Portal de Periódicos da CAPES

HDAC 2 promotes loss of primary cilia in pancreatic ductal adenocarcinoma

2016; Springer Nature; Volume: 18; Issue: 2 Linguagem: Inglês

10.15252/embr.201541922

1469-3178

Tetsuo Kobayashi, Kosuke Nakazono, Mio Tokuda, Yu Mashima, Brian David Dynlacht, Hiroshi Itoh,

Hedgehog Signaling Pathway Studies

Article27 December 2016free access HDAC2 promotes loss of primary cilia in pancreatic ductal adenocarcinoma Tetsuo Kobayashi Corresponding Author Tetsuo Kobayashi [email protected] orcid.org/0000-0003-0698-7224 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Kosuke Nakazono Kosuke Nakazono Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Mio Tokuda Mio Tokuda Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Yu Mashima Yu Mashima Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Brian David Dynlacht Brian David Dynlacht Department of Pathology and Cancer Institute, Smilow Research Center, New York University School of Medicine, New York, NY, USA Search for more papers by this author Hiroshi Itoh Hiroshi Itoh Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Tetsuo Kobayashi Corresponding Author Tetsuo Kobayashi [email protected] orcid.org/0000-0003-0698-7224 Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Kosuke Nakazono Kosuke Nakazono Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Mio Tokuda Mio Tokuda Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Yu Mashima Yu Mashima Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Brian David Dynlacht Brian David Dynlacht Department of Pathology and Cancer Institute, Smilow Research Center, New York University School of Medicine, New York, NY, USA Search for more papers by this author Hiroshi Itoh Hiroshi Itoh Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan Search for more papers by this author Author Information Tetsuo Kobayashi *,1, Kosuke Nakazono1, Mio Tokuda1, Yu Mashima1, Brian David Dynlacht2 and Hiroshi Itoh1 1Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara, Japan 2Department of Pathology and Cancer Institute, Smilow Research Center, New York University School of Medicine, New York, NY, USA *Corresponding author. Tel: +81 743 72 5441; E-mail: [email protected] EMBO Reports (2017)18:334-343https://doi.org/10.15252/embr.201541922 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Loss of primary cilia is frequently observed in tumor cells, including pancreatic ductal adenocarcinoma (PDAC) cells, suggesting that the absence of this organelle may promote tumorigenesis through aberrant signal transduction and the inability to exit the cell cycle. However, the molecular mechanisms that explain how PDAC cells lose primary cilia are still ambiguous. In this study, we found that inhibition or silencing of histone deacetylase 2 (HDAC2) restores primary cilia formation in PDAC cells. Inactivation of HDAC2 results in decreased Aurora A expression, which promotes disassembly of primary cilia. We further showed that HDAC2 controls ciliogenesis independently of Kras, which facilitates Aurora A expression. These studies suggest that HDAC2 is a novel regulator of primary cilium formation in PDAC cells. Synopsis Loss of primary cilia is frequently observed in tumor cells. This study shows that histone deacetylase 2 (HDAC2) contributes to the suppression of primary cilia formation in pancreatic ductal adenocarcinoma (PDAC) cells by controlling Aurora A levels in a Kras-independent manner. Inhibition or depletion of HDAC2 induces primary ciliogenesis in PDAC cells. HDAC2 positively regulates expression of Aurora A kinase. HDAC2 and Kras independently control loss of primary cilia in PDAC cells. Introduction The primary cilium is a hair-like protrusion from the surface of most mammalian cells, functioning as a cellular "antenna" by transducing extracellular signals to the cell body 123. Cylindrical centrioles, which are distinguished as mother and daughter centrioles, compose a centrosome and serve as spindle poles during mitosis, whereas the mother centriole differentiates into a basal body to extend a primary cilium in quiescent cells. Structural and/or functional abnormalities of the cilium are implicated in multiple genetic disorders, collectively termed ciliopathies. Recent studies have shown that defects associated with primary cilia have strong correlations with cancer 45. As primary cilia are important for signaling and are assembled from centrioles that organize spindle poles, it is likely that the absence of the organelle may promote tumorigenesis by aberrant signal transduction and cell cycle regulation. Primary cilia are diminished or lost in multiple cancers, including pancreatic ductal adenocarcinoma (PDAC) 67, renal cell carcinoma 8, basal cell carcinoma 9, breast cancer 101112, ovarian cancer 13, prostate cancer 14, medulloblastoma 15, cholangiocarcinoma 16, glioblastoma 17, and melanoma 18. PDAC accounts for the most frequently occurring pancreatic tumor, which has one of the highest mortality rates with a 5-year survival rate lower than 5% in patients 19. Oncogenic Kras is the most commonly mutated gene in PDACs, which occurs in > 90% of the cells, leading to a constitutively active form of Kras 20. Previously, it has been shown that primary cilia are absent from human PDAC lesions and cultured PDAC cells, independent of proliferation 7. Moreover, inhibition of Kras downstream effectors, MEK and PI3K, has been shown to restore primary cilia formation in PDAC cells, suggesting that ciliogenesis is suppressed by aberrant Kras signaling in a proliferation-independent manner. However, molecular mechanisms that explain how primary ciliogenesis is repressed in PDAC cells remain unclear. In this study, we identified a histone deacetylase, HDAC2, as a regulator of primary cilia formation in PDAC cells. HDAC2 has been known to regulate gene expression by removing acetyl groups from lysine residues within histones 21. We found that expression of Aurora A kinase, previously reported to promote disassembly of primary cilia 22, is positively regulated by HDAC2 in PDAC cells. We further showed that HDAC2 and Kras independently control ciliogenesis by regulating Aurora A expression. These results suggest that HDAC2 contributes to suppression of primary cilia formation by controlling Aurora A levels in a Kras-independent manner in PDAC cells. Results Treatment of HDAC inhibitors restores primary cilia in PDAC cells To clarify how primary cilia formation is suppressed in PDAC cells, we first used Panc1 cells, which possess an oncogenic mutation in Kras 23 and assemble primary cilia with low frequency after induction of quiescence 24. The cells were induced to quiesce to identify proteins that contribute to suppression of primary ciliogenesis in a proliferation-independent manner. HDAC6 is known to play a role in disassembly of primary cilia 22. Therefore, to identify factors that regulate primary cilia formation in PDAC cells, we investigated whether inhibitors of this histone deacetylase (HDAC), or others, have an effect on ciliation in Panc1 cells. We initially treated Panc1 cells with trichostatin A (TSA), an inhibitor of class I, II, and IV HDACs (HDAC1-11) 25, and performed immunofluorescence experiments with antibodies against glutamylated tubulin and Ki67 to quantitate cells that had assembled primary cilia and that were cycling, respectively. Treatment with TSA significantly restored primary cilia formation at 100 and 500 nM (Fig 1A and B). These data suggest that TSA treatment induces primary cilia formation in Panc1 cells. However, we showed that TSA treatment did not significantly impact the cell cycle by determining the percentage of cells with Ki67-positive nuclei, flow cytometry, and expression of a cell cycle marker (Figs 1B and EV1A and B), suggesting that cilium assembly in TSA-treated cells does not occur as a result of cell cycle perturbation. To further pinpoint which HDACs are responsible for repression of primary cilia in Panc1 cells, we next tested other HDAC inhibitors, such as valproic acid (VPA), MS-275, and FK228/depsipeptide. VPA, MS-275, and depsipeptide inhibit class I and IIa (HDAC1-5, 7-9), HDAC1-3, and HDAC1-2, respectively 25. The frequency of ciliation was significantly increased by treatment with VPA, MS-275, or depsipeptide without altering the cell cycle (Figs 1C–E and EV1A and B). We confirmed that the treatment with HDAC inhibitors did not overtly influence cell viability or expression of markers for apoptosis and stress signaling (Fig EV1B and C). These data suggest that HDAC1 and/or HDAC2 could play a role in suppression of primary cilia in Panc1 cells. Figure 1. Treatment with HDAC inhibitors restores primary cilia in Panc1 cells A. Panc1 cells in serum-starved medium were treated with DMSO or 500 nM TSA for 48 h. Cells were visualized with antibodies against glutamylated tubulin (GT335) (green) and Ki67 (red). DNA was stained with Hoechst (blue). Arrows indicate primary cilia. Scale bar, 10 μm. B–E. Panc1 cells in serum-starved medium were incubated with the indicated concentration of (B) TSA, (C) VPA, (D) MS-275, and (E) depsipeptide/FK228 (DP) for 48 h. The percentages of ciliated or Ki67-positive cells were determined by immunostaining with antibodies against glutamylated tubulin and Ki67. Average of three to five independent experiments is shown. Error bars represent standard error of the mean (SEM). *P < 0.05, **P < 0.01 compared with DMSO (B, D, E) or DW (C) (two-tailed Student's t-test). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Depletion or inhibition of HDAC2 does not impact on cell cycle, cell growth, and expression of various signaling markers in serum-starved PDAC cells A–C. Panc1 cells were treated with the indicated inhibitors or siRNAs and cultured in serum-starved medium for 48 h. (A) FACS analysis. Average of five to six (inhibitor) or three (knockdown) independent experiments is shown. (B) Cell extracts were immunoblotted with antibodies against phospho-JNK, JNK, phospho-p38, p38, PARP, and cyclin D1. β-Actin was used as a loading control. (C) MTT cell viability analysis. Average of three (inhibitor) or five (knockdown) independent experiments is shown. Error bars represent SEM. D. Panc1 cells transiently transfected with control, HDAC2#2, or Kras siRNA were cultured in serum-starved medium for 48 h and immunostained with antibodies to (upper row) glutamylated tubulin (green) and Arl13b (red), and (lower row) acetylated tubulin (green) and IFT88 (red). DNA was stained with Hoechst (blue). Scale bar, 10 μm. Arrows indicate primary cilia. Download figure Download PowerPoint Depletion of HDAC2 induces primary ciliogenesis in PDAC cells To determine whether HDAC1 and/or HDAC2 suppress primary ciliogenesis in PDAC cells, we next investigated the consequences of HDAC1 or HDAC2 depletion. We transfected siRNAs and verified that the levels of HDAC1 or HDAC2 were considerably reduced in Panc1 cells (Fig 2A). Ablation of HDAC2 using two different siRNAs led to a significant increase in primary cilia in Panc1 cells without affecting the cell cycle (Figs 2B and EV1A and B). We further verified that silencing of HDAC2 did not affect cell viability and expression of markers for apoptosis and stress signaling (Fig EV1B and C). On the other hand, depletion of HDAC1 did not restore primary cilia formation, suggesting that HDAC2 is responsible for the suppression of primary cilia formation (Fig 2B). Next, we used additional markers against primary cilia, such as acetylated tubulin, IFT88, and Arl13b to further verify that HDAC2 depletion in Panc1 cells induces cilium formation (Fig EV1D). Furthermore, we depleted IFT88, a protein essential for the formation and maintenance of the cilium, in HDAC2-ablated Panc1 cells and found that loss of IFT88 abrogated increased primary cilium formation without impacting on cell cycle (Fig 2C and D). These data collectively indicate that knockdown of HDAC2 promotes bona fide primary cilium assembly in Panc1 cells. Figure 2. Depletion of HDAC2 induces primary ciliogenesis in PDAC cells A, B. Panc1 cells transiently transfected with control, HDAC2#1, HDAC2#2, or HDAC1 siRNA were cultured in serum-starved medium for 48 h. (A) Cell extracts were immunoblotted with antibodies against HDAC1, HDAC2, and Aurora A. β-Actin was used as a loading control. (B) The percentages of cells with primary cilia or Ki67-positive nuclei were determined as described in Fig 1. Average of three to five independent experiments is shown. C, D. Panc1 cells transiently transfected with control, HDAC2#2, IFT88, or HDAC2#2 and IFT88 siRNA were cultured in serum-starved medium for 48 h. (C) Cell extracts were immunoblotted with antibodies against IFT88 and HDAC2. β-Actin was used as a loading control. (D) The percentages of ciliated or Ki67-positive cells were determined as described in Fig 1. Average of three independent experiments is shown. E–G. Panc1 cells treated with control, HDAC2#2, or Kras siRNA were transfected with plasmids expressing GFP and mock, siRNA-resistant (siR-)HDAC2 or siR-HDAC2/H142A and induced to quiescence for 72 h. (E) Cell extracts were immunoblotted with antibodies against HDAC2 and Kras. β-Actin was used as a loading control. (F) Cells were immunostained with an anti-glutamylated tubulin antibody (red). DNA was stained with Hoechst (blue). Arrows indicate primary cilia in GFP-positive cells. Scale bar, 10 μm. (G) The percentages of GFP-positive Panc1 cells with primary cilia were determined. Average of three independent experiments is shown. H, I. KrasPDEC cells transiently transfected with control, mouse HDAC1 (simHDAC1), or mouse HDAC2 (simHDAC2) siRNA were induced to quiescence for 48 h. (H) Cell extracts were immunoblotted with antibodies against HDAC1 and HDAC2. α-Tubulin was used as a loading control. (I) The percentages of ciliated or Ki67-positive cells were determined as described in Fig 1. Average of three independent experiments is shown. Data information: Error bars represent SEM. *P < 0.05, **P < 0.01 compared with siControl (two-tailed Student's t-test). Download figure Download PowerPoint To test whether HDAC2 suppresses primary ciliogenesis through its histone deacetylase activity in PDAC cells, we carried out rescue experiments in which we expressed siRNA-resistant wild-type HDAC2 or a catalytically inactive mutant of HDAC2 (H142A) in serum-starved Panc1 cells depleted of endogenous HDAC2 (Fig 2E). We found that while wild-type HDAC2 canceled ciliation by HDAC2 ablation, the deacetylase mutant did not (Fig 2F and G). These data suggest that histone deacetylase activity of HDAC2 contributes to suppression of primary cilia formation in Panc1 cells. We next verified whether HDAC2 suppresses primary cilia formation in other PDAC cells. To this end, we investigated CFPAC1 cells, which were previously shown to assemble primary cilia with low frequency 24. We observed that TSA treatment or HDAC2 knockdown in CFPAC1 restored primary cilia without affecting proliferation (Fig EV2A and data not shown). We next examined mouse primary Kras/G12D-expressing pancreatic duct epithelial cells (KrasPDEC) that are recognized as potential cells of origin for PDAC 262728. We treated KrasPDEC cells with several HDAC inhibitors, including TSA, VPA, MS-275, and depsipeptide, and found that such treatments restored primary cilia formation without significant alterations in proliferative index (Fig EV2B–D). Similar to Panc1 and CFPAC1 cells, we were able to confirm that the inhibition of HDAC2 induced primary cilia formation in KrasPDEC cells, and depletion of HDAC1 did not impinge upon the frequency of ciliation (Fig 2H and I). These observations collectively suggest that primary cilium formation is commonly suppressed by HDAC2 in PDAC cells. Click here to expand this figure. Figure EV2. Depletion or inhibition of HDAC2 restores primary cilia in PDAC cells A. CFPAC1 cells transiently transfected with control or HDAC2#2 siRNA were cultured in serum-starved medium for 48 h. The percentages of cells with primary cilia or Ki67-positive nuclei were determined as described in Fig 1. Average of three independent experiments is shown. B–D. KrasPDEC cells in serum-starved medium were incubated with the indicated concentration of (B) TSA, (C) VPA, and (D) MS-275 or depsipeptide for 24 h. The percentages of cells with primary cilia or Ki67-positive nuclei were determined as described in Fig 1. Average of three to four independent experiments is shown. E. U87-MG cells transiently transfected with control or HDAC2#2 siRNA were cultured in serum-starved medium for 48 h. The percentages of cells with primary cilia or Ki67-positive nuclei were determined as described in Fig 1. Average of three independent experiments is shown. F, G. RPE1 cells transiently transfected with control, HDAC2#2, IFT88 or HDAC2#2, and IFT88 siRNA were cultured in serum-containing medium for 48 h. (F) Cell extracts were immunoblotted with antibodies against IFT88 and HDAC2. β-Actin was used as a loading control. (G) The percentages of ciliated or Ki67-positive cells were determined as described in Fig 1. Average of three independent experiments is shown. H–J. IMCD3 cells transiently transfected with plasmids expressing GFP and mock, HDAC2 or HDAC2/H142A were cultured without serum for 72 h. (H) Cell extracts were immunoblotted with an antibody against HDAC2. β-Actin was used as a loading control. (I) Cells were immunostained with an anti-Arl13b antibody (red). DNA was stained with Hoechst (blue). Arrows indicate primary cilia in GFP-positive cells. Scale bar, 5 μm. (J) The percentages of GFP-positive IMCD3 cells with primary cilia were determined. Average of three independent experiments is shown. Data information: Error bars represent SEM. *P < 0.05, **P < 0.01 compared with siControl (A, E, G), DMSO (B, D), DW (C), or mock (J) (two-tailed Student's t-test). Download figure Download PowerPoint We further investigated whether HDAC2 generally contributes to suppression of primary cilia. First, we depleted HDAC2 in human glioblastoma U87-MG cells and found that HDAC2 ablation caused primary ciliogenesis (Fig EV2E). We next used human diploid retinal pigment epithelial cells (RPE1), which rarely form primary cilia in cycling cells and assemble primary cilia with high frequency after induction of quiescence. We depleted HDAC2 in proliferating RPE1 cells and found that HDAC2 ablation induced primary ciliogenesis and a simultaneous decrease in Ki67-positive cells (Fig EV2F and G). To test whether a decrease in cycling cells is caused by cilia formation, we performed double knockdown of HDAC2 and IFT88. Silencing of IFT88 significantly canceled cilia formation and a decrease in Ki67-positive cells after HDAC2 ablation (Fig EV2G), suggesting that loss of HDAC2 induces primary cilia formation which does not occur as a result of cell cycle perturbation. Collectively, these results suggest that HDAC2 generally contributes to suppression of primary cilia. To investigate the impact of enforced HDAC2 expression on primary cilia formation, we expressed HDAC2 in mouse inner medullary collecting duct (IMCD3) cells, which assemble primary cilia with high frequency after serum deprivation. We found that ciliation was significantly decreased by HDAC2 expression, but not by the HDAC2 deacetylase mutant (Fig EV2H–J), strongly supporting our conclusion that primary cilia formation is suppressed by HDAC2. HDAC2 positively regulates expression of Aurora A kinase HDAC2 predominantly localizes to the nucleus 21 and is not detected at the centrosome or primary cilium in Panc1 cells (Fig EV3A). This implies that HDAC2 suppresses primary cilia formation by regulating the expression of genes that are involved in the assembly or disassembly of primary cilia. The mitotic kinase, Aurora A, has been reported to promote disassembly of primary cilia in several ciliated cell types 22, and recently, it has been shown to promote loss of primary cilia in ovarian carcinoma 13. In addition, Aurora A expression is elevated in PDAC cells 29, which led us to hypothesize that its expression is regulated by HDAC2 in PDAC cells. We investigated whether Aurora A is involved in repression of primary cilia in Panc1 cells, and found that the treatment of Aurora A inhibitors, PHA-680632 and alisertib, significantly restored primary cilia (Fig EV3B and C). This suggested that Aurora A promotes loss of primary cilia in Panc1 cells. We then examined Aurora A levels in HDAC2-depleted Panc1 cells and found that Aurora A levels were substantially reduced (Figs 2A and 3A). mRNA levels of Aurora A were significantly decreased after ablation of HDAC2 in both Panc1 and CFPAC1 cells and in HDAC inhibitor-treated Panc1 cells (Figs 3B and C, and EV3D). We also observed significant diminution of Aurora A foci at centrosomes after silencing of HDAC2 in Panc1 cells (Fig 3D and E). Moreover, kinase activity of Aurora A immunoprecipitated from HDAC2-depleted Panc1 was remarkably lower than the control (Figs 3F and EV3E). These results collectively suggest that HDAC2 positively regulates levels of Aurora A, likely leading to suppression of primary ciliogenesis in PDAC cells. Click here to expand this figure. Figure EV3. Aurora A contributes to suppress primary ciliogenesis in PDAC cells A. Panc1 cells were visualized with antibodies against glutamylated tubulin (GT335)(green) and HDAC2 (red). DNA was stained with Hoechst (blue). Scale bar, 5 μm. B, C. Panc1 cells in serum-starved medium were treated with the indicated concentration of PHA-680632 (B) or 10 nM alisertib (C) for 48 h. The percentages of cells with primary cilia or Ki67-positive nuclei were determined as described in Fig 1. Average of three independent experiments is shown. D. CFPAC1 cells transiently transfected with control or HDAC2#2 siRNA were cultured in serum-starved medium for 48 h. Relative amount of Aurora A mRNA was determined using quantitative PCR and GAPDH was used as a control. Average of three independent experiments is shown. E. Cell extracts and immunoprecipitated fractions were immunoblotted with an anti-Aurora A antibody. Asterisks and arrowhead indicate IgG and Aurora A, respectively. β-Actin was used as a loading control. F. Panc1 cells transiently transfected with control, HDAC2#2, Kras, or HDAC2#2 and Kras siRNA were cultured in serum-starved medium for 48 h. Cell extracts were immunoblotted with antibodies against Kras, HDAC2, and Aurora A. β-Actin was used as a loading control. G. Panc1 cells in serum-starved medium were treated with the indicated concentration of PHA-680632 for 48 h. Cell extracts were immunoblotted with antibodies against Kras and HDAC2. β-Actin was used as a loading control. Data information: Error bars represent SEM. *P < 0.05, **P < 0.01 compared with DMSO (B, C) or siControl (D) (two-tailed Student's t-test). Download figure Download PowerPoint Figure 3. HDAC2 positively regulates expression of Aurora A kinase A, B. Panc1 cells transiently transfected with control, HDAC2#1, or HDAC2#2 siRNA were cultured in serum-starved medium for 48 h. (A) Cell extracts were immunoblotted with an anti-Aurora A antibody. Relative amount of Aurora A protein was quantified, and β-actin was used as a loading control. Average of three to four independent experiments is shown. (B) Relative amount of Aurora A mRNA was determined using quantitative PCR. GAPDH was used as a control. Average of six independent experiments is shown. C. Panc1 cells in serum-starved medium were treated with the indicated inhibitors for 48 h. Relative amount of Aurora A mRNA was determined as described in panel (B). Average of five to six independent experiments is shown. D, E. Panc1 cells transiently transfected with control or HDAC2#2 siRNA were cultured in serum-starved medium for 48 h. Cells were immunostained with anti-glutamylated tubulin and anti-Aurora A antibodies. (D) DNA was stained with Hoechst (blue). Scale bar, 5 μm. (E) The percentages of Panc1 cells that stain for Aurora A at centrosomes were determined. Average of four independent experiments is shown. F. Panc1 cells transiently transfected with control, HDAC2#2, or Kras siRNA were cultured in medium lacking serum for 48 h. Cell extracts were immunoprecipitated with control rabbit IgG or anti-Aurora A antibody, and the precipitated Aurora A was subjected to in vitro kinase assay. Average of three to four independent experiments is shown. Data information: Error bars represent SEM. *P < 0.05, **P < 0.01 compared with siControl (A, B, E, F) or DMSO (C) (two-tailed Student's t-test). Download figure Download PowerPoint HDAC2 and Kras independently control loss of primary cilia in Panc1 cells A previous study showed that inhibition of Kras effectors, MEK and PI3K, restores primary cilium formation in pancreatic cancer cells 7. In PDAC cells, Aurora A transcription is positively regulated by the ETS2 transcription factor, which is activated by the Kras–MAPK1 pathway 30. Therefore, we asked whether Kras signaling, which is de-regulated in > 90% of PDACs through constitutive activity, suppresses primary cilium formation by inducing Aurora A expression. We found that siRNA-mediated knockdown of Kras in Panc1 cells deprived of serum led to a significant increase in cilium formation without affecting cell cycle, cell viability, or expression of markers for apoptosis and stress signaling (Figs 4A and B, and EV1A–D). We then evaluated the protein and mRNA levels of Aurora A in the Kras-depleted cells and found that loss of Kras led to a significant decrease in levels of this kinase (Figs 4A, C and D, and EV3F). We further observed that Aurora A kinase activity in Kras-depleted cells was lower than control cells (Figs 3F and EV3E). These results suggest that Kras suppresses primary ciliogenesis by augmenting mRNA expression of Aurora A in PDAC cells. However, we do not rule out the possibility that Kras depletion impacts Aurora A level after transcription, as Aurora A protein levels are more dramatically decreased than mRNA level (Fig 4C and D). Since both HDAC2 and Kras positively regulate Aurora A expression, we next tested whether HDAC2 and Kras function in the same pathway to inhibit primary ciliogenesis. To our surprise, we found that combined ablation of Kras and HDAC2 showed significantly enhanced ciliation as compared to singly ablated cells (Fig 4E). Silencing Kras did not have an impact on HDAC2 expression, which was reciprocally confirmed (Figs 4A and EV3F). Furthermore, ectopic expression of HDAC2 did not significantly abrogate ciliation induced by Kras silencing (Fig 2E–G). These results suggest that Kras and HDAC2, at least in part, independently control primary cilia in PDAC cells. Figure 4. Kras and HDAC2 independently suppress formation of primary cilia through Aurora A in Panc1 cells A–D. Panc1 cells transiently transfected with control or Kras siRNA were cultured in serum-starved medium for 48 h. (A) Cell extracts were immunoblotted with antibodies against Kras, HDAC2, and Aurora A. β-Actin was used as a loading control. (B) The percentages of ciliated or Ki67-positive cells were determined as described in Fig 1. Average of four independent experiments is shown. (C) Relative amount of Aurora A protein was quantified, and β-actin was used as a loading control. Average of three independent experiments is shown. (D) Relative amount of Aurora A mRNA was determined as described in Fig 3B. Average of three to four independent experiments is shown. E. Panc1 cells transiently transfected with control, HDAC2#2, Kras, or HDAC2#2 and Kras siRNA were cultured in serum-starved medium for 48 h. The percentages of ciliated or Ki67-positive cells were determined as described in Fig 1. Average of three independent experiments is shown. F–H. Panc1 cells treated with control, HDAC2#2, or Kras siRNA were transfected with plasmids expressing GFP and Flag, Flag-Aurora A, or Flag-Aurora A/D274N and induced to quiescence for 72 h. Cells were immunostained with an anti-glutamylated tubulin antibody (red). (F) Cell extracts were immunoblotted with antibodies against Flag, HDAC2, and Kras. β-Actin was used as a loading control. (G) DNA was stained with Hoechst (blue). Arrows indicate primary cilia in GFP-positive cells. Scale bar, 10 μm. (H) The percentages of GFP-positive Panc1 cells with primary cilia were determined. Average of at least three independent experiments is shown. I. Panc1 cells transiently transfected with control, HDAC2#1, HDAC2#2, Kras, IFT88, HDAC2#1 and IFT88, HDAC2#2 and IFT88, or Kras and IFT88 siRNA were cultured in serum-containing medium for 48 h. The percentages of ciliated or Ki67-positive cells were determined as described in Fig 1. Average of at least three independent experiments is shown.