to grow and form colonies in semisolidmedia (Fig. 3B and C). Cells failed to form any colonies onsoft agarose in the absence of oncogenic ras, regardless of thePRAK status (Fig. 3B). H-rasG12V and N-rasG12D promotedthe formation of a number of small colonies in wild-typecells; however, the colony formation by PRAK-deficient cellstransduced with activated ras was significantly increased inboth size (Fig. 3B) and quantity (Fig. 3C), as compared withthe wild-type cells. These results show that loss of PRAKcooperates with oncogenic ras to induce proliferation andtumorigenesis in hematopoietic cells, suggesting that PRAK,when present in cells, suppresses ras-mediated cell prolifer-ation and oncogenic transformation.

PRAK deficiency abrogates the induction of senescencemarkers by oncogenic rasIt was reported that activated ras induces senescence in

primary splenocytes, which acts as a barrier to lymphomadevelopment (15). Our previous finding that PRAK sup-presses skin carcinogenesis by mediating senescence (17)prompted us to investigate a possible role of PRAK-mediatedsenescence in hematopoietic cell transformation. However,we failed to detect a growth inhibition by oncogenic ras ineither wild-type or PRAK-deficient splenocytes (Fig. 3A).Instead, ras induced an increase in proliferation in these cellpopulations. In addition, neither wild-type nor PRAK-deficient splenocytes displayed elevated percentage of cellspositive for a senescence marker, senescence-associatedb-galactosidase (SA-b-gal), upon transduction of activatedras alleles (data not shown). Nevertheless, oncogenic rasinduced accumulation of other senescence markers, includ-ing DcR2, p16INK4a, and p19ARF (12, 13, 22), and theinduction of these senescence markers by ras was eitherabolished or greatly reduced in PRAK�/� splenocytes (Fig.4A). While the reason why activated ras fails to inducedproliferative arrest and SA-b-gal is unclear, our data suggestthat a PRAK-dependent senescence response may be, at leastpartly, responsible, although it may not be the majormechanism, for the tumor-suppressing function of PRAKin hematopoietic cells.

PRAK deficiency enhances oncogenic ras-induced softagar colony formation in splenocytes throughhyperactivation of the JNK pathwayPrevious studies revealed that p38a negatively regulates

the proliferation of several cell types including fetal myeloidcells and that targeted deletion of p38a enhances theproliferation of these cells and promotes cancer developmentby inducing hyperactivation of the JNK pathway (6). Thesereports raise a possibility that PRAK, as a downstreamsubstrate of p38, may participate in the regulation of the

Table 1. Percentage of tumors of myeloid andlymphoid origins from mice of indicatedgenotypes harboring the Em-N-RasG12D

transgene, determined by immunogenotypingof the tumor cells and the type of infiltratedorgans

Tumor origin %

PRAKþ/þ (n ¼ 15) Myeloid 80.0Lymphoid 20.0

PRAKþ/� (n ¼ 33) Myeloid 90.9Lymphoid 9.1

PRAK�/� (n ¼ 9) Myeloid 77.8Lymphoid 22.2

p53þ/� (n ¼ 22) Myeloid 54.5Lymphoid 45.5






JNK pathway and cell proliferation by p38. We thusexamined the status of JNK activation in primary spleno-cytes transduced with oncogenic ras (Fig. 4A). Indeed, N-RasG12D alone induced a moderate increase in the proteinlevels of phospho-JNK, c-Jun, and a c-Jun downstreamtarget cyclin D1. PRAK deletion alone also caused a weakbut consistent induction of these proteins. However,the combination of N-RasG12D and PRAK deficiency syn-ergistically led to a much higher level of induction of theJNK-c-Jun-cyclin D1 pathway (Fig. 4A). In contrast, PRAKdeletion had no effect on the activating phosphorylation ofextracellular signal-regulated kinase (ERK) and AKTinduced by oncogenic ras (Fig. 4A). Furthermore, treatment

of the splenocytes with a JNK inhibitor SP600125, ortransduction of these cells with shRNAs that effectivesilenced the expression of both JNK1 and JNK2 (Fig.4C, inset), strongly inhibited the induction of soft agarcolony formation by oncogenic ras alone or by the combi-nation of oncogenic ras and PRAK deficiency (Fig. 4Band C). Thus, the induction of colony formation by onco-genic ras and the ability of PRAK deficiency to furtherpromote oncogenic ras-induced colony formation both relyon activation of JNK. In addition, PRAK deficiency alsoenhanced proliferation of Em-N-RasG12D splenocytesin vitro in a JNK-dependent fashion (Fig. 4D). Together,these data suggest that PRAK-mediated inhibition of JNK

Figure 2. Characterization of Tlymphoid and myeloidmalignancies in Em-N-RasG12D

transgenic mice. A, average weightof liver (left) and spleen (right) fromwild-type control mice (Ctrl) or Em-N-RasG12D transgenic mice ofindicated PRAK genotypes withlymphomas or myeloid tumors atterminal illness. Numbers aremean� SEM for indicated number oforgans (n). Statistical significancewas determined by Student t test.�,P < 0.05; ��, P > 0.8, for mice withtumors versuswild-typemice (Ctrl).B, hematoxylin/eosin (H/E) stainingof liver and spleen sections fromnormal wild-type (WT) mice ormyeloid tumor–bearing Em-N-RasG12D transgenic mice withindicated genotypes. C, peripheralblood analysis in normal wild-typemice and myeloid tumor–bearingEm-N-RasG12D transgenic micewith indicated PRAK genotypes,showingmean�SD of white bloodcell count (WBC), lymphocyte andmyeloid cell counts, red blood cellcounts (RBC), and hemoglobinconcentrations (Hb) for 3 mice.�, P < 0.01 for Em-N-RasG12D

transgenic mice versus wild-typemice determined by Student t test.

Yoshizuka et al.





activation contributes to suppression of tumorigenesis inhematopoietic compartments.To gain insights into the mechanism for PRAK-mediated

JNK inhibition, we examined the expression of a leukocyte-specific adaptor protein Grap2. Previous studies show thatGrap2 interacts with and enhances the activity of hemato-poietic progenitor kinase 1 (HPK1), which in turn activatesJNK and promotes proliferation in hematopoietic cells(6, 23). We found that Grap2 expression was induced byoncogenic ras to a much higher level in PRAK�/� spleno-cytes than in wild-type cells (Fig. 4E), suggesting that PRAKinhibits JNK by suppressing the Grap2/HPK1 circuit.We previously showed that in a skin cancer model, PRAK

suppressed carcinogenesis by inducing the tumor-suppres-sing activity of p53 through phosphorylation of p53 at Ser37(17). Oncogenic ras induced total p53 protein levels in bothwild-type and PRAK�/� splenocytes (Fig. 4F, top); however,when the protein loading was adjusted to achieve compa-rable amounts of total p53 levels, we failed to detect anyincrease in the phospho-p53Ser37 level in either wild-type orPRAK�/� splenocytes by Western blot analysis (Fig. 4F,bottom). These indicate that theRas-PRAK-p53Ser37 axis isnot operative in splenocytes, suggesting that PRAK deletionaccelerates ras-mediated hematopoietic cancer developmentthrough a p53Ser37-independent mechanism.

PRAK deficiency leads to hyperactivation of JNK inmiceTo determine whether the hyperactivation of JNK medi-

ated by PRAKdeficiency occurs in vivo, the activated form ofJNK (phospho-JNK-T183/Y185) was analyzed in bothhematopoietic tumors and normal spleens by immunohis-tochemistry. We initially examined hematopoietic tumorsisolated at the terminal illness from the spleens of PRAKþ/þ,PRAKþ/�, and PRAK�/� littermates carrying theN-rasG12D

transgene. Compared with the PRAKþ/þ tumors, theamount of cells positive for phospho-JNK increased inPRAKþ/� tumors and further increased to an even higherlevel in PRAK�/� tumors (Fig. 5A). To rule out thepossibility that the increased phospho-JNK levels wereassociated with infiltrated tumor cells, a group of 6-month-old PRAKþ/þ and PRAK�/� littermates with orwithout the N-rasG12D transgene were examined before anydisease symptom was observed in the N-rasG12D animals.Again, although the N-rasG12D transgene induced anincrease in the number of phospho-JNK–positive cells inboth PRAKþ/þ and PRAK�/�mice as compared with thosewithout the transgene, the induction was much more prom-inent in the PRAK�/� than in the PRAKþ/þ background(Fig. 5B, top). Moreover, in the absence of the N-rasG12D

transgene, PRAK deficiency also significantly, althoughmoderately, augmented the number of phospho-JNK–

Table 3. Immunogenotyping of cells isolated from indicated organs of wild-type mice or myeloid tumor–bearing Em-N-RasG12D;PRAKþ/þ and Em-N-RasG12D;PRAK�/� mice

MarkerCD3þ B220þ CD11bþGR-1� CD11bþGR-1þ

Tissue BM SP BM SP BM SP BM SP

N-ras;PRAKþ/þ 3.9 � 1.2 24 � 6.1 4.6 � 1.0 3.6 � 0.8 30 � 2.3a 66 � 6.7a 53 � 2.1 1.9 � 0.7N-ras;PRAK�/� 3.2 � 0.8 27 � 3.1 3.4 � 1.5 5.1 � 1.8 28 � 2.0a 63 � 7.6a 56 � 3.4 1.7 � 0.7WT 2.7 � 1.0 30 � 4.3 27 � 2.9 56 � 2.8 15 � 0.6 6.1 � 0.5 52 � 3.6 1.4 � 0.3

NOTE: Numbers aremeans of percentage of cells positive for indicated hematopoietic cell markers�SEM for 3 independent analysesfor each organ type from each genotype.Abbreviations: BM, bone marrow; SP, spleen; WT, wild-type.aP < 0.05 for Em-N-RasG12D transgenic mice versus wild-type mice determined by Student t test.

Table 2. Immunogenotyping of cells isolated from indicated organs of wild-type mice or lymphoid tumor–bearing Em-N-RasG12D;PRAKþ/þ and Em-N-RasG12D;PRAK�/� mice

MarkerCD3þ B220þ CD11bþGR-1� CD11bþGR-1þ

Tissue BM SP BM SP BM SP BM SP

N-ras;PRAKþ/þ 61 � 8.7a 82 � 4.7a 2.7 � 1.0 7.4 � 1.7 4.1 � 0.6 3.5 � 0.9 27 � 6.7 1.9 � 0.5N-ras;PRAK�/� 56 � 6.4a 88 � 3.1a 3.3 � 0.5 3.6 � 1.0 2.0 � 0.8 1.4 � 0.5 34 � 7.0 2.3 � 0.6WT 2.7 � 1.0 30 � 4.3 27 � 2.9 56 � 2.8 15 � 0.6 6.1 � 0.5 52 � 3.6 1.4 � 0.3

NOTE: Numbers aremeans of percentage of cells positive for indicated hematopoietic cell markers�SEM for 3 independent analysesfor each organ type from each genotype.Abbreviations: BM, bone marrow; SP, spleen; WT, wild-type.aP < 0.05 for Em-N-RasG12D transgenic mice versus wild-type mice determined by Student t test.






positive cells in spleen (Fig. 5B, top), even though these micedo not develop cancer without N-rasG12D. This observationthus strongly suggests that the positive effect of PRAKdeficiency on JNK activation is not restricted to tumor cells,but occurs also in normal hematopoietic cells and thus servesas the cause, rather than the consequence, of enhancedhematopoietic tumorigenesis. Supporting this notion, theenhancement in JNK activation by PRAK deficiency wasobserved in the spleens of mice harboring the N-rasG12D

transgene in as early as week 9 after birth, a time well beforethe onset of cancer in any mice, as determined by bothimmunohistochemical and Western blot analyses (Fig. 5C).Furthermore, induction of phospho-JNK by the N-rasG12

transgene or PRAK deficiency, and the hyperactivation ofJNK by both, strongly correlated with the increases in thenumber of cells positive for a proliferation marker Ki-67(Fig. 5B, top and bottom), suggesting that activation of JNK

promotes the proliferation of normal hematopoietic cells aswell as tumor cells and contributes to enhanced hemato-poietic cancer development.

DiscussionWe previously showed that PRAK suppresses DMBA-

induced skin carcinogenesis in mice (17). In the currentstudy, we show that PRAK also inhibits hematopoieticcancer development inmice harboring an activated ras allele,indicating that the tumor-suppressing activity of PRAKoperates in multiple tissues. This is consistent with theubiquitous expression pattern of PRAK in tissues includingskin and hematopoietic cells (17, 24). Analysis of thetumors formed in the Em-N-RasG12D transgenic miceindicated that PRAK deficiency accelerated the formationof tumors of both lymphoid and myeloid origins,

Figure 3. PRAK deficiency enhances oncogenic ras-induced proliferation and colony formation in primary splenocytes. A, PRAK deficiency enhancesoncogenic ras-inducedproliferation. Splenocytes isolated fromPRAKþ/þorPRAK�/�micewere transducedwith vector control (VT),H-RasG12V (H-Ras), or N-RasG12D (N-Ras) and plated in triplicates onto 12-well plates at a density of 4 � 104 cells per well in NIH3T3-conditioned splenocyte medium. Cell numberswere counted 6 days later and normalized to the number of vector-transduced cells. Inset, Western blot analysis showing ras, PRAK, and actin expressionlevels in these cell lines. B and C, PRAK deficiency enhances oncogenic ras-induced colony formation on semisolid medium. Splenocytes isolated fromPRAKþ/þ or PRAK�/�mice were transducedwith vector control (VT),H-RasG12V (H-Ras), orN-RasG12D (N-Ras) and plated in triplicates onto 6-well plates at adensity of 1�104 cells perwell inNIH3T3-conditioned splenocytemediumcontaining 0.3% lowmelting point agarose. Cellswere stainedwith 0.02%Giemsain PBS and photographed 3 weeks after seeding (B). Numbers of colonies were quantified using the AlphaView Image Quantification Software (CellBiosciences; C). A and C, numbers are mean � SD for triplicates. Statistical significance was determined by Student t test. ��, P < 0.001; ��, P < 0.0001.

Yoshizuka et al.





suggesting that PRAK serves as a guardian against tumor-igenesis in both hematopoietic lineages. Supporting therole of PRAK in inhibiting hematopoietic cancer devel-opment, hematopoietic cells isolated from PRAK-deficientspleens attained a faster proliferation rate and enhancedability of form colonies on semisolid medium upontransduction of oncogenic ras alleles, as compared withthose from wild-type animals.Enhanced hematopoietic tumorigenesis correlates with

hyperactivation of the JNK pathway by PRAK deficiencyin both mouse spleen tissues and ex vivo cultivated spleno-cytes. in vivo, enhanced JNK activation by PRAK deficiencywas detected in the spleens of Em-N-RasG12D transgenicanimals from well before the disease onset all the way to theterminal illness and in normal spleens from the nontrans-genic littermates. These results suggest that PRAK sup-

presses JNK activity in hematopoietic tumor cells as wellas in normal hematopoietic cells. The promitogenic and pro-oncogenic role of the JNK pathway has been well establishedin multiple cell types including lymphoma cells (6, 25–27).Indeed, we found that JNK activation correlates withenhanced proliferation of hematopoietic cells in vivo andin vitro, as revealed by a higher number of Ki-67–positivecells in spleens and an increased proliferation rate in sple-nocytes, respectively, and that PRAK deficiency promotesoncogenic ras-induced soft agar colony formation in aJNK-dependent manner. These findings suggest that hyper-activation of the JNK pathway plays a key role in theacceleration of hematopoietic cancer development by PRAKdeletion. Supporting this notion, several articles havereported that p38 arrests cell proliferation and suppressestumorigenesis by antagonizing the JNK pathway (6, 28).

Figure 4. PRAKdeficiency enhances oncogenic ras-induced cell proliferation and colony formation on semisolidmediumby hyperactivating the JNKpathway.A, Western blot analysis of PRAKþ/þ and PRAK�/� splenocytes 4 days after transduction with vector control (VT) or N-RasG12D (Ras). B, PRAKþ/þ andPRAK�/� splenocytes were transduced with vector control (VT), H-RasG12V (H-Ras), or N-RasG12D (N-Ras) and plated in triplicates onto 6-well plates at adensity of 1� 104 cells per well in NIH3T3-conditioned splenocyte medium containing 0.3% lowmelting point agarose and 2 mmol/L of SP600125 or DMSO.C, PRAKþ/þ and PRAK�/� splenocytes were cotransduced with pRetroSuper-shJNK1/2 (S1 or S2) or vector control (Ctrl) andH-RasG12V (H-Ras),N-RasG12D

(N-Ras), or vector control (VT) and plated in triplicates onto 6-well plates at a density of 1 � 104 cells per well in NIH3T3-conditioned splenocytemedium containing 0.3% lowmelting point agarose. Inset, Western blot analysis showing knockdown of JNK1 and JNK2 by shRNA (S1 and S2) as comparedwith the control shRNA (Ctrl). B and C, cells were stained with 0.02% Giemsa in PBS 3 weeks later, and numbers of colonies were quantified using theAlphaView Software (Cell Biosciences). Numbers are mean � SD for triplicates. Statistical significance was determined by Student t test. �, P < 0.005;��,P<0.01 forRas-SP600125versusRas-DMSOorRas-shJNKversusRas-Ctrl. D, cell numberswere counted 4daysafter 2�104of Em-N-RasG12D;PRAKþ/þ

or Em-N-RasG12D;PRAK�/� splenocytes were seeded onto 12-well plates and treated with control (DMSO) or 2 mmol/L of SP600125 (SP). Numbersare mean � SD for triplicates. Statistical significance was determined by Student t test. �, P < 0.05 for SP600125 versus DMSO; and ��, P < 0.02 forEm-N-RasG12D;PRAK�/� (DMSO) versus Em-N-RasG12D;PRAKþ/þ (DMSO). E, relative levels of Grap2 mRNA in PRAKþ/þ or PRAK�/� splenocytes 4 daysposttransduction with vector control (VT) or N-RasG12D (Ras) after normalization to GAPDH signals, as determined by quantitative real-time PCR.Numbers are mean � SD for triplicates. F, Western blot analysis of PRAKþ/þ and PRAK�/� splenocytes 4 days after transduction with vector control (VT)or N-RasG12D (Ras). In the bottom 2 panels, sample volumes were adjusted to achieve comparable loading of total p53 protein levels. GAPDH,glyceraldehyde-3-phosphate dehydrogenase.






Interestingly, despite the general mitogenic activity of JNKsshown by multiple studies, it was found that JNK1 nega-tively regulates T-cell receptor-initiated proliferation ofCD4helper (TH) cells (29), suggesting that the function of thispathway may differ in response to distinct stimuli such asoncogenic signals and T-cell receptor activation.In the previous study, we found that PRAK suppresses

skin carcinogenesis by mediating oncogene-induced senes-cence (17). PRAK-mediated senescence may also, at leastpartially, contribute to the suppression of hematopoietictumorigenesis. Although we failed to observe growth arrestin hematopoietic cells transduced with oncogenic ras, at leasta subset of senescence markers were induced in a PRAK-dependent manner. Although we do not understand theprecise reason why activated ras fails to induce growth arrestdespite the obvious PRAK-dependent induction of somesenescence markers, it is possible that induction of senes-cence occurs only in a subpopulation of cells, whereas theremaining cells acquire a higher proliferation rate due to themoderate activation of JNK by oncogenic ras alone. As aresult, the growth arrest in this subpopulation of senescentcells may have been obscured by the increased proliferationof the other cells in the growth curve assay, even though themore sensitive Western blot analysis detected alterations in

senescence markers. It remains to be determined whetherhyperactivation of JNK in PRAK-deficient hematopoieticcells leads to disruption of ras-induced senescence or ras-induced accumulation of senescence markers. However, thefact that activated ras alone causes moderate JNK activationand increased levels of senescence markers at the same timeargues against a role of JNK activation in senescence bypass.Taken together with the well-established role of JNK inpromoting cell proliferation, our data are consistent with thenotion that JNK hyperactivation by PRAK deficiency con-tributes to accelerated tumorigenesis by enhancing cellproliferation, rather than by disrupting senescence, inhematopoietic compartments. On the other hand, PRAK-mediated senescence may only occur in a small subpopula-tion of hematopoietic cells and thus is unlikely to be themajor mechanism underlying the tumor-suppressing func-tion of PRAK in this system.Several recent articles reported hematopoietic malignan-

cies in mice expressing oncogenic N-rasG12D from theendogenous locus (30–32). In these mice, a loxP-STOP-loxP (LSL)-N-rasG12D allele was knocked into the N-raslocus, and its expression was induced specifically in hemato-poietic cells byMx1-Cre. TheMx1-Cre, LSL-NrasG12Dmiceinitially developed an indolent myeloproliferative disorder

Figure 5. PRAKdeficiency leads to hyperactivation of JNK and increased cell proliferation in vivo. Immunohistochemical analysis of phospho-JNK-T183/Y185(A and B, top; and C) and Ki-67 (B, bottom) in spleens of terminally ill Em-N-RasG12D;PRAKþ/þ, Em-N-RasG12D;PRAKþ/�, and Em-N-RasG12D;PRAK�/�

littermates (A), 6-month-old PRAKþ/þ, Em-N-RasG12D;PRAKþ/þ, PRAK�/�, and Em-N-RasG12D;PRAK�/� littermates (B), or 9-week-old Em-N-RasG12D;PRAKþ/þ and Em-N-RasG12D;PRAK�/� littermates (C). Arrows indicate examples of cells positive for phospho-JNK (A, top; C, left). Bar graphs representpositive cells quantified under microscope in 20 randomly chosen 40� fields. Numbers are mean � SD. Statistical significance was determined by unpairedt test. Western blot analysis was conducted to measure the phospho-JNK and JNK levels in spleens of 9-week-old Em-N-RasG12D;PRAKþ/þ andEm-N-RasG12D;PRAK�/� littermates (C, bottom right).

Yoshizuka et al.





(MPD) with elevated white blood cell counts, splenomegaly,and myeloid (CD11bþGR-1þ) infiltration of bone marrowand spleen, and eventually die of a diverse spectrum ofhematologic cancers includingMPD and histiocytic sarcomawith liver and spleen enlargement. Similar to these studies,more than 80% of the Em-N-rasG12Dmice died of histiocyticsarcoma with myeloid infiltration in liver, spleen, and bonemarrow, whereas the remaining developed T-cell lymphoma.However, in contrast to the other model, the myeloid cellsinfiltrating bone marrow and spleen are CD11bþGR-1�,instead of CD11bþGR-1þ, in the myeloid tumor–bearingEm-N-rasG12Dmice. In addition, the myeloid disease in Em-N-rasG12Dmice is not accompanied by increased white bloodcell counts in peripheral blood. These differences are likelydue to the different promoters used to drive N-rasG12D

expression in these studies. Although Mx1 and Em are bothhematopoietic promoters, their specificity may differ indistinct subtypes of hematopoietic cells, leading to differen-tial activation of N-ras in these cells. In addition, the endog-enous N-ras promoter and the Em promoter may drivedifferent expression levels of N-rasG12D. Furthermore, assuggested by Wang and colleagues for the Mx1-Cre, LSL-NrasG12D mice (32), the genesis of histiocytic sarcoma withliver involvement may require simultaneous expression ofoncogenic N-ras in both hematopoietic cells and the hepaticmicroenvironment. While this is also likely to be true for theEm-N-rasG12D mice, our finding that PRAK deficiencypromotes JNK-dependent proliferation and colony forma-

tion of primary splenocytes suggests that the cell autonomouseffect of N-rasG12D in hematopoietic cells at least partlycontributes to enhanced tumor formation in this model.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interests were disclosed.

Authors' ContributionsConception and design: N. Yoshizuka, J. Han, P. SunDevelopment of methodology: N. YoshizukaAcquisition of data (provided animals, acquired and managed patients, providedfacilities, etc.): N. Yoshizuka, M. Lai, R. Liao, R. CookAnalysis and interpretation of data (e.g., statistical analysis, biostatistics, compu-tational analysis): N. Yoshizuka, M. Lai, J. Han, P. SunWriting, review, and/or revision of the manuscript: N. Yoshizuka, P. SunAdministrative, technical, or material support (i.e., reporting or organizing data,constructing databases): M. Lai, R. LiaoStudy supervision: C. Xiao, P. Sun

AcknowledgmentsThe authors thank Dr. A. W. Harris (Walter and Eliza Hall Institute of Medical

Research, Parkville, Victoria, Australia) for providing the Em-N-RasG12D strain andDr. Carol Prives for an antibody against mouse p53 phosphorylated at S37.

Grant SupportThis work was supported by grants from NIH (CA106768, CA131231 and

RR025744 to P. Sun and an international collaborative grant from NSF China(30828019 to P. Sun and J. Han).

The costs of publication of this article were defrayed in part by the payment of pagecharges. This article must therefore be herebymarked advertisement in accordance with18 U.S.C. Section 1734 solely to indicate this fact.

Received November 28, 2011; revised April 3, 2012; accepted April 3, 2012;published OnlineFirst June 4, 2012.

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2012;10:810-820. Published OnlineFirst June 4, 2012.Mol Cancer Res Naoto Yoshizuka, Maoyi Lai, Rong Liao, et al. Development by Antagonizing the JNK Pathway

-Induced Hematopoietic CancerrasPRAK Suppresses Oncogenic

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