STK33 Promotes Growth and Progression of Pancreatic Cancer ... · Molecular and Cellular Pathobiology STK33 Promotes Growth and Progression of Pancreatic Cancer as a Critical Downstream

Molecular and Cellular Pathobiology

STK33 Promotes Growth and Progression ofPancreatic Cancer as a Critical DownstreamMediator of HIF1aFanyang Kong1,2, Xiangyu Kong2, Yiqi Du2, Ying Chen1,3, Xuan Deng4, Jianwei Zhu2,Jiawei Du5, Lei Li2, Zhiliang Jia1, Dacheng Xie5, Zhaoshen Li2, and Keping Xie1

Abstract

The serine/threonine kinase STK33 has been implicated incancer cell proliferation. Here, we provide evidence of acritical role for STK33 in the pathogenesis and metastaticprogression of pancreatic ductal adenocarcinoma (PDAC).STK33 expression in PDAC was regulated by the hypoxia-inducible transcription factor HIF1a. In human PDAC speci-mens, STK33 was overexpressed and associated with poorprognosis. Enforced STK33 expression promoted PDAC

proliferation, migration, invasion, and tumor growth, where-as STK33 depletion exerted opposing effects. Mechanisticinvestigations showed that HIF1a regulated STK33 via directbinding to a hypoxia response element in its promoter. Inshowing that dysregulated HIF1a/STK33 signaling promotesPDAC growth and progression, our results suggest STK33 as acandidate therapeutic target to improve PDAC treatment.Cancer Res; 77(24); 6851–62. �2017 AACR.

IntroductionPancreatic ductal adenocarcinoma (PDAC) is the seventh lead-

ing cause of cancer-related deaths worldwide, with incidence ratesmatchingmortality rates (1, 2). Its poor prognosis is reflected by a5-year survival rate around 7%–8% despite significant improve-ment of treatment and development of new treatmentmodalities,such as chemotherapies and immunotherapies (1, 3–6). Pancre-atic cancer progression involves a complex regulatory networkconsisting of many signaling pathways. Previous studies uncov-eredmultiple mechanisms underlying PDAC pathogenesis (7, 8).However, the molecular mechanisms that trigger PDAC growthand progression remained largely unknown.

The tumor microenvironment has a key role in the initiationand progression of PDAC, and hypoxia is a prominent phys-icochemical feature of tumor microenvironments (9–11).

PDAC is hypoxic, primarily owing to its hypovascular nature,driving its drug resistance and high malignancy (12–14). Hyp-oxia activates signaling pathways to balance out the abnormaltumor vasculature and increases oxygen consumption associ-ated with dysregulated tumor cell proliferation. At the molec-ular level, this adaptation relies primarily on the transcriptionfactor hypoxia-inducible factor (HIF)-1a. It is a master regu-lator of cellular and systemic homeostatic response to hypoxia,which transcriptionally regulates many genes involved in ener-gy metabolism, angiogenesis, and apoptosis as well as genesinvolved in oxygen delivery or metabolic adaptation to hypoxia(15–18). Although previous studies verified multiple HIF1atarget genes, novel and potentially important candidate targetsof HIF1a in tumor development and progression are contin-ually being identified.

Serine/threonine kinase 33 (STK33) belongs to a family ofcalcium/calmodulin–dependent kinases. STK33 is expressed in avariety of normal tissues, including the testis, fetal lung and heart,and retina; however, it is weakly expressed in the normal pancreas(19). Researchers have observed elevated expression of STK33 in avariety of cancers (20–28). Over the past decade, many studiesfocused on the "synthetic lethality" function of STK33 in tumorswith KRas mutations, suggesting that STK33 is a druggable targetfor Kras-mutant cancer patients, when therapeutic targeting ofKRas remains one of the biggest challenges in cancer research (20,22). Other studies refuted this conclusion, though, challengingthe necessity of STK33 expression in mutant KRas-driven cancerdevelopment (27). Recent biofunctional analysis of STK33 dem-onstrated that it plays an active role in promoting tumor initia-tion, progression, malignancy, and resistance to therapy. Thesestudies using BRD-8899, an effective kinase inhibitor of STK33,identified kinase-dependent and -independent regulatory effectsof STK33 on a variety of malignant tumors (23, 26). However, theexpressionof andpotential roles for STK33 inPDACdevelopmentand progression have yet to be examined.

1Department of Gastroenterology, Hepatology and Nutrition, The University ofTexas MD Anderson Cancer Center, Houston, Texas. 2Department of Gastroen-terology, Changhai Hospital, Shanghai, P.R. China. 3Department of Pathology,Changhai Hospital, Shanghai, P.R. China. 4Department of Laboratory Medicine,Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, P.R.China. 5Department of Oncology and Tumor Institute, Shanghai East Hospital,Shanghai Tongji University, Shanghai, P.R. China.

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

F. Kong, X. Kong, and Y. Du are the co-first authors of this article.

Corresponding Authors: Keping Xie, Department of Gastroenterology, Hepa-tology and Nutrition, The University of Texas MD Anderson Cancer Center, Unit1466, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-794-5073; Fax:713-745-3654; E-mail: [email protected]; and Zhaoshen Li, Departmentof Gastroenterology, Changhai Hospital, Shanghai, P.R. China. Phone: 8621-3116-1344; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-0067

�2017 American Association for Cancer Research.

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In this study, we found that STK33 was highly expressed inPDACs and significantly correlated with poor prognosis. Hypoxiainduced STK33 expression in PDAC cells, and STK33 was a directtarget of and transcriptionally regulated by HIF1a. Therefore, thisnovel HIF1a/STK33 signaling pathway critically regulates PDACgrowth and metastasis and is a novel PDAC biomarker for use innew treatment modalities.

Materials and MethodsHuman tissue specimens and IHC analysis

The expression of STK33 and HIF1a was analyzed usingtissue microarrays (TMA) containing 100 primary PDAC and78 normal adjacent pancreatic tissue specimens (SuperChip).The characteristics of the TMAs were listed in SupplementaryTables S1 and S2. IHC analyses of these specimens were con-ducted with anti-STK33 (catalog no. ab206296) and anti-HIF1a(catalog no. ab113642) antibodies (Abcam) as described pre-viously (29). Overall survival (OS) was defined as the intervalfrom the date of surgery to the date of death. All analyses forhuman subjects were approved by the institutional reviewboard of The University of Texas MD Anderson Cancer Center(Houston, TX) and conducted in accordance with the Declara-tion of Helsinki. Written informed consent was obtained fromall subjects wherever necessary.

Cell culture and hypoxic conditionsThe HPNE, 293T, and human PDAC cell lines AsPC-1, BxPC-3,

Capan-2, PANC-1, and SW1990 were purchased from the ATCC.The human PDAC cell line FG and murine pancreatic cancer celllines Panc02 and Panc02-H7 were described previously (30). Allthe cell lines were obtained between 2012 and 2015 and authen-ticated by ATCC using short tandem repeat profiling analysis,routinely tested for mycoplasma contamination within the last 6months by using Hoechst staining and PCR, and used at passagenumbers <10 for this study after reception or thawing in ourlaboratory. All of these cell lines were maintained in plastic flasksas adherent monolayers in Eagle Minimal Essential Mediumsupplemented with 10% FBS, sodium pyruvate, nonessentialamino acids, L-glutamine, and a vitamin solution (Flow Labora-tories). All cells were incubated at 37�C in a humidified atmo-sphere with 5% CO2. For hypoxic culture, cells were placed in ahypoxia incubator in an atmosphere consisting of 94% N2, 5%CO2, and 1% O2.

Plasmids and siRNAsThe plasmids pcDNA3.0-STK33 (pSTK33) and pcDNA3.0-

hemagglutinin (HA)-HIF1a were purchased from OriGene andAddgene. siRNAs for STK33 (siSTK33) andHIF1a (siHIF1a) weresynthesized by Invitrogen. A negative control siRNA (Invitrogen)and control pcDNA3.0 vector were used. Transfection of plasmidsand siRNAs into PDAC cells was performed using Lipofectamine2000 CD transfection reagent (Invitrogen). For transient trans-fection, cells were transfected with plasmids or siRNA at differentconcentrations as indicated for 48 hours before performance offunctional assays.

Western blot analysisStandard Western blotting was carried out using whole-cell

protein lysates; primary antibodies against STK33 (catalogno. #4F7; Abnova), HIF1a (catalog no. ab113642; Abcam),

and hemagglutinin (catalog no. 26183-1MG; Thermo FisherScientific); and a secondary antibody (anti-rabbit IgG or anti-mouse IgG; Santa Cruz Biotechnology). Equal protein sampleloading was monitored using an anti-actin antibody.

AnimalsMale and female pathogen-free athymic nude mice and

C57BL/6 mice were purchased from the National CancerInstitute (Bethesda, MD). The animals were maintained infacilities approved by AAALAC International in accordancewith the current regulations and standards of the U.S. Depart-ment of Agriculture and Department of Health and HumanServices. All animal studies have been conducted in accordancewith the Institutional Animal Care and Use Committee of TheUniversity of Texas MD Anderson Cancer Center.

Tumor growth and metastasisHuman PDAC cells (1 � 106) in 0.1 mL of Hank balanced salt

solution were injected subcutaneously into the right scapularregions of nude mice. The resulting tumors were measured everyweek. Tumor-bearing mice were killed by cervical dislocationwhen they became moribund or on day 35 after injection, andtheir tumors were removed and weighed. Panc02 and Panc02-H7cells were subcutaneously injected into syngeneic C57BL/6 mice(1 � 105 cells per mouse). Mice were killed 21 (Panc02) or 18(Panc02-H7) days after tumor injections, and their tumors wereremoved and weighed.

To quantitatively measure metastasis, an experimental livermetastasis model was used. Specifically, tumor cells (5 � 105)were injected intravenously into another group of mice via theileocolic vein. All mice were sacrificed by cervical dislocation onday 28 after injection or when they appeared to be moribund.Their livers were then removed, and surface metastases on thelivers were counted after dissecting them into their individuallobes. Every surfacemetastasis was examined by two investigatorswho were unaware of the experimental protocol and scoredseparately.

Tumor cell migration/invasion assayBoth cell scratch–wound (horizontal migration) assays and

modified Boyden chamber (vertical invasion) were performed todetermine the migratory ability and invasiveness of PDAC cellswith altered STK33 expression as described previously (31). Forthe cell scratch–wound assays, cells were grown in 6-well platesuntil confluent. A wound was generated on the surface of theresulting cell monolayer via scraping with the 10-mL tip of apipette. After that, the cells were incubated for 12–48 hours. Thecells in the wounded monolayer were photographed at differenttime points, and cell migration was assessed by measuring gapsizes in multiple fields. For the Boyden chamber assay, 24-welltissue culture plates with 12-cell culture inserts (Millipore) wereused. Each insert contained an 8-mm pore-size polycarbonatemembrane with a precoated thin layer of a basement membranematrix (ECMatrix for the invasion assay) or without a coatedmatrix (for the migration assay). Ten percent FBS-containingmedium was placed in the bottom chambers to act as a chemoat-tractant. Cells (5 � 105) in a 300-mL volume of serum-freemedium were placed in the top chambers and incubated at 37�Cfor 48 hours. Cells on the bottom surface of the polycarbonatemembrane, which had invaded the ECMatrix and migrated

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through the membrane, were stained, counted, and photo-graphed under a microscope.

Reverse transcription-PCRReverse transcription-PCR analysis of STK33mRNA expression

was performed using total RNA. Total RNA was purified using anRNeasy PlusMini Kit (catalog no. 74134;Qiagen), and cDNAwassynthesized using an iScript cDNA Synthesis Kit (catalog no. 170-8890; Bio-Rad). The cDNA product was subjected to PCR ampli-fication with PCR primers to detect each factor. PCR primers usedin this report were summarized in Supplementary Table S3.

Construction of STK33 promoter reporter plasmidsA 719-bp DNA fragment containing the STK33 50 sequence

from -722 to -4 relative to the transcription initiation site wassubcloned into a pGL4.16-basic vector (Promega). The resultingfull-length reporter plasmid, which contained multiple hypoxiaresponse elements (HRE), was designated pGL4.16-634 (thepromoter reporter plasmid was named by the start site of theHRE). Deletion mutation reporters for this plasmid (pGL4.16-436, and pGL4.16-347) were then generated. All of these con-structswere verifiedby sequencing the inserts andflanking regionsof the plasmids.

Promoter reporter and dual luciferase assayPDAC cells were transfected with STK33 promoter reporters,

siRNAs, or specific gene expression plasmids. The STK33 promot-er activity in these cells was normalized via cotransfection of ab-actin/Renilla luciferase reporter containing a full-length Renillaluciferase gene. The resulting luciferase activity in the cells wasquantified using a dual luciferase assay system (Promega) 24hours after transfection.

Chromatin immunoprecipitation assayPDAC cells (2 � 106) were prepared for a chromatin immu-

noprecipitation (ChIP) assay using a ChIP assay kit (Millipore)according to the manufacturer's protocol. The resulting precipi-tated DNA specimens were analyzed using PCR to amplify frac-tions of the STK33 promoter. The PCR products were resolvedelectrophoretically on a 2% agarose gel and visualized usingethidium bromide staining.

Statistical analysisA two-tailed x2 test or the Fisher exact test was used to deter-

mine the significance of differences among covariates. All in vitroexperiments were performed in triplicate and at least three times.Datawerepresented either asmeans� SD fromone representativeindependent experiment of three with similar results or means �SEM from three independent experiments. The significance of thein vitro and in vivo data was determined using the Student t test(two-tailed) or one-way ANOVA. In all of the tests, P values lessthan 0.05 were considered statistically significant. The SPSS soft-ware program (version 17.0; IBM Corporation) was used forstatistical analysis.

ResultsDirect association of elevated expression of STK33 withpathologic features of PDACs

To determine the roles of STK33 in PDACpathogenesis, we firstinvestigated STK33 protein expression in the 100 primary PDAC

specimens, 78 tumor-adjacent pancreatic tissue specimens, andnormal pancreatic tissue specimens in the TMAs. The clinicopath-ologic characteristics of the TMAs are listed in SupplementaryTable S1. As shown in Fig. 1A and B, elevated STK33 expressionwas associated with poor histologic differentiation (total, P <0.001). We observed that the STK33 protein expression in PDACspecimens wasmuch higher than that in normal tissue specimens(Supplementary Fig. S1). Western blot analysis of the expressionof STK33 in paired normal pancreatic tissue and PDAC specimensconfirmed this finding (Fig. 1C). Furthermore, we found thatpatients with high STK33 expression had shorter OS than didthose with low expression (P < 0.001; Fig. 1D). We also investi-gated the relationship of STK33 protein expression with age, sex,tumor size, T category, and regional lymph node metastasis andfound no statistically significant differences in expression accord-ing to these variables (P > 0.05; Supplementary Table S2). Thesefindings indicated that STK33 plays critical roles in PDACdevelopment and progression and may be a valuable biomarkerfor this disease.

The subcellular localization of STK33 in PDACsWe examined STK33 expression and its subcellular location

in PDAC and tumor-adjacent tissue specimens using IHC,observing that STK33 was located in both the cytoplasm andnucleus (Supplementary Fig. S1). Also, we confirmed the local-ization of STK33 in PDAC cells using immunofluorescentstaining and found that STK33 was mainly localized in thecytoplasm in AsPC-1 and Capan-2 cells but in the nuclei in FG,BxPC-3, PANC-1, and SW1990 (Supplementary Fig. S2). Inter-estingly, both PDAC cell lines with cytoplasmic (AsPC-1) andnuclear (PANC-1) STK33 expression exhibited a remarkableupregulation of nuclear STK33, but only slight changeof cytoplasmic STK33, which may indicated a special role ofnuclear expression of STK33 in PDAC progression (please seeSupplementary Fig. S3A). To determine the clinical significanceof cytoplasmic and nuclear expression of STK33, we furtheranalyzed its relationships with clinical parameters. Resultsshowed that patients with nuclear expression of STK33 hadsignificantly shorter OS survival durations (P ¼ 0.000; Fig. 1E),whereas the expression levels of cytoplasmic STK33 expressionwere not significantly correlated with OS of PDAC patients(Supplementary Fig. S3B).

Promotion of PDAC growth by STK33 expression in vitro andin vivo

To determine the effect of STK33 expression on PDAC growth,we induced its expression in PDAC cell lines. STK33 was highlyexpressed in FG, PANC-1, and SW1990 cells but expressed atrelatively low levels in AsPC-1, BxPC-3, and Capan-2 cells (Fig.2A). As shown in Fig. 2B, pSTK33 effectively induced overexpres-sionof STK33 inAsPC-1 andCapan-2 cells, and siSTK33markedlydownregulated expression of STK33 in SW1990 and PANC-1cells. Consistent results were obtained when AsPC-1 andCapan-2were transfectedwith siSTK33 and PANC-1 and SW1990were transfected with pSTK33 (Supplementary Fig. S4). We theninvestigated the effect of STK33 on PDAC biology using pSTK33and siSTK33#2.We found that overexpression of STK33 inAsPC-1and Capan-2 cells led to increased monolayer growth, whereasknockdown of expression of STK33 resulted in decreased cellmonolayer growth (Fig. 2C).

HIF1a/STK33 Signaling in PDAC Progression

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In animal models, transfection with pSTK33 consistently pro-moted the growth of AsPC-1 cells in the subcutis, and knockdownof STK33 expression inhibited the growth of SW1990 cells (Fig.2D–F). The impacts of altered expression of STK33 on tumorgrowth in xenograft model were further confirmed in syngeneicmouse model of mouse pancreatic cancer (Fig. 2G). These datademonstrated that STK33 promoted the growth of PDACs in vitroand in vivo, supporting that STK33may function as anoncogene inPDAC cases.

Promotion of PDAC invasion and migration by STK33 in vitroand in vivo

To determine the effect of STK33 expression on PDAC migra-tion and invasion, we transfected AsPC-1 and Capan-2 cells withpSTK33 and PANC-1 and SW1990 cells with siSTK33#2 for 48hours. We had wounded the transfected cells via scratching, andmaintained them for at least 12 hours. The results demonstratedthat forced expression of STK33 strongly promoted the flatteningand spreading of AsPC-1 (Fig. 3A) and Capan-2 (Fig. 3B) cells,

Figure 1.

Expression of STK33 in PDACs and itsassociation with clinicopathologicfeatures of PDAC. TMA PDACspecimens were immunostained for aspecific anti-STK33 antibody. A,Representative images of STK33expression in PDAC specimens withdifferent differentiation grades. B,Association of elevated expression ofSTK33 with differentiation grade inPDACs. C, Western blot analysis ofSTK33 expression in tumor (T) andpaired peritumoral tissue (N)specimens obtained from five PDACpatients. STK33, 58 kDa; b-actin,42 kDa. D, STK33 expression andpatient survival. Patients weregrouped according to high and lowSTK33 expression. High STK33expression was associated with poorOS. E, Nuclear STK33 expression andpatient survival. Nuclear expression ofSTK33 increased the probability ofpoor prognosis for PDAC.

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Figure 2.

Effect of altered STK33 expression on PDAC growth in vitro and in vivo. A, Western blot analysis showing the expression of STK33 protein in PDAC cell lines.B, Verification of the efficiency of STK33 overexpression vectors and siRNAs in PDAC cell lines. AsPC-1 and Capan-2 cells were transfected with pSTK33 orcontrol vectors, and SW1990 and PANC-1 cells were transfected with siSTK33#1, siSTK33#2, or control siRNAs. STK33, 58 kDa; b-actin, 42 kDa. C, Assessmentof PDAC cell growth in vitro by Cell Counting Kit-8 (CCK-8) at the indicated time points. D–F, AsPC-1 cells transfected with pSTK33 and SW1990 cellstransfectedwith siSTK33#2were injected subcutaneously into nudemice (1� 106 cells permouse, fivemice per group). Tumorweights (D), tumor growth curves (E),and gross tumors (F) are shown. The formula to calculate the tumor volume is ab2/2; a represents the longest diameter of the tumor and b represents thediameter perpendicular to the longest. G, STK33 promoted tumor growth in C57BL/6 mice. Panc02 cells transfected with pSTK33 and Panc02-H7 cellstransfected with siSTK33#2 were subcutaneously injected into syngeneic C57BL/6mice. Gross tumors are shown and tumor weights of each group are representedby means � SD. � , P < 0.05.






whereas downregulation of expression of STK33 attenuated theflattening and spreading of SW1990 (Fig. 3C) and PANC-1 (Fig.3D) cells. Boyden chamber assay results confirmed these findings.Both the migratory ability and invasiveness of STK33-transfectedAsPC-1 (Fig. 3A) and Capan-2 (Fig. 3B) cells were much greaterthan those of control cells, whereas those of siSTK33-transfected

SW1990 (Fig. 3C) and PANC-1 (Fig. 3D) cells were markedlyattenuated. We then confirmed the in vitro results in mousemodels of PDAC, finding that enforced expression of STK33markedly increased liver metastasis of AsPC-1 cells (Supplemen-tary Fig. S5A), whereas knockdown of expression of STK33 abro-gated liver metastasis of SW1990 cells (Supplementary Fig. S5B).

Figure 3.

Influence of STK33 expression on PDAC cell migration and invasion in vitro and metastasis in vivo. AsPC-1 (A), Capan-2 (B), SW1990 (C), and PANC-1 (D)cellswere transfectedwith pSTK33, siSTK33, control vectors, or siRNAs (mock) for 48 hours. For a cell scratch–wound assay, cells in each groupwere placed in 6-wellplates, wounded via scratching, and maintained at 37�C for at least 12 hours. Cell cultures were photographed, and cell migration was assessed bymeasuring the cell-free areas in multiple fields (the inset numbers indicate the percentage mean gap areas � SD in triplicate). The migration and invasion ofAsPC-1, Capan-2, SW1990, and PANC-1 cells were determined as described in Materials and Methods. The data represent the means � SEM in triplicate fromone representative experiment of threewith similar results. � ,P <0.05 in comparisons of pSTK33-treated, siSTK33-treated,mock, and control groups (Student t test).

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Similarly, enforced expression of STK33 markedly increased lungmetastasis of AsPC-1 cells (Supplementary Fig. S6A), whereasknockdown of expression of STK33 abrogated lung metastasis ofSW1990 cells (Supplementary Fig. S6B). These data further con-firmed the oncogenic role of STK33 in progression andmetastasisof PDAC.

STK33 expression is upregulated under hypoxic conditions inPDAC cells

To determine whether STK33 expression can be induced byhypoxia, we exposed AsPC-1, Capan-2, and PANC-1 cells tohypoxic conditions (1% O2) for up to 48 hours. We found thatthe STK33 expression of PDAC cells at the mRNA and protein

levels increased markedly under these conditions and HIF1aprotein expression increased rapidly in these cell lines (Fig. 4Aand B). We also examined the expression of HIF1a and STK33in PDAC cells after treatment with CoCl2, a known HIF1aactivator. We found that the treatment increased the levels ofHIF1a and STK33 expression in a dose-dependent manner inAsPC-1 and Capan-2 cells (Fig. 4C and D). Furthermore,Matrigel invasion assay demonstrated that knockdown ofSTK33 expression partially inhibited the invasion of AsPC-1and Capan-2 cells under hypoxic conditions (Fig. 4E). Theseresults suggested that hypoxia induces STK33 expression inPDAC cells and HIF1a is involved in this regulatorymechanism.

Figure 4.

Hypoxia-induced STK33 expression in PDAC cells. A, Real-time analysis of STK33 mRNA expression in PDAC cells under hypoxic conditions (1% O2) for 0, 6, 12, 24,and 48 hours. B, Western blot analysis of the expression of STK33 and HIF1a in PDAC cells under hypoxic conditions (1% O2) from up to 48 hours. HIF1a, 93 kDa;STK33, 58 kDa; b-actin, 42 kDa. C, Real-time PCR analysis of STK33mRNA expression in PDAC cells under treatment with 0, 100, 150, 200, 250, or 300 mmol/L CoCl2for 24 hours. D, Western blot analysis of STK33 and HIF1a expression in PDAC cells after treatment with CoCl2 for 24 hours. All of the data are presentedas the means � SEM from three independent experiments. HIF1a, 93 kDa; STK33, 58 kDa; b-actin, 42 kDa. E, Matrigel invasion assay results for PDAC cellsafter silencing of STK33 under normoxic or hypoxic (1% O2) conditions. The STK33-knockdown efficiency was determined using a Western blot assay (HIF1a,93 kDa; STK33, 58 kDa; b-actin, 42 kDa). Data are presented as the means � SEM from three independent experiments. ^, Statistically significant when comparedwith the normoxia group (P < 0.05); #, statistically significant when compared with the hypoxia group (P < 0.05).






Close relationship between HIF1a and STK33 expression inPDAC cells

To identify the mechanisms underlying STK33 overexpressionin PDAC cells, we investigated the effect of altered expression ofHIF1a, the best characterized regulator in hypoxic microenviron-ments, on expression of STK33 in PDAC cells. As shown in Fig. 5A,overexpression of HIF1a in AsPC-1 and Capan-2 cells markedlyupregulated the expression of both STK33 mRNA and protein,whereas knockdown of expression of HIF1a decreased them inSW1990 and PANC-1 cells. Subsequently, we performed IHCanalysis of HIF1a expression using a TMA with specimensobtained from the same PDAC patient cohort used for STK33staining. On the basis of immunostaining for HIF1a and STK33,we observed a close correlation between the protein expressionlevels for STK33 and HIF1a (Fig. 5B). Importantly, statisticalanalysis revealed that the expression of STK33 was positivelycorrelated with that of HIF1a in the PDAC specimens (R ¼0.68; P ¼ 0.000; Fig. 5C). Double fluorescence staining assayrevealed a colocalization of STK33 and HIF1a in PDAC tissues(Supplementary Fig. S7). Moreover, similar to the results on theimpact of STK33 expression on patient survivals (SupplementaryFig. S8A), elevated expression of HIF1a in this study was corre-lated with a reduced OS durations (P ¼ 0.001 and P ¼ 0.000,according to two different groupingmethods; Supplementary Fig.S8B and S8C). It was also demonstrated that the combination ofSTK33 andHIF1a expression increased our ability to predict poorprognosis for PDAC (P ¼ 0.0097; Supplementary Fig. S8D).

Tounderstand the role of STK33 inHIF1a–mediated PDACcellmigration, we overexpressed STK33 in HIF1a–knockdownSW1990 and PANC-1 cells. Results showed that STK33 over-expression at least partially rescued the inhibitory effect ofHIF1a knockdown on SW1990 and PANC-1 cell migration,suggesting that STK33 was involved in HIF1a–mediated migra-tion (Fig. 5D).

Transcriptional regulation of STK33 expression by HIF1a inPDAC cells

To further explore the mechanism of regulation of STK33expression by HIF1a, we analyzed the STK33 promoter sequence50-CGTG-30 for potential HREs, which were described previously(32). Sequence analysis of the STK33 promoter uncovered threeputative HREs located at -347 (HRE1), -436 (HRE2), and -634(HRE3) bp relative to the transcriptional start site of STK33(Fig. 6A). We then generated the full-length STK33 promoterpSTK33-634 and deletion mutants of it. To determine whetherHIF1a regulates STK33 expression at the transcriptional level, wecotransfected the deletion mutant reporters with or withoutHIF1a expression vectors into 293T cells. Luciferase reporter assayresults demonstrated that deletion of the region from -520 to -399bp, covering the HRE2 site, markedly reduced the promoteractivity of STK33 induced by HIF1a (Fig. 6B). To further deter-mine whether HIF1a regulates STK33 promoter transcriptionalactivity in PDACs, we cotransfected a pGL4.16-634 reporter withHIF1a expression vectors or siRNA into PDAC cells. As shownin Fig. 6C, increased HIF1a expression in AsPC-1 and Capan-2cells activated STK33 promoter activity, whereas knockdown ofHIF1a expression in SW1990 and PANC-1 cells attenuated theSTK33 promoter activity. To provide more evidence that HIF1abounddirectly to the STK33promoter, we conducted aChIP assayusing chromatin prepared from AsPC-1 and SW1990 cells. Theresults confirmed thatHIF1a directly bound to theHRE2 site (Fig.

6D) rather than other HRE sites in the STK33 promoter in PDACcells. These data strongly suggested that HIF1a bound to theSTK33 promoter and transcriptionally regulated STK33 expres-sion in PDAC cells.

DiscussionIn this study, we investigated the biologic effects of and under-

lying molecular basis for STK33 expression in PDACs and delin-eated the clinical significance of the newly identified HIF1a/STK33 regulatory pathway. We found that expression of STK33was frequently higher in tumors than in nontumorous adjacenttissue. We also demonstrated a positive correlation betweenSTK33 expression and clinicopathologic features of PDACpatients, particularly that increased STK33 expression predictspoor tumor differentiation and decreased survival OS durations.In vitro and in vivo experiments demonstrated that STK33 had apotent oncogenic role in promoting PDAC proliferation, migra-tion, and invasion. Our mechanistic studies revealed that STK33overexpression in PDACs was mainly regulated by HIF1a, whichbinds directly to HREs on the STK33 promoter. Given theseresults, we concluded that STK33 is a direct target of HIF1a inPDACs, constituting a new signaling axis and promoting PDACdevelopment and progression.

Recent studies have demonstrated elevated expression ofSTK33 in a variety of cancers, including hepatocellular carcinoma,large-cell lung cancer, and hypopharyngeal squamous cell carci-noma.However, the expression and biologic function of STK33 inPDACs during tumorigenesis has been seldom studied. We foundthat STK33 expression was markedly increased in PDAC cell linesand specimens. Analysis of the relationship of clinicopathologiccharacteristics of the PDAC patients from whom the TMA speci-mens were obtained with STK33 expression demonstrated thatthe expressionwas associatedwith tumordifferentiation andpoorprognosis. Previous studies demonstrated that STK33 is localizedmainly in the cytoplasm of cells in different organs (19). Our IHCresults demonstrated that STK33 localized to the nuclei andcytoplasm of the PDAC cells. Interestingly, nuclear expression ofSTK33 appeared to be associated with reduced OS durations.Further studies are required to determine the mechanism ofSTK33 protein subcellular localization and translocation ofSTK33 to the cytoplasm and nucleus. These results indicated thatSTK33 may function as an oncogene and play important roles inPDAC development and progression. In our biologic studies, wefound that elevated expression of STK33 markedly enhancedPDAC proliferation, migration, and invasion in vitro and tumorgrowth in vivo,whereas silencing of STK33markedly decreased theability of PDAC cells to proliferate, migrate, and invade andinhibited the growth of PDACs in vivo. All of these findings werein accordance with those of previous studies, strongly supportingthat STK33 functions as an oncogene in PDACs.

KRas is frequently mutated in many types of cancer cells (33).Clinical observations and functional studies suggested thatKRas isan attractive therapeutic target for PDAC treatment. However,effective KRas inhibitors have yet to be found (34–36). Research-ers identified STK33 as having a synthetic lethality function in thecontext of in cells expressing mutant KRas using high-throughputRNA interference (21). Also, authors reported that knockdown ofSTK33 expression induced apoptosis in KRas-dependent cancercell lines but not in cells wild-type for KRas (21, 22). Becausetherapeutic targeting of R has been a great challenge to date, this

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Figure 5.

Coexpression of STK33 and HIF1a in PDAC cells. AsPC-1 and Capan-2 cells were transfected with a HA-HIF1a vector or control vector for 48 hours, andSW1990 and PANC-1 cells were transfected with siHIF1a#1 and siHIF1a#2 or control siRNA for 48 hours. A, Total RNA and protein lysates were harvested,and the expression of STK33 and HIF1a in the lysates was determined using real-time PCR and Western blotting (HIF1a, 93 kDa; STK33, 58 kDa; b-actin, 42 kDa)and anti-HA antibodywas used to detect the expression of HA-HIF1a fusion protein.B, IHC stains of the same TMAPDAC sections for STK33with a specific anti-HIF1aantibody. Representative images of PDAC sections with STK33 and HIF1a staining are shown (�100 magnification in the main images; �400 magnificationin the insets). C, Assessment of the positive correlation between HIF1a and STK33 expression in PDAC specimens (n ¼ 98) using Pearson correlationcoefficient analysis. Some of the dots on the graph represent more than one specimen. D, Matrigel invasion assay results for PDAC cells after silencing HIF1awith or without transfection of pSTK33. The transfection efficacy and biofunction according to Western blot (HIF1a, 93 kDa; STK33, 58 kDa; b-actin, 42 kDa)and Boyden chamber analysis are shown. siNC, nontargeting siRNA. The experiments were performed independently three times. ^, Statistically significantwhen compared with the siNC group (P < 0.05); #, statistically significant when compared with the siHIF1a group (P < 0.05).






finding led authors to propose that inhibitors of STK33 kinaseactivity would selectively kill Kras-mutant cells in cancer patients.However, over the past decade, this conclusion was opposed bymany researchers, as they obtained no likely results in theirlaboratories, so the effect of anti-KRas agents that inhibit STK33

remains controversial (23, 26, 37). Answering this question issignificant to PDAC, because the vast majority of pancreaticcancers harbor KRas mutations (�90%). In this study, we deter-mined the biologic function of STK33 in KRas-mutant cell lines(AsPC-1, Capan-2, PANC-1, and SW1990). However, a deficiency

Figure 6.

Direct binding of HIF1a to the STK33 promoter.A, Three HREs located at different sites in the STK33 promoter sequence.B, STK33 promoter reporters (pGL4.16-634,-436, and -347) were transfected into 239T cells in triplicate with HIF1a expression or control vectors for 24 hours. The STK33 promoter activity was thenexamined using a dual luciferase assay kit. The promoter activities of the treated groups relative to those of the control groups are shown.C,AsPC-1 andCapan-2 cellswere cotransfected with pGL4.16-634, a HA-tagged HIF1a expression vector, or a control vector. PANC-1 and SW1990 cells were cotransfected with pGL4.16-634,siHIF1a, or control siRNA. The promoter activities in the cells determined using a dual luciferase assay are shown. The experiments were performedindependently three times. D, Results of ChIP-real-time PCR and ChIP-PCR assay conducted using chromatins isolated from AsPC-1 and SW1990 cells. The PDACcells were transfected with HA-HIF1a and exposed to normoxia (N) or hypoxia (H) for 24 hours. A specific anti-HA antibody was used, and normal IgG wasused as a control. One percent of the total cell lysates was subjected to PCR before immunoprecipitation (input control). The experiments were performedthree times independently. �, P < 0.05.

Kong et al.





of evidence from the use of wild-type PDAC cell models in ourstudy leaves the validity of correlation of STK33 and Ras expres-sion in PDACs uncertain.

Researchers discovered STK33 in the course of sequencing thehuman chromosome 11 region 11p15 and mouse chromosome 7(38). Ample studies have proven that STK33 has potent oncoge-nicity and preliminarily identified its protumor mechanism. As akinase, SKT33 exhibited autophosphorylation and directly phos-phorylated the intermediate filament protein vimentin, whichfunctions as a structural component of and modulates signaltransduction in cancer cells (20, 39). Authors also reported thatthe kinase-independent effect of STK33 supports STK33 as a criticalregulatorof cancer cell proliferationandmigration (23,26).Usingatransgenic mouse model of hepatocellular carcinoma, Yang andcolleagues (26) found that STK33 bound directly to the transcrip-tional factor C-myc and increased its transcriptional activity, whichcouldnotbe inhibitedby treatmentwith theSTK33kinase inhibitorBRD-8899.However, theC-terminusof STK33played an inhibitoryrole in regulating this interaction and significantly inhibited hepa-tocellular carcinoma proliferation in vitro and tumor growth in vivo.In addition, Azoitei and colleagues (22) found that the HSP90/CDC37chaperon complexbound toand stabilizedSTK33 incancercells by preventing its degradation from by the proteasome-medi-ated pathway. However, the exploration of potential mechanismsof STK33 in regulating PDAC biology has been limited.

Increasing evidence indicates that hypoxia is an important driv-ing force for PDAC progression (7, 40–44). HIF1a, a subunit ofHIF-1, functions as a master regulator of cellular and systemichomeostatic response to hypoxia (45–48). HIF1a has long beenknown to be a transcriptional factor, inducing the expression ofmultiple target genes in favor of the tolerance of PDAC cells tohypoxia, by recognizing HREs (50-RCGTG-30). Investigators foundthat in a sustained hypoxic environment, HIF1a was stabilizedand prevented from undergoing protein degradation in anubiquitin/proteasome pathway (49). In this study, we foundthat in PDAC cells, the expression of STK33 increased markedlyunder hypoxic conditions (1% O2) and that expression ofHIF1a was upregulated. We confirmed these results in ourtreatment of PDAC cells with CoCl2 at different concentrations.Furthermore, PDAC cells exhibited enhanced migration underhypoxic but not normoxic conditions, and this enhancementwas attenuated considerably by knockdown of STK33 expres-sion. These results suggested that HIF1a is involved in upre-gulation of STK33 expression in PDAC cells.

We found four lines of evidence in this study verifying thepresence of the HIF1a/STK33 signaling pathway in PDACs andsupporting its role in regulation of PDAC progression and metas-tasis. First, we found that HIF1a and STK33 were concomitantlyoverexpressed in pancreatic tumor specimens. Second, overex-pression of HIF1a upregulated the expression of STK33 at boththe mRNA and protein level, whereas reduced expression ofHIF1a did the opposite. Third, HIF1a bound directly to the

STK33 promoter region and regulated expression of STK33 at thetranscriptional level. Fourth, overexpression of STK33 rescuedinhibition of PDACmigration by HIF1a knockdown. All of theseresults strongly supported that STK33 expression in PDACs wastranscriptionally regulated by HIF1a.

In summary, we obtained both clinical and experimentalevidence identifying STK33 as an important oncogene in PDACsand found that its expression was frequently increased in PDACcell lines and specimens. Biologically, STK33 expression promot-ed PDACproliferation,migration, and invasion in vitro and tumorgrowth in vivo. Mechanistically, the expression of STK33 in PDACswas transcriptionally activated by HIF1a, an important regulatorof hypoxic microenvironments. Thus, this study is fundamentallyimportant, as we not only identified a novel molecular mecha-nism of PDAC growth and progression but also identified theaberrant HIF1a/STK33 signaling pathway as a promising newmolecular target for novel therapeutic modalities that inhibitPDAC metastasis and progression. The mechanism of nucleartranslocation of STK33 and its potential relationship with KRasmutations in PDAC cells warrant further exploration.

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

Authors' ContributionsConception and design: F. Kong, X. Deng, D. Xie, Z. Li, K. XieDevelopment of methodology: Y. Chen, X. Deng, L. Li, D. XieAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Kong, X. Kong, J. Zhu, J. Du, L. Li, Z. LiAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Kong, X. Kong, Y. Du, J. Du, K. XieWriting, review, and/or revision of the manuscript: F. Kong, X. Kong, Y. Du,K. XieAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F. Kong, J. Du, Z. Jia, Z. Li, K. XieStudy supervision: Z. Li, K. Xie

AcknowledgmentsWe thank Don Norwood for editorial assistance.

Grant SupportThis work was supported by grants R01CA172233, R01CA195651, and

R01CA198090 from the National Cancer Institute, NIH (to K. Xie) andgrant NSFC no. 81402017 and Shanghai Yang Fan Project # 14YF1405600(to X. Kong) and grant NSFC no. 81402425 (to L. Li) from the National NaturalScience Foundation of China.

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 January 11, 2017; revised July 6, 2017; accepted October 5, 2017;published OnlineFirst October 16, 2017.

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Kong et al.




2017;77:6851-6862. Published OnlineFirst October 16, 2017.Cancer Res Fanyang Kong, Xiangyu Kong, Yiqi Du, et al.

αa Critical Downstream Mediator of HIF1STK33 Promotes Growth and Progression of Pancreatic Cancer as

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