Protein Kinase D1 Mediates Anchorage-dependentand -independent Growth of Tumor Cells via the ZincFinger Transcription Factor Snail1*□S
Received for publication, April 10, 2012, and in revised form, July 10, 2012 Published, JBC Papers in Press, July 12, 2012, DOI 10.1074/jbc.M112.370999
Tim Eiseler‡§, Conny Köhler‡1, Subbaiah Chary Nimmagadda‡1, Arsia Jamali‡¶1, Nancy Funk‡, Golsa Joodi‡¶,Peter Storz�, and Thomas Seufferlein§2
From the ‡Department for Internal Medicine I, University Clinic Halle, Martin Luther University Halle-Wittenberg, Ernst-GrubeStrasse 40, 06120 Halle (Saale), Germany, ¶Student Scientific Research Center, Tehran University of Medical Sciences,1417613151 Tehran, I.R. of Iran, �Department of Cancer Biology, Mayo Clinic Comprehensive Cancer Center, Mayo Clinic,Jacksonville, Florida 32224, and §Department of Internal Medicine I, Ulm University, Albert Einstein Allee 23,D-89081 Ulm, Germany
Background: The protein kinase D (PKD) family is involved in the control of cell motility and proliferation.Results: PKD1 controls growth of cancer cells through phosphorylation of Snail1 at Ser-11.Conclusion: Only PKD1, but not PKD2, mediates isoform-specific control of pancreatic cancer cell proliferation throughSnail1.Significance:We demonstrate for the first time isoform-specific control of pancreatic cancer growth by a single phosphoryla-tion of a substrate.
Wehere identify protein kinase D1 (PKD1) as amajor regula-tor of anchorage-dependent and -independent growth of cancercells controlled via the transcription factor Snail1. Using FRET,we demonstrate that PKD1, but not PKD2, efficiently interactswith Snail1 in nuclei. PKD1 phosphorylates Snail1 at Ser-11.There was no change in the nucleocytoplasmic distribution ofSnail1 usingwild type Snail1 and Ser-11 phosphositemutants indifferent tumor cells. Regardless of its phosphorylation status orfollowing co-expression of constitutively active PKD, Snail1waspredominantly localized to cell nuclei. We also identify a novelmechanism of PKD1-mediated regulation of Snail1 transcrip-tional activity in tumor cells. The interaction of the co-repres-sors histone deacetylases 1 and 2 as well as lysyl oxidase-likeprotein 3 with Snail1 was impaired when Snail1 was not phos-phorylated at Ser-11, which led to reduced Snail1-associatedhistonedeacetylase activity.Additionally, lysyl oxidase-like pro-tein 3 expression was up-regulated by ectopic PKD1 expression,implying a synergistic regulation of Snail1-driven transcription.Ectopic expression of PKD1 also up-regulated proliferationmarkers such as Cyclin D1 and Ajuba. Accordingly, Snail1 andits phosphorylation at Ser-11 were required and sufficient tocontrol PKD1-mediated anchorage-independent growth andanchorage-dependent proliferation of different tumor cells. Inconclusion, our data show that PKD1 is crucial to supportgrowth of tumor cells via Snail1.
The protein kinase D (PKD)3 family of serine/threoninekinases consists of three members: PKD1 (PKC�), PKD2, andPKD3. They share similar structural features and often phos-phorylate the same substrates (1–7). The protein kinase D fam-ily has been implicated in the regulation of proliferation of dif-ferent cells including pancreatic cancer cells (6–11). We havepreviously identified protein kinase D as a major regulator ofcancer cell motility and invasion (2–5). However, it is unclearwhether these functions are regulated by all PKD isoforms in asimilar fashion and via the same PKD targets or substrates.Therefore, we investigated how PKD1 and PKD2, two PKD iso-forms that mediate vital functions in pancreatic tumor growthand angiogenesis, are involved in the regulation of pancreaticcancer cell growth (12–14). We initiated a bioinformaticsscreening approachusing Scansite (15) to identify putative PKDphosphorylation consensus motifs in potentially relevant PKDsubstrates and identified (in accordance with Du et al. (16))Snail1 as a putative PKD substrate. Snail1 is an important zincfinger transcription factor controlling the epithelial-mesenchy-mal transition and tumor growth (17, 18). Snail1 transcriptionalactivity can be mediated by regulation of protein stability vialysyl oxidase-like proteins (LOXLs) (19, 20). LOXL isoforms 2and 3 interact with Snail1 to modify critical lysine residues andthereby stabilize the protein (19). Snail1 repressor activity isalso modulated by phosphorylation of 6 residues via glycogensynthase kinase 3�, inducing nuclear export and �-Trcp-con-trolled ubiquitin-dependent degradation (20, 21). Snail1 tran-scriptional repression is mediated by recruitment of a Sin3A-histone deacetylase 1 and 2 (HDAC1-HDAC2) complex. Thisinteraction is critical for Snail1 repressor function and depen-
* This work was supported, in whole or in part, by National Institutes of HealthGrants R01CA140182 and R01GM86435 (to P. S.). This work was also sup-ported by Deutsche Krebshilfe Grant 109222, Wilhelm-Roux-ProgramGrants FKZ 23/19 and FKZ 23/08 (to T. E.), Florida Department of Health Bank-head-Coley Cancer Research Program Grant FLA07BN-08 (to P. S.), and Ger-man Federal Ministry of Education and Research (Bundesministerium für Bil-dung und Forschung) Grant NGFN plus/PKB-01GS08209-4 (to T. S.).
□S This article contains supplemental Figs. 1–5 and Table 2.1 These authors contributed equally to this work.2 To whom correspondence should be addressed. Tel.: 49-731-50044501; Fax:
3 The abbreviations used are: PKD, protein kinase D; LOXL, lysyl oxidase-like protein; KD, kinase-dead; qPCR, quantitative real time PCR; BME,basement membrane extract; ANOVA, analysis of variance; HDAC, his-tone deacetylase.
dent on the N-terminal SNAG domain of Snail1 (22), which isadjacent to the PKD phosphorylation consensus in the protein.Thus, the aim of this studywas to identify how phosphorylationof Snail1 by PKD regulates Snail1 activity, tumor cell growth,and invasive features and to determine whether Snail1 phos-phorylation by PKDs is isoform-specific.
EXPERIMENTAL PROCEDURES
Cell Culture—Panc89 (pancreatic ductal adenocarcinoma),Panc1 (pancreatic ductal adenocarcinoma), HEK293T, andHeLa cells were maintained in RPMI 1640 medium supple-mented with 10% FCS and penicillin/streptomycin. Panc1 cellswere transfected using Turbofect (Fermentas), and siRNAswere transfected using Oligofectamine (Invitrogen). Experi-ments in HeLa cells were performed using HeLa Monster rea-gent (Mirus). Panc1, HEK293T, and HeLa cells were acquiredfrom ATCC. Stable Panc89 cells used in this study weredescribed previously (4, 5). For production of lentiviruses, 6 �106 HEK293T cells were transfected using Lipofectamine 2000(Invitrogen). Virus supernatants were harvested after 48 h andused for transduction of stable Panc89 cell lines. Cells weresubsequently subjected to puromycin selection to generatesemistable cell lines used in assays.Plasmids, Antibodies, and Dye Reagents—GFP-tagged ex-
pression constructs for PKD1, PKD1KD (K612W), PKD2-GFP,and PKD2KD-GFP have been described previously (5, 23).Snail1-FLAG and Snail1-GFP constructs (21) were acquiredfrom Addgene. Snail1S11A/S11E-FLAG and Snail1S11A/S11E-GFP mutants were generated by site-directed mutagene-sis (QuikChange II kit, Stratagene) using the following primers:Snail1S11A forward, 5�-CTC-GTC-AGG-AAG-CCC-GCC-GAC-CCC-AAT-CGG-AAG; Snail1S11A reverse, 5�-CTT-CCG-ATT-GGG-GTC-GGC-GGG-CTT-CCT-GAC-GAG;Snail1S11E forward, 5�-CTC-GTC-AGG-AAG-CCC-GAG-GAC-CCC-AAT-CGG-AAG; and Snail1S11E reverse, 5�-CTT-CCG-ATT-GGG-GTC-CTC-GGG-CTT-CCT-GAC-GAG. Mu-tations were verified by sequencing. Short hairpin RNAsagainst lacz, PKD1, and PKD2 were described previously (4).Ajuba, Snail1, and Cyclin D1 antibodies were acquired fromCell Signaling Technology. Anti-FLAG M2, anti-Actin AC40and anti-Tubulin were from Sigma-Aldrich. LOXL3 antibodieswere purchased from Abnova and Sigma-Aldrich. Anti-GFPantibody was acquired from Roche Applied Science. HDAC1and HDAC2 antibodies were from Abcam. Quantitative realtime PCR (qPCR) primers were obtained from Qiagen. PKD1C20 antibody was acquired from Santa Cruz Biotechnology.PKD2 antibody was obtained from Calbiochem. Non-targetshRNA control (scrambled, shc002), sh_Snail1 1 (NM_005985.2-136s1c1), and sh_Snail1 2 (NM_005985.2-504s1c1)were from Sigma-Aldrich. Immunofluorescence secondaryantibodies were purchased from Invitrogen. pMotif antibodywas a gift from Peter Storz (Mayo Clinic).Total Cell Lysates and Co-immunoprecipitation—Total cell
lysates and co-immunoprecipitations were performed asdescribed previously (3, 5, 24). In brief, total cell lysates wereeither prepared by solubilizing cells in radioimmune precipita-tion assay buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1 mM
EDTA, 1% Nonidet P-40, 0.25% deoxycholate, 0.1% SDS plus
complete protease and PhosStop inhibitors (Roche AppliedScience)) or 2% SDS lysis buffer (10mMHepes, 150mMNaCl, 1mM EDTA, pH 6.8 plus inhibitors). Lysates were clarified bycentrifugation at 13,000 � g for 10 min. For immunoprecipita-tion, equal amounts of proteins were incubated with specificantibodies for 1.5 h at 4 °C. Immune complexes were collectedwith protein G-Sepharose (GE Healthcare) for 30 min at 4 °Cand washed three times with lysis buffer (20 mM Tris, pH 7.4, 5mg MgCl2, 150 mM NaCl, 1% Triton X-100). Precipitated pro-teins were released by boiling in sample buffer and subjected toSDS-PAGE. The proteins were blotted onto nitrocellulosemembranes (Pall Corp., Germany). After blocking with 2%BSAin TBSwith Tween 20, blots were probedwith specific antibod-ies. Proteins were visualized by HRP-coupled secondary anti-bodies using ECL (Thermo Fisher). Quantitative analysis ofWestern blots was done by measuring integrated band densityusing NIH ImageJ. Values shown represent -fold change inrespect to control.qPCR—Quantitative real timePCRswere performed in aBio-
Rad iQ5 cycler with SYBRGreen. Total RNAwas isolated usingan RNeasy minikit (Qiagen). We used 400 ng of total RNA forcDNA synthesis. Quantitative real time PCR analysis was per-formed in three replicas and in at least three independentexperiments using qPCR primers for GAPDH (control),LOXL1–3, and Cyclin D2 (Qiagen). Results were calculatedusing the��Ctmethod normalized toGAPDHand vector con-trol cells.Three-dimensional Basement Membrane Extract (BME) Cell
Culture—Three-dimensional BME culture was performed byseeding 10,000 cells of stable Panc89 cell lines (4, 5) in BME(growth factor-reduced, phenol red-free; Cultrex, R&D Sys-tems, Trevigen). Tumor cell clusters were documented after 16days at 10� magnification (see Fig. 8A) or 32 days (see Fig. 8G)at 8� magnification using a Keyance microscope. Diameters oftumor clusters in images were quantified in perpendicular direc-tions for each cluster using spacial calibration of images (NIHImageJ). For statistical analysis, conditions were compared usingfrequency distribution histograms. Statistical significancewas cal-culated using two-tailed unpaired Student’s t test.Immunofluorescence ConfocalMicroscopy andAcceptor Pho-
tobleach Fluorescence Resonance Energy Transfer (FRET)—HeLa cells were transfected withHeLaMonster and seeded at adensity of 150,000 cells/well on glass coverslips. After adhesionovernight, cells were fixed with 4% formaldehyde at room tem-perature for 20 min, washed, quenched with 0.1 M glycine, andthen permeabilized with 0.1% Triton X-100. Samples wereblocked and stained in PBS supplemented with 5% FCS, 0.05%Tween 20. Primary and secondary Alexa Fluor dye antibodies(Invitrogen) were incubated for 2 h, respectively. Samples weremounted after extensive washing in Fluoromount-G (SouthernBiotechnology) and analyzed by a confocal laser scanningmicroscope (TCS SP5, Leica) equipped with respective 63�Plan Apo oil immersion objectives. Images were acquired insequential scan mode, and processing was done using NIHImageJ. Scale bars represent 10 �m. Acceptor photobleachFRET experiments were performed in transiently transfectedHeLa cells processed as stated above. FRET measurementswere performed by acquiring pre- and postbleach images of
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donor and acceptor using the Leica acceptor photobleach FRETmacro. Thresholded percent FRETvalueswere depicted using aseven-color look-up table. Quantitative FRET analysis was per-formed by calculatingmean FRET efficiency and S.E. for n� 18cells and two independent conditions (PKD1 versus PKD2).Statistical significance (****, p � 0.0001) was calculated usingtwo-tailed unpaired Student’s t test.Soft Agar Assays—Anchorage-independent growth was
measured using soft agar colony formation assays. StablePanc89 cells expressing the indicated constructs were seeded at10,000 cells/well in 6-well plates in 0.5% soft agar (Bacto Agar,BD Biosciences) with 0.5% agar bottom layers in three replicatewells per condition and in at least three independent experi-ments. Colonies were documented at 10� magnification usinga Keyance microscope after 13 days (see Fig. 6B) or 10 days,respectively (see Fig. 7, A and B). For transiently transfectedPanc1 cells, 50,000 cells/well in 6-well plates were seededand documented after 6 days (see Fig. 6C). Results were cal-culated by quantifying the average number of colonies pervisual field at 10� or 4� magnification, whereas for tran-sient expression in Panc1 cells, the entire well was counted.Statistical analysis was performed using one-way ANOVAwith Bonferroni multiple comparison post-testing or Stu-dent’s unpaired t testing.Cell Proliferation Assays—Cell proliferation assays were per-
formedwith transiently transfectedHeLa cells. After 24 h, 5000cells were seeded in 100 �l of standard growth medium in trip-licate replicas per condition in 96-well culture plates for timepoints T0, T24, T48. After adhesion overnight, T0 cells werefixed and stained with crystal violet (0.5% in H2O, 20% (v/v)methanol) for 20 min at room temperature. After extensivewashing, plates were dried, and additional plates were pro-cessed after 24 and 48 h in the same manner. To quantify celldensity, crystal violet was dissolved in 100 �l of methanol/well,and adsorption was measured at 550 nm using a Tecan1000plate reader. Doubling time was calculated using linear regres-sion (Prism software). Cell densities in graphs are shown formean A550 values in triplicate replicas �S.E.HDAC Activity Assays—HDAC activity assays were per-
formed using a fluorometric kit (Cayman Chemical Co.). 3 �
106 HeLa cells were seeded in 10-cm dishes with two dishes percondition. Cells were lysed after 48 h according to the manu-facturer’s instructions. Assays were performed in black 96-wellplates in triplicate replicas per condition. To measure HDACactivity, 10 �l of crude nuclear extract were used after normal-ization of protein content by a BCA kit. Deacetylation of a spe-cific HDAC substrate was measured at 455 nm (excitation, 360nm) using a TecanM1000 reader. Assays were further normal-ized for GFP transgene expression in crude nuclear extracts(Snail1-GFP and Snail1S11A-GFP) by measuring GFP fluores-cence at 535 nm (excitation, 475 nm). Statistical analysis wasperformed using one-way ANOVA with Bonferroni multiplecomparison post-testing.Statistic Analyses—Statistical analysis was performed using
Prism software version 5.00 forWindows (GraphPad Software,SanDiego, CA). Statistical significance in graphs is indicated byasterisks (*, p� 0.05 to 0.01; **, p� 0.01 to 0.001; ***, p� 0.001;****, p � 0.0001).
RESULTS
Following a bioinformatics screen, we identified (in accord-ance with Ref. 16) Snail1 as a putative PKD substrate andmapped the respective phosphorylation site to Ser-11.Mapping of PKD1 Phosphorylation Sites in Snail1—Fig. 1A
depicts a structural overview of Snail1 with the putative PKDphosphorylation site located at Ser-11 directly adjacent to itsSNAG domain (amino acids 1–9). The potential phosphoryla-tion site LVRKPS* matches the published PKD phosphoryla-tion consensus sequence LXRXXS* and partially matchesthe PKD phosphosubstrate antibody recognition sequence(pMotif; LXR(Q/K/E/M)(M/L/K/E/Q/A)S*) (25, 26). Usinganti-pMotif antibody, we investigated Snail1 in vivo phosphor-ylation by PKD1 (Fig. 1B). Active PKD1 enhanced phosphory-lation of Snail1, whereas Snail1 phosphorylation was barelydetectable in cells expressing catalytically inactive PKD1KD. Inaddition, phosphorylation of Snail1 was absent when Ser-11was replaced byAla (S11A) even in the presence of active PKD1.Thus, in accordance with data published by Du et al. (16),Ser-11 is a PKD phosphorylation site in vivo, and it is the onlyPKD phosphorylation site in Snail1 (Fig. 1B). Next, we wanted
FIGURE 1. Mapping of PKD phosphorylation sites in Snail1 in vivo. A, structural overview of Snail1. The N-terminal SNAG domain, serine-proline-rich region,destruction box, nuclear export sequence (NES), and C2H2 zinc fingers are shown. The putative PKD phosphorylation consensus motif of Snail1 Ser-11 and theconsensus sequence of the phospho-PKD substrate motif antibody (pMotif) are shown below the graph. B, mapping of Snail1 phosphorylation at Ser-11 in vivo.Blots depict immunoprecipitates (IP) of FLAG-Snail1 from HeLa cells co-expressing Snail1-FLAG and Snail1S11A-FLAG constructs with active (CA) and kinase-inactive (KD) PKD1. Control blots on the right-hand side display transgene expression. Phosphorylation of Snail1 at Ser-11 was probed using pMotif antibodyand reprobed with anti-FLAG M2. endogen, endogenous.
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to assess the upstream regulation of Snail1 by PKD isoforms 1and 2. To determinewhether both isoformswould interactwithSnail1 in intact cells, we performed co-localization and FRETstudies.Only PKD1 Interacts Efficiently with Snail1 in the Nuclei of
HeLa Cells—For co-localization and FRET studies, we usedtransiently transfected HeLa cells ectopically expressingSnail1-FLAG together with PKD1-GFP or PKD2-GFP, respec-tively. Both PKD1 and -2 were localized to nuclei of HeLa cellsand co-localized with FLAG-tagged Snail1 (Fig. 2, A and B). Tofurther characterize this co-localization and to determine apotential interaction, we performed acceptor photobleachFRET studies. Fig. 2A displays a representative FRET experi-ment for PKD1-GFP and Snail1-FLAG. Panels A� and B�depict donor pre- and postbleach states, whereas panels D�and E� show acceptor pre- and postbleach images, respec-tively. The relative increase in donor fluorescence intensity
is marked by arrowheads in postbleach images (panel B�).Percent FRET values indicating interaction of the two pro-teins are shown in panel F� depicted by a seven-color look-uptable (Fig. 2A, panels A�–F�). Similar experiments were per-formed for PKD2-GFP and Snail1-FLAG (Fig. 2B, panelsA�–F�). Active PKD2 is known to phosphorylate nuclear sub-strates (27). However, interaction of wild type PKD2 andSnail1 was barely detectable. Fig. 2C displays the statisticalanalysis of mean FRET efficiency and S.E. for PKD1-GFP(n � 18) and for PKD2-GFP (n � 17) cells. Mean FRETefficiency dropped markedly by 3.38-fold for PKD2 (3.8 �1.1%) as compared with PKD1 (12.9 � 1.1%) with some cellsdisplaying no interaction at all (Fig. 2B). (FRET efficiencyvalues for all experiments are shown in supplemental Table2.) These data indicate that PKD1 preferably interacts withSnail1 and suggests that interaction between PKDs andSnail1 is isoform-specific. We further verified these findings
FIGURE 2. PKD1- and PKD2-GFP co-localize with Snail1-FLAG in nuclei of HeLa cells. Only PKD1-GFP is capable of efficiently interacting with Snail1-FLAGin nuclei, whereas interaction efficiency is significantly reduced by 3.38 times for PKD2. A, panels A�–F�, acceptor photobleach FRET experiment in HeLa cellsco-expressing PKD1-GFP and Snail1-FLAG labeled with anti-FLAG M2 and Alexa Fluor 546 antibodies. Panels A� and B� depict donor pre- and postbleach,whereas panels D� and E� display acceptor pre- and postbleach states, respectively. Bleached regions of interest (ROIs) are shown in panel C�. Percent FRET valuesare depicted in panel F�, and FRET is represented by a thresholded seven-color look-up table (LUT). B, acceptor photobleach FRET experiment in HeLa cellsco-expressing PKD2-GFP and Snail1-FLAG. Panels A� and B�) depict donor pre- and postbleach, whereas panels D� and E� display acceptor pre- and postbleachstates, respectively. Bleached regions of interest are shown in panel C�. Percent FRET values are depicted in panel F�. Images shown are of a single confocalsection. The scale bar represents 10 �m. C, statistical analysis of acceptor photobleach FRET experiments displayed in A and B. The graph depicts mean FRETefficiency and S.E. for PKD1 (n � 18 cells) and PKD2-GFP (n � 17 cells) experiments. FRET efficiency values for all experiments are shown in supplemental Table2. Statistical significance (****, p � 0.0001) was calculated using a two-tailed unpaired Student’s t test. D, endogenous Snail1 and PKD1 interact. Anti-PKD1 andnonspecific IgGs were used for immunoprecipitation (IP) from Panc89 vector cells. Immunoprecipitations were subsequently probed for the presence ofendogenous Snail1 using specific antibodies. Error bars in graphs represent S.E.
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by co-precipitation experiments with endogenous Snail1and PKD1 (Fig. 2D).Du et al. (16) reported that phosphorylation of Snail1 at
Ser-11 by PKDs regulates its nuclear export by interaction with14-3-3� proteins in epithelial cell lines including C4-2 tumorcells. However, the authors conceded that tumor cellsmay havedifferent mechanisms for regulating Snail1 transcriptionalactivity. We investigated how subcellular localization of Snail1was altered when Ser-11 was phosphorylated by PKD1 in twoepithelial cancer cell lines, Panc1 humanpancreatic cancer cellsand HeLa cervical cancer cells.Subcellular Localization of Snail1, Snail1S11A, and
Snail1S11E Is Not Changed—To first investigate how Snail1phosphorylation would impact its subcellular localization, weperformed localization studies with Snail1 and the S11A and
S11E phosphosite mutants in HeLa and Panc1 cells, respec-tively. There was no detectable change in subcellular localiza-tion using the phosphosite mutants compared with Snail1 wildtype (WT) in HeLa cells (Fig. 3A) and Panc1 cells (data notshown). We quantified subcellular distribution in three exper-iments using HeLa cells and found that Snail1 predominantlylocalized in nuclei independently of its phosphorylation statusin more than 99% of at least 1000 cells quantified per condition(Fig. 3A, panels A�–O�). We also did not observe any change inthe subcellular localization of wild type Snail1 upon co-expres-sion with constitutively active PKD1 in both HeLa (Fig. 3B) andPanc1 cells (data not shown). Similar data were obtained withendogenous Snail1 in both cell lines expressing active PKD1(supplemental Fig. 1A). In addition, there was no change in thesubcellular localization of predominantly cytoplasmic endoge-
FIGURE 3. Phosphorylation of Ser-11 by PKD does not alter subcellular distribution of Snail1. A, Snail1-GFP (panels A�–E�), Snail1S11A-GFP (panels F�–J�),and Snail1S11E-GFP (panels K�–O�) are predominantly localized to the nucleus independently of Snail1 Ser-11 mutation status. B, co-expression of constitu-tively active PKD1.CA-GFP (A�) with wild type Snail1-FLAG (B� and E�) does not alter subcellular localization of Snail1 (A�–E�). Nuclei were stained with DAPI.Images depict single confocal sections. The scale bar represents 10 �m.
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nous Snail1 in non-transformed, immortalized HEK293T cellsupon expression of active PKD1 or kinase-inactive PKD1KD(supplemental Fig. 1B). According to Du et al. (16), Snail1should have exhibited nuclear localization in cells expressingPKD1KD in this setting. Thus, in the cell lines examined in thisstudy, 14-3-3� binding to a consensus surrounding Ser-11 doesnot seem to be the relevant mechanism for Snail1 subcellularlocalization. This prompted us to investigate further molecularmechanisms to explain the function of Snail1 phosphorylationby PKD1.Regulation of Snail1-mediated Transcriptional Activity by
Lysyl Oxidase-like Family Members 2 and 3—In addition toHDAC1 and -2 (22), LOXL2 and -3 are known Snail1 interac-tion partners, enhancing Snail1 protein stability and Snail1-de-pendent regulation of marker genes (19). To investigate theirrole in the regulation of Snail1 transcriptional activity down-stream of PKD1, we initially screened a panel of pancreatic can-cer cell lines including Panc89 cells stably expressing GFP vec-tor or PKD1-GFP (4, 5) as well as HeLa cells for the presence ofSnail1, PKD1, and the LOXL3 isoform (Fig. 4A). Snail1 andLOXL3 proteins were present in HeLa, Panc1, MiaPaca,Panc89, and the stable Panc89 cell lines. PKD1was expressed atdifferent levels in all cell lines. Snail1 was strongly expressed inHeLa, Panc1, and both stable Panc89 cell lines, prompting us touse these cells for further analyses. Using qPCR, we additionallytested which LOXL isoforms were present in these stablePanc89 GFP vector or PKD1-GFP cells and whether a furtherupstream regulation by PKD1 may be involved. Both LOXL2and -3 isoformswere expressed in Panc89 cells. To our surprise,the expression of LOXL3, but not of LOXL2, was significantlyup-regulated by 5.5� 0.36-fold in cells expressing PKD1 (Fig. 4,B and C), suggesting a PKD1-dependent synergistic regulationof Snail1 activity via LOXL3. Next, we investigated how phos-phorylation at Ser-11 would impact co-regulation by HDACs.Thus, we initially performed co-localization studies of WTSnail1 and Snail1-S11A with the published co-regulatorHDAC1 (22) in HeLa cells. Both Snail1-FLAG and Snail1S11A-FLAG co-localized with the endogenous co-repressor HDAC1in the nuclei (Fig. 4D). We were further able to demonstrateinteraction of Snail1-FLAG with HDAC1 in the nuclei byacceptor photobleach FRET (data not shown).Binding of HDAC1, HDAC2, and LOXL3 Is Impaired by
Snail1S11A, Reducing HDACActivity—To study themolecularimpact onHDACbinding following phosphorylation of Ser-11,
we performed co-immunoprecipitation studies with phospho-site mutants. The Snail co-repressors HDAC1 and -2 interactwith Snail1 in transcriptional complexes to regulate the expres-sion of target genes (22). LOXL2 and -3 isoforms also act asco-regulators, modifying Snail1-mediated transcriptional reg-ulation by enhancing its stability. The interaction of LOXL2with Snail1 has been shown to be dependent on the N-terminalpart of Snail1, which contains the SNAG domain (amino acids1–9) adjacent to the Ser-11 phosphorylation site, and this partis also essential for interaction with HDAC transcriptional co-repressors (19, 28). Thus, we performed co-immunoprecipita-tion experiments in HeLa cells following co-expression ofFLAG-tagged HDACs with Snail1 phosphosite mutants as wellas with endogenous HDAC1 and -2 (22). Strikingly, the inter-action of both HDAC1 (data not shown) and -2 with Snail1 wasdecreased upon expression of the Snail1S11A mutant but notupon expression of SnailS11E (Fig. 4E). Fig. 4Fdepicts the resultof three independent co-immunoprecipitation experiments forco-expressed HDAC2. HDAC2 binding was significantlyreduced with the S11Amutant and almost returned to the wildtype level with S11E. HDAC1 demonstrated the same overallpattern of regulation (data not shown).We also investigated theinteraction of endogenousHDACswith wild type Snail1 as wellas its S11A and S11Emutant proteins. Fig. 4,G andH, show thatbinding of endogenousHDAC1 andHDAC2 to Snail1S11Awasreduced as compared with wild type Snail1 from 1 to 0.53 timesintegrated band density for HDAC1 and to 0.58 times forHDAC2, whereas it was increased for the S11E mutant to 1.29times for HDAC1 and 1.24 times for HDAC2. Additional co-immunoprecipitation experiments with endogenous HDAC1and -2 also using other tags may be found in supplemental Fig.2, A–D. Thus, phosphorylation of Snail1 Ser-11 by PKD1 islikely to be required for the stable interactionwith its co-repres-sors (Fig. 4, E–H). In accordance with these data, LOXL3 inter-action with Snail1S11A was also decreased as observed in co-immunoprecipitation experiments. Integrated band densitieswere reduced from 1 for WT to 0.3 times for the Snail1S11Amutant (supplemental Fig. 2E).Wenext assessed how the phos-phorylation-dependent interaction of Snail1 with its co-regula-tors modulates HDAC transcriptional regulatory activity.Snail1-dependent HDAC Activity and Regulation of Prolifer-
ation Markers—We performed HDAC activity assays mea-suring WT Snail1- as well as Snail1S11A-associated histonedeacetylation to verify results of co-immunoprecipitation
FIGURE 4. A, Snail1, LOXL3, and PKD1 are expressed in a subset of pancreatic cancer cell lines, HeLa cells, and stable Panc89 cells expressing GFP vector as wellas PKD1-GFP. 200 �g of total cell lysates were probed with specific antibodies. B, expression and upstream regulation of the Snail1 co-regulator lysyl oxidase-like proteins 2 and 3 in stable Panc89 cell lines. LOXL3, but not LOXL2, is up-regulated by ectopic PKD1. The graph displays -fold change in regulation relativeto respective vector controls. qPCR for LOXL2 and LOXL3 was performed on RNA isolated from stable Panc89 cells expressing GFP and PKD1-GFP. Fourindependent experiments were quantified in triplicate replicas. Results were normalized to GAPDH and calculated according to the ��Ct method. Statisticalsignificance was calculated using one-way ANOVA with Dunnett’s multiple comparison post-testing (***, p � 0.05). C, LOXL3 expression is up-regulated instable PKD1-GFP Panc89 cells. 250 �g of total cell lysates were probed for LOXL3 using specific antibodies. D, regulation of Snail1 activity by phosphorylationat Ser-11. Co-localization of Snail1-FLAG (panels A�–C�) and Snail1S11A-FLAG (panels D� and E�) with their endogenous co-repressor HDAC1 in HeLa nuclei isshown. Images depict single confocal sections. The scale bar represents 10 �m. E, mutation Snail1S11A impairs interaction of Snail1 with co-expressedFLAG-HDAC2, whereas binding is reconstituted with Snail1S11E. Proteins were probed with respective specific antibodies in Western blots. F, statisticalanalysis of three independent co-precipitation experiments in E. -Fold change in HDAC2 co-precipitation with Snail1 and mutants was calculated fromintegrated band densities of Western blots. Significance was calculated using Student’s t test. G, co-immunoprecipitation (IP) of endogenous HDAC1 withSnail1-, Snail1S11A-, and Snail1S11E-GFP from HeLa total cell lysates. Endogenous HDAC1 was probed with specific antibodies, and immunoprecipitationswere reprobed for Snail1 expression by anti-Snail1 antibody. H, co-immunoprecipitation of endogenous HDAC2 with Snail1-, Snail1S11A-, and Snail1S11E-GFPin HeLa total cell lysates. Endogenous HDAC2 was probed with specific antibodies, and immunoprecipitations were reprobed for Snail1 expression. Error barsin graphs represent S.E.
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experiments. GFP vector, Snail1-GFP, or Snail1S11A-GFP con-structs were ectopically expressed in HeLa cells for 48 h, andcrude nuclear extracts were prepared using an HDAC activityassay kit (Cayman Chemical Co.). Equal amounts of extractwere used in assays, and results were further normalized toGFP-Snail1 transgene expression present in nuclear lysates. Inline with interaction studies, statistical analysis of three inde-pendent HDAC assays demonstrated reduced activity in cellsexpressing the Snail1S11A mutant by 21.37% compared withWT Snail1 (Fig. 5A). Expression of Snail1 transgene controls incrude nuclear lysates is shown in supplemental Fig. 3A. BecauseSnail1-associated HDAC activity contributes only partially tothe total HDAC activity as demonstrated by inhibition with the
HDAC inhibitor trichostatin (Fig. 5A), the extent of activityreduction by the S11A mutant is remarkable and also matchesthe markedly reduced HDAC1/2 and LOXL3 binding (Fig. 4,E–H, and supplemental Fig. 2E). Because we were interested inthe role of Snail1 in the control of pancreatic cancer growth, wenext investigated whether HDAC activity also translated intoexpression of marker proteins known to be involved in prolif-eration (Fig. 5, B and C). Thus, we investigated proliferationmarkers regulated downstream of Snail1 and PKD1 in Panc1and the stable Panc89 cell lines. Panc1 cells were transientlytransfected with GFP vector, Snail1-GFP, or Snail1S11A-GFPand changes in Cyclin D1 expression levels were observed (29–31). Cyclin D1 was markedly up-regulated by Snail1-GFP,
FIGURE 5. Snail1-dependent histone deacetylase activity and regulation of proliferation markers. A, Snail1S11A reduces Snail1-associated HDAC activityas compared with wild type Snail1. HDAC activity was measured using a fluorometric assay kit. Crude nuclear extracts from 10 � 106 HeLa nuclei werenormalized for protein expression, and HDAC activity was measured in triplicate wells per condition in 96-well plates (Tecan infinity M1000) for GFP vector,Snail1-GFP, and Snail1S11A-GFP. For Snail1-GFP and Snail1S11A-GFP, results were further normalized to GFP transgene expression levels in crude lysates. Thegraph depicts the combined statistical analysis of three experiments. Statistical significance was calculated using one-way ANOVA with Bonferroni multiplecomparison post-testing. Expression of transgenes in HeLa crude nuclear extracts and loading controls are shown in supplemental Fig. 3C. B, Snail1S11Aimpairs Snail1-mediated proliferation marker protein expression in Panc1 cells. Panc1 cells were transfected with GFP, Snail1-GFP, and Snail1S11A-GFP. CyclinD1 and Ajuba markers involved in the regulation of proliferation were probed in 60 �g of total cell lysates with specific antibodies. Transgenes were probedwith anti-Snail1 antibody. Actin was used as a loading control. C, PKD1 and PKD1KD-GFP regulate proliferation marker protein levels in a pattern similar to thatof phosphosite mutants. The expression levels of Cyclin D1 and Ajuba were probed with respective antibodies in 60 �g of total cell lysates of stable Panc89 cells.Transgenes were detected with anti-GFP antibody. Tubulin was used as a loading control. D, expression of the Snail target gene Cyclin D2 is prominentlyup-regulated by ectopic PKD1, but not PKD2 expression. The graph displays fold change in regulation relative to respective vector controls. Quantitativereal-time PCR for Cyclin D2 was performed on RNA isolated from stable Panc89 cells expressing GFP, PKD1-GFP, and PKD2-GFP. Four independent experimentswere quantified in triplicate replica. Results were normalized to GAPDH and calculated according to the ��CT-method. Statistical significance (**, p � 0.05) wascalculated using one-way Anova with Bonferroni multiple comparison post-testing. Error bars in graphs represent S.E.
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whereas expression of Snail1S11A reduced Cyclin D1 expres-sion (vector, 1-fold; Snail1-GFP, 2.7-fold; Snail1S11A-GFP,1.48-fold integrated band density; Fig. 4B). We further testedthe effects of the phosphomimetic Snail1S11E mutant onCyclin D1 expression. Indeed, Cyclin D1 was markedly up-reg-ulated by Snail1S11E (supplemental Fig. 3B). To substantiateour findings, we additionally investigated a second proliferationmarker, Ajuba. Interestingly, we found Ajuba to be a down-stream target of Snail1. Ajuba is known to regulate cell cycleprogression andG2/M transition by enhancingAuroraAkinaseactivity through direct interaction (32–34). Thus, Ajuba isinvolved in mitotic checkpoint control (34). Additionally,Aurora A and B kinases are overexpressed in cancer tissues andare potentially tumorigenic (32). Ajuba protein levels were up-regulated byWT Snail1, whereas its expression was reduced by
Snail1S11A (vector, 1-fold; Snail1-GFP, 2.22-fold; Snail1S11A-GFP, 1.78-fold integrated band density; Fig. 5B). To furthervalidate our results, we assessed the regulation of the samemarkers in Panc89 cells stably expressing either vector, PKD1-GFP, or kinase-inactive PKD1KD-GFP (Fig. 5C). In line withFig. 5B, Cyclin D1 was up-regulated 3.9-fold by PKD1-GFP,whereas its expression dropped 2.74-fold upon expression ofPKD1KD-GFP. The expression of Ajuba was up-regulated byPKD1-GFP 2.9-fold compared with only 1.6-fold by PKD1KD-GFP.Thus, PKD1-mediated regulation of proliferationmarkersis similar to that of Snail1 and its phosphosite mutants. How-ever, PKD1KD-GFP was not capable to act fully as a dominantnegative construct in these experiments. Thismay be explainedin line with the literature (23) by a prominent localization ofPKD1KD-GFP at the trans-Golgi network as evidenced by
FIGURE 6. A, PKD1, as opposed to PKD2, enhances anchorage-independent growth in soft agar experiments. We seeded 10,000 cells of stable Panc89 cell linesexpressing GFP, PKD1-GFP, PKD1KD-GFP, PKD2-GFP, and PKD2KD-GFP in triplicate wells in 0.5% soft agar and documented assays after 13 days. A, the graphdepicts the average number of colonies and S.E. per visual field documented at 10� magnification for five independent experiments. Statistical significance(****, p � 0.0001) was calculated using a two-tailed unpaired Student’s t test. B, panels A�–E�, examples of soft agar colonies documented for quantification. Thescale bar represents 100 �m. C, Snail1 expression enhances anchorage-independent growth of Panc1 cells as compared with vector control, whereasSnail1S11A reduces the number of colonies with respect to wild type Snail1. We transiently transfected 50,000 Panc1 cells and subsequently seeded cells in0.5% soft agar in triplicate wells per assay and in three experiments. Assays were documented after 6 days. The graph depicts the average number of coloniesand S.E. per well at 10� magnification. Statistical significance (****, p � 0.0001) was calculated using one-way ANOVA with Bonferroni multiple comparisonpost-testing. Representative transgene expression and images of colonies are shown in supplemental Fig. 4, A and B. Error bars in graphs represent S.E.
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strong co-localization with trans-Golgi network markerTGN46 (supplemental Fig. 3C).To further assess whether the regulation of downstream tar-
gets by PKDs was indeed isoform-specific, we examined theregulation of another Snail target, Cyclin D2 (18), by qPCR inPanc89 cells expressing PKD1- or PKD2-GFP, respectively. Inline with our previous findings, Cyclin D2 expression was up-regulated 1329-fold by wild type PKD1, whereas its expressionwas only up-regulated 90.8-fold by wild type PKD2, only 7% ofthe effect of PKD1. These data confirm the selective regulationof Cyclin D2 by PKD1 as compared with PKD2 (Fig. 5D).We next investigated whether these biochemical data would
translate into biological readouts. At first, soft agar assays wereperformed to identify changes in anchorage-independentgrowth mediated by PKD1 and -2 isoforms or the respectivekinase-inactive proteins.PKD1, but Not PKD2, Enhances Anchorage-independent
Growth in Panc89 Cells—Fig. 6A shows the statistical analysisof five independent soft agar experiments performed with
Panc89 cells stably expressing GFP vector, PKD1-GFP, kinase-inactive PKD1 (PKD1KD-GFP), PKD2-GFP, or kinase-inactivePKD2 (PKD2KD-GFP), respectively (4, 5). In line with ourFRET studies and the biochemical data shown above, only wildtype PKD1 significantly increased the average number of colo-nies per visual field by 31.4 times as compared with GFP vectorcells. PKD1KD reduced the number of colonies as comparedwith PKD1-GFP by 3.6 times but still had a minor effect on
FIGURE 7. Snail1 is a necessary and sufficient mediator of PKD1-regulated anchorage-independent growth and proliferation in pancreatic cancercells. Stable Panc89 cells expressing GFP vector and PKD1-GFP were transduced with lentiviruses expressing non-target shRNA (scrambled; Sigma-Aldrich),sh_Snail1 1 (NM_005985.2-136s1c1, Sigma-Aldrich), and sh_Snail1 2 (NM_005985.2-504s1c1, Sigma-Aldrich) and subjected to antibiotic selection. Then weused 10,000 cells of stable cell lines expressing the respective constructs and shRNAs and seeded cells in triplicate wells in 0.5% soft agar. Assays weredocumented after 10 days at 4� magnification for colony counting. A, the graph depicts the combined average number of colonies per visual field of threeexperiments with six images at 4� magnification per well and three replicate wells per experiment. B, exemplary images (panels A�–F�) used for quantificationof colony numbers at 4� magnification. The scale bar represents 100 �m. Table 1 displays average differences (%) in colony number between conditions.Statistical significance (****, p � 0.0001) was calculated using one-way ANOVA with Bonferroni multiple comparison post-testing. C, control blots for knock-down efficacy of endogenous Snail1 with sh_Snail1 1 and 2 in stable Panc89 cells. Snail1 expression levels were probed in 60 �g of total cell lysates usinganti-Snail1 antibody. Tubulin was used as a loading control. Error bars in graphs represent S.E.
TABLE 1Regulation of anchorage-independent growth by PKD1 and Snail1shRNAsRelative differences (%) in colony numbers are indicated by positive and negativevalues, respectively.
Colonies �/�Change
%Vector_sh_scramble to PKD1-GFP_sh_scramble �84.6Vector_sh_scramble to Vector_sh_Snail1 1 �60.4Vector_sh_scramble to Vector_sh_Snail1 2 �53.6PKD1-GFP_sh_scramble to PKD1-GFP_sh_Snail1 1 �68.5PKD1-GFP_sh_scramble to PKD1-GFP_sh_Snail1 2 �82.8
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anchorage-independent growth of Panc89 cells, which corre-lates well with the data on proliferationmarker expression (Fig.5B). In contrast, PKD2 had no effect on anchorage-indepen-dent growth (Fig. 6, A and B, panels D� and E�). Fig. 6B, panels
A�–E�, show representative colonies documented for quantifi-cation. In addition to the significant increase in colony number,colony size was also markedly increased upon expression ofPKD1 (Fig. 6B, panel B�). Thus, our results again indicate a
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PKD1 isoform-specific regulation of anchorage-independentproliferation in pancreatic cancer cells. We additionally per-formed soft agar experiments with transiently transfectedPanc1 cells expressing GFP vector, WT Snail1-GFP, and theS11A mutant construct (Fig. 6C). In line with previous experi-ments performed with PKD1, anchorage-independent growthin Panc1 cells was significantly enhanced by WT Snail1 (3.65times) as compared with vector control (****, p � 0.0001) andreduced by 44% upon expression of the S11Amutant comparedwith WT Snail1 (****, p � 0.0001) (Fig. 6C). Colonies docu-mented for the respective conditions are depicted in supple-mental Fig. 4A (panels A�–C�), and transgene expression oftotal cell lysates is shown in supplemental Fig. 4B.Snail1 Is Required to Mediate PKD1-regulated Effects on
Anchorage-independent Growth—To demonstrate that PKD1-mediated Snail1 phosphorylation is required for PKD1-inducedanchorage-independent growth of pancreatic cancer cells, weperformed soft agar assays with Panc89 cells stably expressingvector or PKD1-GFP with two different shRNAs against Snail1as well as a non-targeting scrambled control. Fig. 7A displaysthe summarized statistical analysis of three soft agar assayswithPanc89 cells. PKD1 expression increased anchorage-indepen-dent growth as compared with vector cells by 84.6%. In vectorcells, knockdown of Snail1 reduced the average number of col-onies per visual field by 60.4% for sh_Snail1 1 and 53.6% forsh_Snail1 2. For PKD1-expressing cells, Snail1 knockdownreduced the number of colonies by 68.5% for sh_Snail1 1 and82.8% for sh_Snail1 2 (Table 1). We also quantified colony size.PKD1 expression enhanced colony size, and this was reducedby knockdown of Snail1 (data not shown). Examples of imagesused for quantification of colony numbers at 4� magnificationare shown in Fig. 7B for all conditions (panels A�–F�). Therespective knockdown controls for Snail1 in the stable cell linesare shown in Fig. 7C. In conclusion, these data indicate thatSnail1 as a downstream target of PKD1 is required to regulateanchorage-independent growth of pancreatic cancer cells bySer-11 phosphorylation.PKD1 Enhances Whereas PKD1KD Inhibits Panc89 Tumor
Cluster Growth in Three-dimensional BMECulture—To inves-tigate a PKD1-dependent regulation of anchorage-dependenttumor cluster growth and proliferation, we performed three-dimensional BME culture using stable Panc89 cells expressingPKD1- and PKD1KD-GFP. Vector, PKD1-GFP, and PKD1KD-
GFP cells were seeded in BME and documented after 16 days ofgrowth. Fig. 8A displays representative examples of tumor cellclusters used for the assessment of three-dimensional growth(diameter). In line with the soft agar assays, the average size oftumor cell clusters with stable ectopic expression of PKD1-GFPwas significantly increased by 10.1% as compared with GFPvector-expressing cells (***, p � 0.0005) (Fig. 8, A and B).PKD1KD-GFP significantly reduced the average cluster diam-eters by 10.3% when compared with vector controls (****, p �0.0001) (Fig. 8B), indicating that PKD1 is also involved in theregulation of anchorage-dependent growth of pancreatictumor cell clusters. Fig. 8, C and D, depict the respective fre-quency distribution histograms of tumor cluster diameters forPKD1-GFP and PKD1KD compared with GFP control cells.These data demonstrate that PKD1 expression resulted in ahigher percentage of larger clusters, whereas PKD1KD-ex-pressing cells formed smaller colonies. To corroborate thesedata, we performed proliferation assays with HeLa cells toinvestigate a general regulation of anchorage-dependent prolif-eration by Snail1 Ser-11 phosphorylation. GFP vector, WTSnail1, and the Snail1S11A mutant were transiently expressedin HeLa cells, and proliferation was quantified by measuringA550 values of crystal violet-stained cells at time points T0, T24,and T48 h. 48 h after transfection WT Snail1 markedlydecreased doubling times from 56.25 to 35.65 h (vector versusSnail1-GFP), enhancing proliferation, whereas expression ofSnail1S11A had virtually no effect on the doubling time (52.17h) (Fig. 8E). Transgene expression is shown in supplementalFig. 5. Thus, PKD1-dependent phosphorylation of Snail1 atSer-11 is involved in controlling anchorage-dependent and -in-dependent growth and proliferation in two-dimensional andthree-dimensional environments. To further validate our dataon the regulation of proliferation by PKD1, we performed len-tivirus-mediated knockdown experiments in GFP vector cellsfollowed by three-dimensional BME culture (Fig. 8, F–H). Clus-ters were documented after 32 days (Fig. 8G). In line with allprevious data, knockdown of PKD1 resulted in drasticallyreduced cluster sizes (diameters) of 38.3% in PKD1 knockdowncells (Fig. 8H). Specific knockdown of PKD1, but not PKD2, wasverified by isoform-specific antibodies (Fig. 8F). Frequency dis-tribution histograms show a shift to smaller cluster diametersfollowing knockdown of PKD1 (Fig. 8I). Taken together, thesefindings indicate that PKD1 enhances proliferation and
FIGURE 8. Three-dimensional growth in BME. A, panels A�–C�, 10,000 single cells of stable Panc89 cell lines expressing GFP, PKD1-GFP, and PKD1KD-GFP wereseeded in a BME gel and documented in assays after 16 days. The scale bar represents 100 �m. B, PKD1 significantly enhances clusters growth, whereas PKD1KDdecreases cluster size. The average diameter of tumor cell clusters was quantified in perpendicular directions for each cluster using spacial calibration of images(for vector, n � 150; for PKD1-GFP, n � 161; and for PKD1KD-GFP, n � 181). The graph depicts average diameters and S.E. of three experiments. C, frequencydistribution histograms of structure diameters for vector versus PKD1-GFP. D, frequency distribution histogram of structure diameters for vector versusPKD1KD-GFP. E, Snail1 enhances whereas S11A mutation inhibits proliferation in HeLa cells after 48 h. The combined analysis of three independent prolifer-ation assays was performed with transiently transfected cells expressing vector, Snail1-GFP, and Snail1S11A-GFP. Cells were seeded after 24 h at a density of5000 cells/well in triplicate replicas in 96-well plates. Cell density was quantified by measuring A550 of crystal violet-stained cells dissolved in methanol at timepoints T0, T24, and T48 h. The graph depicts the relative mean intensities for the respective cell lines after 24 and 48 h, respectively. Statistical significance wascalculated using an unpaired Student’s t test. Doubling times were calculated using linear regression (GraphPad Prism). Representative transgene expressionis shown in supplemental Fig. 5. F, Panc89 GFP vector cells were transduced with lentiviruses expressing scrambled control shRNA (Sigma-Aldrich) andsh_PKD1 (NM_002742.x-2978s1c1, Sigma-Aldrich). A PKD1 knockdown was probed using a specific anti-PKD1 antibody in semistable cell lines followingselection. Blots were reprobed for PKD2 expression, and Actin was used as a loading control. G, semistable Panc89 vector sh_scramble- and sh_PKD1-expressing cells were seeded at 10,000 single cells in BME gel and documented after 32 days. The scale bar represents 100 �m. H, knockdown of PKD1significantly reduces clusters growth (diameter). The average diameter of tumor cell clusters was quantified in perpendicular directions for sh_scramble (n �45) and sh_PKD1 (n � 84). The graph depicts average diameters and S.E. of three experiments. Numbers in the graph denote -fold change in percent.I, frequency distribution histogram for knockdown of PKD1 versus scrambled shRNA control. Knockdown of PKD1 significantly reduces cluster sizes in the BMEmatrix. Error bars in graphs represent S.E.
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anchorage-dependent growth of tumor cell clusters in three-dimensional culture. By contrast, PKD1KD-GFPor knockdownof PKD1 significantly inhibited proliferation, and this wasmediated by phosphorylation of Snail1 at Ser-11.
DISCUSSION
PKDs are involved in the regulation of important cellularfeatures such as proliferation (10, 11, 13, 35–37), motility, andinvasiveness (2–5) of different tumor types. However, specificand detailed functions for distinct PKD isoforms have not beenaddressed so far. In a previous work, Ochi et al. (38) have pro-posed a function for PKD1 in the regulation of anchorage-de-pendent growth. However, the properties of distinct PKD iso-forms were not directly compared or addressed by inhibitorsthat are not isoform-specific. Thus, it is as yet unclear whetherPKD isoforms act in a redundant or specific fashion in tumors.Our findings indicate that PKD1, as opposed to PKD2, regu-
lates the expression of marker proteins involved in a hyperpro-liferative phenotype such as Cyclins D1 and D2 (29, 31) as wellas Ajuba (33, 34) via phosphorylation of Snail1 at serine 11 inpancreatic cancer cells. Our data also suggest that phosphory-lation at this site is necessary for efficient binding of vital Snail1co-repressors such as HDAC2 modulating Snail1-dependentHDAC activity. In contrast to Du et al. (16), Snail1 phosphory-lation at Ser-11 did not affect nucleocytoplasmic shuttling ofthe protein. Thismay be explained by 14-3-3� down-regulationin many tumor cells by different mechanisms (39) includingpromotor methylation and inhibition downstream of p53mutations, thereby facilitating cancer formation by many
routes (40). Indeed, PKD1KD was not able to induce nuclearlocalization of primarily cytoplasmic Snail1 in non-trans-formed, immortalized HEK293T cells (supplemental Fig. 1B).Here we propose a different mechanism for the regulation ofSnail1 function by PKD1 in tumor cells: the phosphorylation-dependent binding of co-repressors such as HDAC2 to Snail1.In addition to regulation of HDAC activity, we identifiedanother regulatory mechanism induced by PKD1 that affectsSnail1 function: PKD1 is required for up-regulation of LOXL3,which can stabilize the Snail1 protein (Fig. 4, A, B, and C, andsupplemental Fig. 2E).In conclusion, our data demonstrate that PKD1 enhances
proliferation (10, 36) of pancreatic and other cancer cells, andthis regulation is mediated by Snail1 via phosphorylation atSer-11. Snail1 is therefore required and sufficient for PKD1-driven proliferation and anchorage-independent growth of dif-ferent tumor cells. An overview of PKD1-mediated Snail1 reg-ulation and control of biological effects is depicted in Fig. 9.Thus, PKD1 expression could be relevant for primary tumors
to drive proliferation and initiate epithelial-mesenchymal tran-sition, preparing cells for the dissemination phase. At laterstages, however, when cells are invading the surroundingmatrix or tumor stroma, loss of PKD1 activity could even bebeneficial because loss of PKD1 enables cells to acquire a highmotility phenotype via the regulation of Actin-regulatory pro-teins such as Cortactin and Slingshot1L. This is also furthersupported by reports showing a reduced expression of PKD1 ina number of invasive tumor cells and tumor tissues (2).
Acknowledgments—We thank Dr. A. Hausser (University of Stutt-gart) for providing PKD1 and -2 expression constructs.We thank Pro-fessor Peter Scheurich (University of Stuttgart) and the University ofHalle Cell Sorting Core facility for sorting of stable Panc89 cell lines.
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FIGURE 9. Overview of PKD1-mediated Snail1 regulation. PKD1 phosphor-ylation of Snail1 Ser-11 is necessary for efficient binding of co-repressorsHDAC1 and -2 as well as LOXL3. Expression of LOXL3, acting as a functionaltranscriptional co-activator, is also up-regulated by PKD1, implying a positivesynergistic activation of Snail1. Snail1 phosphorylation at Ser-11 by PKD1enhances Snail1 marker protein expression involved in proliferation andanchorage-independent growth. This regulation is necessary as well as suffi-cient to modulate hyperproliferation in Panc89 pancreatic ductal adenocarci-noma cells and other cell lines. 2D, two-dimensional; 3D, three-dimensional.
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Snail1 Mediates PKD1-induced Growth of Cancer Cells
32380 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 287 • NUMBER 39 • SEPTEMBER 21, 2012
