Primary cilia and aberrant cell signaling in epithelial ovarian cancer
© Egeberg et al.; licensee BioMed Central Ltd. 2012
Received: 2 November 2011
Accepted: 1 May 2012
Published: 10 August 2012
Ovarian cancer is the fourth leading cause of cancer-related deaths among women in Denmark, largely due to the advanced stage at diagnosis in most patients. Approximately 90% of ovarian cancers originate from the single-layered ovarian surface epithelium (OSE). Defects in the primary cilium, a solitary sensory organelle in most cells types including OSE, were recently implicated in tumorigenesis, mainly due to deregulation of ciliary signaling pathways such as Hedgehog (Hh) signaling. However, a possible link between primary cilia and epithelial ovarian cancer has not previously been investigated.
The presence of primary cilia was analyzed in sections of fixed human ovarian tissue as well as in cultures of normal human ovarian surface epithelium (OSE) cells and two human OSE-derived cancer cell lines. We also used immunofluorescence microscopy, western blotting, RT-PCR and siRNA to investigate ciliary signaling pathways in these cells.
We show that ovarian cancer cells display significantly reduced numbers of primary cilia. The reduction in ciliation frequency in these cells was not due to a failure to enter growth arrest, and correlated with persistent centrosomal localization of aurora A kinase (AURA). Further, we demonstrate that ovarian cancer cells have deregulated Hh signaling and platelet-derived growth factor receptor alpha (PDGFRα) expression and that promotion of ciliary formation/stability by AURA siRNA depletion decreases Hh signaling in ovarian cancer cells. Lastly, we show that the tumor suppressor protein and negative regulator of AURA, checkpoint with forkhead-associated and ring finger domains (CHFR), localizes to the centrosome/primary cilium axis.
Our results suggest that primary cilia play a role in maintaining OSE homeostasis and that the low frequency of primary cilia in cancer OSE cells may result in part from over-expression of AURA, leading to aberrant Hh signaling and ovarian tumorigenesis.
Epithelial ovarian cancer (EOC) belongs to a heterogeneous group of neoplasms that exhibit a wide range of molecular defects, affecting cell survival, proliferation, differentiation and migration. EOC is the most lethal of the gynecologic malignancies, accounting for more than 90% of all ovarian malignancies, and is mainly a disease of postmenopausal women . The high mortality rate of EOC is primarily due to difficulties in diagnosing early stages of the disease. Most patients (approximately 75%) present with advanced stage (III/IV) tumors, for which the five-year survival rate is below 46% . This is not surprising given the size and location of the ovaries, making them not readily accessible by pelvic examination unless significantly enlarged. Improvements in surgical techniques and chemotherapy regiments over the last three decades have resulted in improvements in ovarian cancer treatment; however, despite these advances most patients treated for EOC eventually develop disease recurrence [2, 3].
The etiology behind EOC is poorly understood, although invagination clefts and inclusion cysts lined with ovarian surface epithelium (OSE) have been pointed out as hot spots for initiation of neoplastic processes in EOC [4–6]. Further, a number of recent studies have indicated that EOC is linked to aberrant cell signaling, including Hedgehog (Hh) and platelet-derived growth factor (PDGF) signaling as well as over-expression of aurora A kinase (AURA) and deregulated expression of the novel tumor suppressor protein, checkpoint with forkhead-associated and ring finger domains (CHFR) [7–19]. Consequently, targeted agents against Hh pathway components, PDGFR and AURA have been explored recently in the management of ovarian cancer and recurrent disease .
Hh signaling regulates cell proliferation and differentiation in numerous tissues during embryonic and fetal development and remains active in the adult body where it is involved in the maintenance of stem cell populations [21–23]. Hh signaling depends on a fine-tuned intracellular signal mediated by the repressor or activator forms of the transcription factors GLI2 and GLI3, and is mainly based on a positive feedback loop via GLI1 and a negative feedback loop via Patched-1 (PTCH1) transcription [24, 25]. It is, in particular, these feedback loops that are found disturbed in EOC specimens [7–10].
PDGFR signaling regulates cell growth and survival, transformation, migration and wound healing . Several reports document a change in the expression level of the alpha form of PDGFR (PDGFRα) compared to normal OSE cells and that this expression is associated with high tumor grade, high proliferation index, and poor survival rate [11–14].
AURA is a major mitotic kinase involved in centrosome maturation, mitotic entry, and spindle assembly . AURA maps to a chromosomal region frequently shown to be amplified in human ovarian cancer [15, 16, 18], and several studies have identified elevated AURA kinase activity and/or increased protein level as common characteristics in ovarian cancer [15–17, 28].
CHFR is a novel player in the genesis and progression of EOC . CHFR has multiple functions in checkpoints during mitosis, such as regulation of the G2/M transition by its inherent ubiquitin ligase activity and targeting of key proteins, such as AURA, to the proteasome [29–32]. Nevertheless, a better understanding of the multiple signaling pathways associated with ovarian tumorigenesis is needed in order to identify new ways to target signaling pathways in EOC and in this way increase the efficiency of ovarian cancer treatment and minimize recurrent disease.
Recent research showed that primary cilia may play a critical role in tumorigenesis and cancer progression by functioning as a tumor suppressor organelle that regulates cell proliferation, differentiation, polarity, and migration [33, 34]. Primary cilia are microtubule-based organelles emanating from the distal end of the mother centriole located beneath the plasma membrane during growth-arrest . Reception and transduction by the cilium of chemical and mechanical signals from the extracellular environment is made possible by specific receptors and ion channels located in or near the ciliary membrane. Here signaling pathways regulated by receptor tyrosine kinases, G-protein-coupled receptors, notch receptors, receptors for extracellular matrix proteins and TRP ion channels, including Hh, Wnt and PDGFRα signaling [35–39], are coordinated. The functional importance of the primary cilium is reflected by a number of severe genetic diseases and developmental disorders caused by dysfunction of cilia, commonly referred to as ciliopathies [40, 41]. Recent studies have associated some cancers with loss of primary cilia resulting in deregulated cell proliferation, and others with deregulated ciliary signaling [42–49]. As an example, Wong et al.  demonstrated a role of the primary cilium as an important modulator of Hh signaling in basal cell carcinoma development. They showed that loss of primary cilia in mouse skin cells with a constitutive active Gli2 accelerated tumorigenesis due to disruption in Gli2/Gli3 processing, leading to an altered Gli2 activator/Gli3 repressor ratio . Furthermore, over-expression of an activated form of GLI2 was shown to activate Hh target genes in two prostate cancer cell lines without primary cilia, while over-expression of an activated form of Smoothened (SMO) was not [47, 50]. Cilium resorption can occur as a physiological consequence of cell cycle progression, but, as outlined above, any alteration in physiological ciliary formation or function can have disastrous effects. Interestingly, AURA, which is found to be highly over-expressed in a variety of human cancers [18, 51–53], was recently proposed to regulate disassembly of primary cilia upon mitogenic stimulation . The proposed molecular mechanism includes co-localization of AURA and the scaffolding protein HEF1 at the ciliary basal body and subsequent phosphorylation and activation of the tubulin deacetylase HDAC6, leading to destabilization and resorption of the ciliary axoneme . Although AURA is frequently over-expressed or deregulated in human ovarian cancer cells [15–18, 28], it is unknown whether this correlates with defective primary cilia in these cells.
In this report, we investigated the occurrence of functional primary cilia in growth-arrested normal human OSE cells and two different human ovarian adenocarcinoma cell lines (SK-OV3 and OVCAR3; referred to in the text as cancer OSE cell lines) with the focus on the correlation between centrosomal AURA levels and the presence or absence of cilia and cilia-related signaling pathways. We show that the majority (>60%) of normal growth-arrested OSE cells display primary cilia with PDGFRα and Hh signaling components. In contrast, the fraction of growth-arrested cancer OSE cells with primary cilia was less than 20%, and these cells displayed aberrant Hh signaling and down-regulated expression and/or glycosylation of PDGFRα. We also show that AURA is up-regulated in cancer OSE cells and that RNAi-induced depletion of AURA in these cells leads to a modest, but significant, increase in the number of ciliated cells and partial restoration of Hh signaling. Finally, we show that CHFR localizes to the ciliary basal body in OSE cells. These results suggest that primary cilia play a role in maintaining OSE homeostasis and that the low frequency of primary cilia in cancer OSE cells may result in part from over-expression of AURA, leading to aberrant Hh signaling and ovarian tumorigenesis.
Characterization and isolation of human OSE cells in cultures
We next characterized the wt and cancer OSE cell lines using antibodies specific for different cytoskeletal proteins and OSE markers in immunofluorescence microscopy (IFM) and western blot (WB) analysis. Consistent with IHC analysis of OSE in ovarian tissue sections (Figure 1B-D), cultured wt OSE cells were positive for cytokeratin-8 and −18 (CK8 and CK18) as well as vimentin and N-cadherin, and negative for E-cadherin (Figure 2A-C). In contrast, only a very few CK8 and CK18 positive cells were observed in the SK-OV3 cell line, whereas OVCAR3 cells were positive for both. However, OVCAR3 cells were negative to vimentin staining. Furthermore, both cancer cell lines expressed E-cadherin, which localized to the cell borders, whereas anti-N-cadherin stained a punctuated material within the cells (Figure 2A-C). These findings correlate well with previous reports indicating that ovarian cancer cells display a more classical epithelial phenotype compared to normal OSE cells [59, 64, 65].
Reduced frequency of primary cilia in cultures of human cancer OSE cells
Construction of the ciliary axoneme requires intraflagellar transport (IFT), a bidirectional transport system driven by motor protein complexes that bring axonemal precursors to the growing tip of the cilium and return turnover products to the base . Since IFT20 and IFT88 are required for effective ciliogenesis [67–69], we assessed the sub-cellular localization and expression of these proteins in the wt and OSE cell cultures grown in the presence or absence of serum. Similar to findings in other cell types [67–69], IFT20 was localized at the Golgi apparatus and IFT88 at the base and tip of primary cilia in wt OSE cells (Figure 3D). Further, WB analysis of lysates from wt and cancer OSE cell cultures grown with or without serum demonstrated that IFT88 and IFT20 are expressed at similar levels in all three OSE cell lines (Figure 3E). Thus the reduced frequency of ciliated cells in cancer OSE cells is unlikely to result from lack of these IFT proteins.
Hedgehog and PDGFRα signaling are associated with OSE primary cilia and are disrupted in cancer OSE cells
We next investigated the localization and expression of PDGFRα in OSE cultures by IFM and WB analysis. PDGFRα was previously shown to be up-regulated during growth arrest  and to localize to primary cilia in fibroblasts [74, 75] and other cell types . However, ciliary localization of PDGFRα has not previously been reported for OSE cells. As shown in Figure 5D, we found that PDGFRα localizes to primary cilia of wt OSE cells and PDGFRα is up-regulated during growth arrest in these cells (Figure 5E). In contrast, SK-OV3 and OVCAR3 cells display a markedly lower level of PDGFRα protein and no increase in PDGFRα level is observed upon serum depletion in these cell lines (Figure 5E). As described elsewhere , the PDGFRα antibody used recognizes two protein bands in WB analysis; a high-molecular weight protein band representing the mature and fully glycosylated form and a low-molecular weight protein band representing the immature and only partly glycosylated form of the receptor. Notice that in OVCAR3 cells only the low-molecular weight form of the receptor (#2) is detectable in WB analysis (Figure 5E). These data indicate that PDGFRα signaling via primary cilia during growth arrest likely is perturbed in cancer OSE cells, although this requires further investigations.
The level of aurora A kinase is reduced at the ciliary base in normal OSE cells and up-regulated in cancer OSE cells with defective primary cilia
Consistent with the results of WB and RT-PCR analyses (Figure 6B,C), IFM analysis revealed that the centrosomal pool of AURA was clearly diminished in serum-depleted, ciliated wt OSE cells compared to non-starved cells (Figure 6D). Similarly, we observed that centrosomes of the few ciliated cancer OSE cells lacked AURA (data not shown). However, in serum-depleted SK-OV3 and OVCAR3 cells, centrosomes (marked with anti-EB3 and anti-pericentrin) mostly lacked primary cilia (stained with anti-Acet.tub) and were clearly AURA positive (Figure 6E; see also Figure 3C). The over-expression and localization of AURA to centrosomes in growth-arrested cancer OSE cells suggest that AURA may play a role in suppressing ciliogenesis and/or promoting ciliary disassembly in cancer OSE cells.
The tumor suppressor protein, CHFR, localizes to the base of OSE primary cilia
In the mouse, the tumor suppressor protein, Chfr, is known to inhibit AurA by ubiquitination and proteasomal degradation . The potential involvement of AURA in regulating cilia assembly or disassembly in human OSE cells (see above) prompted us to investigate whether CHFR is associated with the centrosome/cilium axis in these cells. To this end, we generated a polyclonal rabbit antibody against human CHFR (see Methods for details). WB analysis of lysates of cultured, serum-starved hTERT-RPE1 or NIH3T3 cells demonstrated that the CHFR antiserum recognizes a single band of about 73 kDa equivalent to the predicted size of endogenous CHFR (73.4 kDa for isoform 1) (Additional file 1: Figure S1A, B), and by WB analysis the CHFR antibody also recognized exogenous green fluorescent protein (GFP)-tagged CHFR expressed stably in serum-starved hTERT-RPE1 cells (Additional file 1: Figure S1C). Further, both endogenous and CHFR and GFP-tagged CHFR localized to the base of primary cilia in serum-starved hTERT-RPE1 cells (Additional file 1: Figure S1D, E). In serum-starved wt OSE cells the CHFR antibody predominantly labeled the base of primary cilia, but no clear localization of the antibody was observed in interphase or mitotic wt OSE cells (Figure 6F). However, in hTERT-RPE1 cells CHFR was detected at centrosomes in growth-arrested as well as cycling cells (data not shown), suggesting that the lack of detection of CHFR at centrosomes of mitotic OSE cells could be due to low abundance of the protein. These data conflict with previous studies showing that over-expressed, epitope-tagged CHFR displays a predominantly nuclear localization [78–80], but are in agreement with studies showing that endogenous CHFR localizes to cytoplasm and centrosomes during interphase growth [31, 81, 82] and to spindle poles during mitosis . This is the first report on CHFR localization to primary cilia, and future studies might reveal if CHFR takes part in the signaling machinery that regulates ciliary disassembly.
Depletion of AURA increases the frequency of primary cilia and reduces Hh signaling in cancer OSE cells
In this study, we investigated the occurrence of primary cilia in human wt and cancer OSE cells with a focus on the correlation between AURA and the presence or absence of primary cilia with functional Hh signaling and expression of PDGFRα. Our results show that EOC cells are mostly devoid of primary cilia, and we suggest that this in part may be due to increased expression of AURA in these cells. These findings are in agreement with other studies on cancer cells, such as pancreatic adenocarcinoma cells, basal cell carcinoma cells, and clear cell renal cell carcinomas that also have reduced frequency of primary cilia [44, 46, 83, 84], which in some cases can be explained by over expression of AURA . However, in contrast to, for example, pancreatic cancer cells that may not form cilia because the cells fail to enter growth arrest , cancer OSE cells, such as SK-OV3, enter growth arrest upon serum depletion at a level comparable to that of wt OSE cells. The vast majority of OVCAR3 cells also entered growth arrest upon serum depletion, although not as many as SK-OV3 cells. Thus, lack of cilia in these cells seems not to be caused merely by a failure of the cells to become quiescent, suggesting that ovarian cancer cells have defects in the regulatory proteins that control ciliary assembly and/or disassembly.
How is ciliary formation perturbed in cancer OSE cells? Initially, we investigated the expression and localization of two IFT proteins, IFT20 and IFT88, essential for the assembly of primary cilia and found no obvious difference between normal human OSE cells and the two ovarian cancer cell lines. In contrast, we observed a dramatic decrease in the expression of AURA in growth-arrested wt OSE cells compared to growth-arrested SK-OV3 and OVCAR3 cells. Although SK-OV3 and OVCAR3 cells differ, for example in regard to morphology and ability to enter growth arrest, both cell lines maintained a high level of AURA at centrosomes in cells not forming primary cilia. Since AURA has been implicated in ciliary disassembly , we suggest that high levels of AURA at the centrosomal region suppress ciliary formation and/or promote ciliary disassembly in growth-arrested cancer OSE cells. This may have dire consequences for regulation of signaling pathways that are coordinated by primary cilia such as Hh and PDGFRα signaling,which, when aberrantly regulated, are associated with EOC [7–14]. Indeed, we here show that PDGFRα and essential components of the Hh pathway, including SMO, PTCH1 and GLI2, localize to primary cilia of wt OSE cells and that cancer OSE cells display increased basal expression of Hh responsive genes. Further, in cancer OSE cells, there is a defect in expression and/or glycosylation of PDGFRα, in that SK-OV3 cells are not up-regulating PDGFRα expression during growth arrest, and that both up-regulation and glycosylation of the receptor is hampered in OVCAR3 cells. Previously, up-regulation of PDGFRα during growth arrest was shown to be blocked in Tg737 orpk mouse embryonic fibroblasts, which have a hypomorphic mutation in IFT88 and, therefore, form no or very short primary cilia . We suggest that defects in ciliary formation due to over-expression and centrosomal localization of AURA in cancer OSE cells in a similar way may perturb proper Hh signaling as well as PDGFRα expression and function leading to homeostatic imbalance of the ovarian surface epithelium.
In order to investigate AURA function in the formation of primary cilia in more detail, we conducted siRNA knockdown of AURA in growth-arrested SK-OV3 cells, since these cells entered growth arrest upon serum depletion at a level comparable to that of wt OSE cells. AURA knockdown increased the number of ciliated cancer OSE cells albeit to a small, but significant, extent, and this was accompanied by a lower level of the full-length activator form of the GLI2 protein, involved in Hh signaling. These results are similar to previous results reported for, for example, renal cancer cells that lack the von Hippel-Lindau tumor suppressor protein; in these cells, it was shown that siRNA-mediated inhibition of the HEF1-AURA pathway caused a significant increase in the frequency of ciliated cells, whereas over expression of AURA or HEF1 in control renal cells promoted cilia loss . Thus over-expression of AURA and loss of primary cilia may be a common characteristic of several types of cancers, in which a moderate restoration of ciliary formation is associated in part with a reduction in aberrant Hh signaling. The fact that AURA siRNA did not fully restore ciliary formation in cancer OSE cells, suggests that the cells were not completely depleted for AURA and/or that the function of other regulatory proteins in ciliary assembly and maintenance is disrupted.
A number of proteins have been suggested to play a role in regulating AURA activity and/or expression. A prominent example is the tumor suppressor protein, CHFR, which has been implicated in multiple human cancers, including EOC [19, 85, 86]. Originally, CHFR was shown to function as a mitotic checkpoint protein required for tumor suppression, partly through ubiquitination and targeting of AURA for degradation in the proteasome [31, 32, 87]. In concurrence with previous findings that CHFR localizes at centrosomes in interphase cells  and at spindle poles in mitotic cells , we found that CHFR localizes to the centrosomal region at the base of primary cilia and in some cases along the length of the cilium in growth-arrested OSE and RPE cells. This is the first report on the localization of this tumor suppressor protein to primary cilia, and although speculative at this point, we suggest that CHFR may function at the cilium to promote cilia stability through inactivation of AURA.
Several proteins are known to interact with AURA during mitosis, but AURA partners and downstream targets at other cell cycle stages are less investigated. In a seminal work by Pugacheva et al.  it was shown that ciliary disassembly in RPE cells is in part coordinated by AURA-mediated activation of HDAC6, a tubulin deacetylase that promotes destabilization of microtubules [88–90]. In contrast, Sharma et al.  used the same cell type to show that inhibition of HDAC6 followed by increased level of microtubule acetylation did not affect cilia stability in concurrence with the findings that HDAC6-deficient mice are viable and have no phenotypes associated with known ciliopathies . Similarly, we find that knockdown of AURA by siRNA did not affect the overall level of acetylated tubulin as judged by WB analysis, suggesting that tubulin deacetylase(s) is not the major target for AURA-induced ciliary disassembly or inhibition of ciliary assembly in OSE cells.
In this work we have established a new platform from which to investigate cellular processes and signaling pathways in ovarian cancer using primary cultures of human OSE cells as well as cultures of human ovarian cancer cell lines. We show that EOC, which comprises the vast majority of human ovarian cancers, is associated with defects in formation of primary cilia that control signaling pathways in ovarian homeostasis such as Hh and PDGFRα signaling. We also show that reduced frequency of primary cilia in cancer cells correlates with overexpression of AURA and persistent localization of AURA to the centrosome in growth arrested cells devoid of primary cilia. We further show that the tumor suppressor protein, CHFR, which inactivates AURA and when mutated or expressed at low levels causes ovarian tumorigenesis, is a centrosomal protein that localizes to the ciliary base in growth arrested wt OSE cells. Future analysis should focus on how CHFR and AURA interact at the primary cilium to control downstream targets in ciliary assembly, disassembly and function.
Collection of human ovaries and tissue sectioning
Healthy human ovaries were sent to the Laboratory of Reproductive Biology at the University Hospital of Copenhagen for cryopreservation (Cryopreservation of ovarian tissue has been approved by the Minister of Health in Denmark and by the ethical committee of the municipalities of Copenhagen and Frederiksberg, journal number KF/01/170/99) from women about to initiate chemotherapy for malignancies other than ovarian cancer. Tissue specimens were dissected into appropriate tissue blocks and fixed for 12 to 24 hours at 4°C in Bouin’s fixatives. The specimens were dehydrated with graded alcohols, cleared in xylene, and embedded in paraffin wax. Serial sections, 5 μm thick, were cut and placed on silanized glass slides. Representative sections of each series were stained with H & E.
OSE cells were obtained by scraping the surface of the ovaries with a surgical blade as described elsewhere . The cells were collected in Iscove’s modified Dulbecco’s medium (Invitrogen, Taastrup, Denmark) with 1% penicillin-streptomycin (Invitrogen), immediately followed by centrifugation at 300 x g for five minutes at room temperature. The cell pellet was resuspended in OSE growth medium (Minimum Essential Medium α-medium [Invitrogen], 15% fetal bovine serum [FBS; Invitrogen], 1% Glutamax™-1 [Invitrogen], 1% Minimum Essential Medium non-essential amino acids solution [Invitrogen], 1% insulin-transferrin-selenium supplement [Invitrogen], 1% penicillin-streptomycin, and 3.3 mU/ml follicle-stimulating hormone/luteinizing hormone (Menopur, Ferring, Kiel, Germany), and placed in a 35-mm culture dish coated with 0.1% gelatin (Sigma, St. Louis, Missouri, USA). The cultures were incubated at 37°C in 5% CO2 in air and left undisturbed for at least 48 hours. Medium was changed at intervals of two to three days. The ovarian cancer cell lines OVCAR3 (ATCC-HTB-161) and SK-OV3 (ATCC-HTB-77) were purchased from the American Type Culture Collection (Manassas, Virginia, USA). The cancer cell lines were cultured in OSE growth medium on a gelatin coating as described above. Passing of cells was performed by trypsination. The cell lines were maintained by passaging continuously on a weekly basis. Cells were examined at a sub-confluent stage in the presence of serum (0 hour or interphase cells) or at confluency followed by serum starvation for indicated time periods to induce growth arrest.
Culture, transfection, and selection of stable hTERT-RPE1 cells expressing GFP-CHFR was performed essentially as described . For generation of GFP-CHFR expressing cells, plasmid pEGFP-C1 (Clontech, Mountain View, California, USA) containing full-length CHFR coding sequence (kind gift from Kenneth B. Schou, Danish Cancer Society, Copenhagen, Denmark) was used. The culture of NIH3T3 cells was done as described previously .
Immunohistochemical (IHC) analysis
Primary antibodies applied in this study
Aurora A/AlK (1 G4)
kind gift from Greg Pazour
kind gift from Greg Pazour
IFM, immunofluorescence microscopy; IgG, immunoglobulin G; IHC, immunohistochemistry; WB, western blot
Immunofluoresence microscopy (IFM) analysis
Cells were grown on 12-mm sterile HCl-cleansed coverslips coated with 0.1% gelatin. The coverslips were washed in ice cold PBS and fixed with either 4% paraformaldehyde (PFA; PFA-fix), 4% PFA and methanol (PFA + MeOH-fix) or with 3% PFA in Brinkley Reassembly Buffer 80 (and methanol (mix-fix). For PFA-fix, cells were fixed for 15 minutes at room temperature, washed twice in PBS, and then permeabilized with 0.2% Triton X-100 and 1% BSA in PBS for 12 minutes. For PFA + MeOH-fix, cells were first fixed with 4% PFA for 10 minutes, washed twice in PBS, and fixed again for 5 minutes in ice-cold methanol. After removal of the methanol, the coverslips were allowed to air dry for a short period followed by permeabilization with 0.2% Triton X-100 and 1% BSA in PBS for 12 minutes. For mix-fix, cells were fixed with 3% PFA in Brinkley Reassembly Buffer 80 (80 mM PIPES pH 6.9, 1 mM EGTA, 1 mM MgCl2) for two minutes, washed in ice cold PBS, and fixed again for two minutes in ice-cold methanol. After removal of the methanol, the coverslips were allowed to air dry for a short period followed by rehydration in PBS. To avoid unspecific antibody binding, coverslips (all kinds of fixation and permeabilization) were incubated in blocking buffer (2% BSA in PBS) for 30 minutes at room temperature or overnight at 4°C before transfer to a moisture chamber. The coverslips were subsequently incubated with primary antibodies diluted in blocking buffer for 90 minutes at room temperature or overnight at 4°C (see Table 1 for list of primary antibodies used) followed by 4 x 5 minutes wash in blocking buffer and incubation in dark for 45 minutes with fluorochrome-conjugated secondary antibodies (Alexa Flour 350, Alexa Flour 488, and Alexa Flour 568, all from Invitrogen) diluted 1:600 in blocking buffer. Staining of F-actin with rhodamine-coupled phalloidin (Invitrogen, 1:100) was done concomitantly with secondary antibody incubation. Hereafter, coverslips were washed once in blocking buffer and briefly incubated with DAPI. After washing in PBS, coverslips were mounted on microscope slides in anti-fade mounting solution, sealed with nail-polish and analyzed by microscopy as described for IHC. Cilia frequency was determined by quantifying the number of ciliated and non-ciliated cells of a minimum number of 130 cells for each sample in at least three replicates.
SDS-PAGE and western blot analysis
SDS-PAGE and WB analysis was carried out essentially as previously described . Cell lysates were prepared in boiling 0.1% SDS lysis buffer supplemented with EDTA-free protease inhibitor cocktail (Roche, Mannheim, Germany) and 1 mM Na3VO4. Lysates were sonicated and centrifuged to precipitate cell debris, and protein concentrations were measured using a DC Protein Assay (Bio-Rad, Hercules, California, USA) according to the manufacturer’s instruction. Proteins were separated under reducing and denaturing conditions by SDS-PAGE) using 10% Bis-Tris precast gels (Invitrogen) followed by electrophoretic transfer to nitrocellulose membranes (Invitrogen). Membranes were incubated for at least 30 minutes at room temperature or overnight at 4°C in 5% nonfat dry milk in Tris Buffered Saline with Tween (5% milk-TBST; 10 mM Tris–HCl (pH 7.5), 120 mM NaCl, 0.1% Tween 20), before incubation with primary antibodies for two hours at room temperature or overnight at 4°C in moisture chambers (see Table 1 for antibodies used in WB analysis). Antibodies were diluted in 5% milk-TBST as indicated below. Membranes were washed several times in TBST followed by incubation with alkaline phosphatase-conjugated secondary antibodies (Sigma) in 5% milk-TBST for 45 minutes at room temperature. Blots were washed in TBST and protein bands were visualized using BCIP/NBT Phosphatase Substrate (KPL, Gaithersburg, Maryland, USA). After air drying, the developed blots were scanned and processed with Adobe Photoshop version 6.0.
PCR and primers
Primers applied in this study
Primer sequence (5′ → 3′)
AURA (from )
AURA knockdown in SK-OV3 cells was performed using ready-to-use custom synthesized siRNA (Thermo Scientific, Lafayette, Colorado, USA) against human AURA (target sequence: GAACUUACUUCUUGGAUCA) or scrambled oligonucleotides (mock) with similar GC content, both at 50 nM, and DharmaFECT transfection reagent (Thermo Scientific) according to the manufacturer’s instructions. Cells were transfected at 60% confluency. The day after siRNA treatment, the cells were serum depleted as described above and were used for experiments 72 hours after siRNA treatment. Cilia quantifications were always accompanied by parallel WBs against AURA to verify knockdown.
CHFR antibody production
For production of rabbit polyclonal antibodies specific for human CHFR, a maltose binding protein (MBP)-CHFR fusion protein was produced in Escherichia coli. The sequence corresponding to the entire coding region of CHFR (Genbank ID AF170724.1) was amplified by PCR from plasmid pEGFP-C1 (Clontech) containing full-length CHFR coding sequence (kind gift from Kenneth B. Schou, Danish Cancer Society, Copenhagen, Denmark) using forward (5′-CAGAATTCATGGAGCGGCCCGAG-3′) and reverse (5′-AAGGTCGACTTAGTTTTTGAACCTTGTCTG-3′) primers with recognition sites for EcoRI and SalI, respectively, and Herculase DNA polymerase from Stratagene (La Jolla, California, USA). The PCR product was purified and cloned into pMalC2 (New England Biolabs, Ipswich, Massachusetts) using standard procedures and the ligated DNA transformed into competent E. coli DH10α cells. Resulting plasmids were control sequenced by Eurofins MWG Operon. Production and purification of MBP-CHFR fusion protein was carried out essentially as described previously  and purified MBP-CHFR fusion protein used for polyclonal rabbit antibody production by Yorkshire Bioscience Ltd (Heslington, York, United Kingdom). The resulting CHFR rabbit antiserum was stored in saturated ammonium sulfate solution at 4°C until use.
All experiments were repeated three or more times and data are presented as representative individual experiments or as mean values plus SD. The data were tested for significance using one-way analysis of variance (ANOVA) or Student’s t-test. The level of significance was set at P < 0.05 (*), P < 0.01 (**), P < 0.001 (***).
analysis of variance
aurora A kinase
bovine serum albumin
checkpoint with forkhead-associated and ring finger domains
differential interference contrast microscopy
epithelial ovarian cancer
fetal bovine serum
green fluorescent protein
maltose binding protein
ovarian surface epithelium
phosphate buffered saline
proliferating cell nuclear antigen
platelet-derived growth factor
platelet-derived growth factor receptor
phosphorylated retinoblastoma protein
reverse transcriptase polymerase chain reaction
tris buffered saline with tween
STC and LBP acknowledge funding from the Danish Natural Science Research Council (09–070398 and 10–085373), the Lundbeck Foundation (R54-A5642 and R54-A5375), and Nordforsk (27480). DLE was supported by a scholarship from the Danish Cancer Society (A312), and RM was supported by a Fulbright Scholarship. The authors would like to thank Mrs. Anni Bech Nielsen and Mr. Søren Lek Johansen for excellent technical assistance, Jacob. M. Schrøder for generation of hTERT-RPE1 cells stably expressing GFP-CHFR, and Ms. Pernille Ebbesen, Ms. Pernille Nilsson and Ms. Caroline Røddick for help on localization studies of GFP-CHFR in RPE cells.
- Jemal A, Siegel R, Xu J, Ward E: Cancer statistics, 2010. CA Cancer J Clin. 2010, 60: 277-300. 10.3322/caac.20073.View ArticlePubMed
- Aletti GD, Gallenberg MM, Cliby WA, Jatoi A, Hartmann LC: Current management strategies for ovarian cancer. Mayo Clin Proc. 2007, 82: 751-770.View ArticlePubMed
- Jelovac D, Armstrong DK: Recent progress in the diagnosis and treatment of ovarian cancer. CA Cancer J Clin. 2011, 61: 183-10.3322/caac.20113.PubMed CentralView ArticlePubMed
- Aoki Y, Kawada N, Tanaka K: Early form of ovarian cancer originating in inclusion cysts. A case report. J Reprod Med. 2000, 45: 159-161.PubMed
- Deligdisch L, Einstein AJ, Guera D, Gil J: Ovarian dysplasia in epithelial inclusion cysts. A morphometric approach using neural networks. Cancer. 1995, 76: 1027-1034. 10.1002/1097-0142(19950915)76:6<1027::AID-CNCR2820760617>3.0.CO;2-6.View ArticlePubMed
- Feeley KM, Wells M: Precursor lesions of ovarian epithelial malignancy. Histopathology. 2001, 38: 87-95. 10.1046/j.1365-2559.2001.01042.x.View ArticlePubMed
- Bhattacharya R, Kwon J, Ali B, Wang E, Patra S, Shridhar V, Mukherjee P: Role of hedgehog signaling in ovarian cancer. Clin Cancer Res. 2008, 14: 7659-7666. 10.1158/1078-0432.CCR-08-1414.View ArticlePubMed
- Chen X, Horiuchi A, Kikuchi N, Osada R, Yoshida J, Shiozawa T, Konishi I: Hedgehog signal pathway is activated in ovarian carcinomas, correlating with cell proliferation: its inhibition leads to growth suppression and apoptosis. Cancer Sci. 2007, 98: 68-76. 10.1111/j.1349-7006.2006.00353.x.View ArticlePubMed
- Liao X, Siu MK, Au CW, Wong ES, Chan HY, Ip PP, Ngan HY, Cheung AN: Aberrant activation of hedgehog signaling pathway in ovarian cancers: effect on prognosis, cell invasion and differentiation. Carcinogenesis. 2009, 30: 131-140.View ArticlePubMed
- Schmid S, Bieber M, Zhang F, Zhang M, He B, Jablons D, Teng NN: Wnt and Hedgehog Gene Pathway Expression in Serous Ovarian Cancer. Int J Gynecol Cancer. 2011, 21: 975-980. 10.1097/IGC.0b013e31821caa6f.PubMed CentralView ArticlePubMed
- Apte SM, Bucana CD, Killion JJ, Gershenson DM, Fidler IJ: Expression of platelet-derived growth factor and activated receptor in clinical specimens of epithelial ovarian cancer and ovarian carcinoma cell lines. Gynecol Oncol. 2004, 93: 78-86. 10.1016/j.ygyno.2003.12.041.View ArticlePubMed
- Henriksen R, Funa K, Wilander E, Backstrom T, Ridderheim M, Oberg K: Expression and prognostic significance of platelet-derived growth factor and its receptors in epithelial ovarian neoplasms. Cancer Res. 1993, 53: 4550-4554.PubMed
- Lassus H, Sihto H, Leminen A, Nordling S, Joensuu H, Nupponen NN, Butzow R: Genetic alterations and protein expression of KIT and PDGFRA in serous ovarian carcinoma. Br J Cancer. 2004, 91: 2048-2055. 10.1038/sj.bjc.6602252.PubMed CentralView ArticlePubMed
- Matei D, Emerson RE, Lai YC, Baldridge LA, Rao J, Yiannoutsos C, Donner DD: Autocrine activation of PDGFRalpha promotes the progression of ovarian cancer. Oncogene. 2006, 25: 2060-2069. 10.1038/sj.onc.1209232.View ArticlePubMed
- Gritsko TM, Coppola D, Paciga JE, Yang L, Sun M, Shelley SA, Fiorica JV, Nicosia SV, Cheng JQ: Activation and overexpression of centrosome kinase BTAK/Aurora-A in human ovarian cancer. Clin Cancer Res. 2003, 9: 1420-1426.PubMed
- Hu W, Kavanagh JJ, Deaver M, Johnston DA, Freedman RS, Verschraegen CF, Sen S: Frequent overexpression of STK15/Aurora-A/BTAK and chromosomal instability in tumorigenic cell cultures derived from human ovarian cancer. Oncol Res. 2005, 15: 49-57.PubMed
- Landen CN: Lin YG, Immaneni A, Deavers MT, Merritt WM, Spannuth WA, Bodurka DC, Gershenson DM, Brinkley WR, Sood AK: Overexpression of the centrosomal protein Aurora-A kinase is associated with poor prognosis in epithelial ovarian cancer patients. Clin Cancer Res. 2007, 13: 4098-4104. 10.1158/1078-0432.CCR-07-0431.View ArticlePubMed
- Tanner MM, Grenman S, Koul A, Johannsson O, Meltzer P, Pejovic T, Borg A, Isola JJ: Frequent amplification of chromosomal region 20q12-q13 in ovarian cancer. Clin Cancer Res. 2000, 6: 1833-1839.PubMed
- Gao Y, Lou G, Zhang GM, Sun XW, Ma YY, Yang YM, Liu G: CHFR promoter hypermethylation and reduced CHFR mRNA expression in ovarian cancer. Int J Biol Markers. 2009, 24: 83-89.PubMed
- Campos SM, Ghosh S: A current review of targeted therapeutics for ovarian cancer. J Oncol. 2010, 201: 149362-
- Jiang J, Hui C: Hedgehog signaling in development and cancer. Dev Cell. 2008, 15: 801-812. 10.1016/j.devcel.2008.11.010.View ArticlePubMed
- Lai K, Kaspar BK, Gage FH, Schaffer DV: Sonic hedgehog regulates adult neural progenitor proliferation in vitro and in vivo. Nat Neurosci. 2003, 6: 21-27. 10.1038/nn983.View ArticlePubMed
- Liu S, Dontu G, Mantle ID, Patel S, Ahn N, Jackson KW, Suri P, Wicha MS: Hedgehog signaling and Bmi-1 regulate self-renewal of normal and malignant human mammary stem cells. Cancer Res. 2006, 66: 6063-6071. 10.1158/0008-5472.CAN-06-0054.PubMed CentralView ArticlePubMed
- Lipinski RJ, Gipp JJ, Zhang J, Doles JD, Bushman W: Unique and complimentary activities of the Gli transcription factors in Hedgehog signaling. Exp Cell Res. 2006, 312: 1925-1938. 10.1016/j.yexcr.2006.02.019.View ArticlePubMed
- Rohatgi R, Milenkovic L, Scott MP: Patched1 regulates hedgehog signaling at the primary cilium. Science. 2007, 317: 372-376. 10.1126/science.1139740.View ArticlePubMed
- Heldin CH, Westermark B: Mechanism of action and in vivo role of platelet-derived growth factor. Physiol Rev. 1999, 79: 1283-1316.PubMed
- Fu J, Bian M, Jiang Q, Zhang C: Roles of Aurora kinases in mitosis and tumorigenesis. Mol Cancer Res. 2007, 5: 1-10. 10.1158/1541-7786.MCR-06-0208.View ArticlePubMed
- Chung CM, Man C, Jin Y, Jin C, Guan XY, Wang Q, Wan TS, Cheung AL, Tsao SW: Amplification and overexpression of aurora kinase A (AURKA) in immortalized human ovarian epithelial (HOSE) cells. Mol Carcinog. 2005, 43: 165-174. 10.1002/mc.20098.View ArticlePubMed
- Kang D, Chen J, Wong J, Fang G: The checkpoint protein Chfr is a ligase that ubiquitinates Plk1 and inhibits Cdc2 at the G2 to M transition. J Cell Biol. 2002, 156: 249-259. 10.1083/jcb.200108016.PubMed CentralView ArticlePubMed
- Privette LM, Petty EM: CHFR: a novel mitotic checkpoint protein and regulator of tumorigenesis. Transl Oncol. 2008, 1: 57-64.PubMed CentralView ArticlePubMed
- Privette LM, Weier JF, Nguyen HN, Yu X, Petty EM: Loss of CHFR in human mammary epithelial cells causes genomic instability by disrupting the mitotic spindle assembly checkpoint. Neoplasia. 2008, 10: 643-652.PubMed CentralView ArticlePubMed
- Yu X, Minter-Dykhouse K, Malureanu L, Zhao WM, Zhang D, Merkle CJ, Ward IM, Saya H, Fang G, van Deursen J, Chen J: Chfr is required for tumor suppression and Aurora A regulation. Nat Genet. 2005, 37: 401-406. 10.1038/ng1538.View ArticlePubMed
- Mans DA, Voest EE, Giles RH: All along the watchtower: is the cilium a tumor suppressor organelle?. Biochim Biophys Acta. 2008, 1786: 114-125.PubMed
- Plotnikova OV, Golemis EA, Pugacheva EN: Cell cycle-dependent ciliogenesis and cancer. Cancer Res. 2008, 68: 2058-2061. 10.1158/0008-5472.CAN-07-5838.PubMed CentralView ArticlePubMed
- Satir P, Pedersen LB, Christensen ST: The primary cilium at a glance. J Cell Sci. 2010, 123: 499-503. 10.1242/jcs.050377.PubMed CentralView ArticlePubMed
- Christensen ST, Clement CA, Satir P, Pedersen LB: Primary cilia and coordination of receptor tyrosine kinase (RTK) signaling. J Pathol. 2011, 226: 172-184.PubMed CentralView ArticlePubMed
- Goetz SC, Anderson KV: The primary cilium: a signalling centre during vertebrate development. Nat Rev Genet. 2010, 11: 331-344. 10.1038/nrg2774.PubMed CentralView ArticlePubMed
- Christensen ST, Ott CM: Cell signaling. A ciliary signaling switch. Science. 2007, 317: 330-331. 10.1126/science.1146180.View ArticlePubMed
- Wallingford JB, Mitchell B: Strange as it may seem: the many links between Wnt signaling, planar cell polarity, and cilia. Genes Dev. 2011, 25: 201-213. 10.1101/gad.2008011.PubMed CentralView ArticlePubMed
- Fliegauf M, Benzing T, Omran H: When cilia go bad: cilia defects and ciliopathies. Nat Rev Mol Cell Biol. 2007, 8: 880-893. 10.1038/nrm2278.View ArticlePubMed
- Hildebrandt F, Benzing T, Katsanis N: Ciliopathies. N Engl J Med. 2011, 364: 1533-1543. 10.1056/NEJMra1010172.PubMed CentralView ArticlePubMed
- Han YG, Kim HJ, Dlugosz AA, Ellison DW, Gilbertson RJ, Alvarez-Buylla A: Dual and opposing roles of primary cilia in medulloblastoma development. Nat Med. 2009, 15: 1062-1065. 10.1038/nm.2020.PubMed CentralView ArticlePubMed
- Moser JJ, Fritzler MJ, Rattner JB: Primary ciliogenesis defects are associated with human astrocytoma/ glioblastoma cells. BMC Cancer. 2009, 9: 448-10.1186/1471-2407-9-448.PubMed CentralView ArticlePubMed
- Seeley ES, Carriere C, Goetze T, Longnecker DS, Korc M: Pancreatic cancer and precursor pancreatic intraepithelial neoplasia lesions are devoid of primary cilia. Cancer Res. 2009, 69: 422-430. 10.1158/0008-5472.CAN-08-1290.PubMed CentralView ArticlePubMed
- Yuan K, Frolova N, Xie Y, Wang D, Cook L, Kwon YJ, Steg AD, Serra R, Frost AR: Primary cilia are decreased in breast cancer: analysis of a collection of human breast cancer cell lines and tissues. J Histochem Cytochem. 2010, 58: 857-870. 10.1369/jhc.2010.955856.PubMed CentralView ArticlePubMed
- Wong SY, Seol AD, So PL, Ermilov AN, Bichakjian CK, Epstein EH, Dlugosz AA, Reiter JF: Primary cilia can both mediate and suppress Hedgehog pathway-dependent tumorigenesis. Nat Med. 2009, 15: 1055-1061. 10.1038/nm.2011.PubMed CentralView ArticlePubMed
- Zhang J, Lipinski RJ, Gipp JJ, Shaw AK, Bushman W: Hedgehog pathway responsiveness correlates with the presence of primary cilia on prostate stromal cells. BMC Dev Biol. 2009, 9: 50-10.1186/1471-213X-9-50.PubMed CentralView ArticlePubMed
- Montani M, Heinimann K, von Teichman A, Rudolph T, Perren A, Moch H: VHL-gene deletion in single renal tubular epithelial cells and renal tubular cysts: further evidence for a cyst-dependent progression pathway of clear cell renal carcinoma in von Hippel-Lindau disease. Am J Surg Pathol. 2010, 34: 806-881. 10.1097/PAS.0b013e3181ddf54d.View ArticlePubMed
- de Andrea CE, Wiweger M, Prins F, Bovee JV, Romeo S, Hogendoorn PC: Primary cilia organization reflects polarity in the growth plate and implies loss of polarity and mosaicism in osteochondroma. Lab Invest. 2010, 90: 1091-1101. 10.1038/labinvest.2010.81.View ArticlePubMed
- Zhang J, Lipinski R, Shaw A, Gipp J, Bushman W: Lack of demonstrable autocrine hedgehog signaling in human prostate cancer cell lines. J Urol. 2007, 177: 1179-1185. 10.1016/j.juro.2006.10.032.View ArticlePubMed
- Reiter R, Gais P, Jutting U, Steuer-Vogt MK, Pickhard A, Bink K, Rauser S, Lassmann S, Höfler H, Werner M, Walch A: Aurora kinase A messenger RNA overexpression is correlated with tumor progression and shortened survival in head and neck squamous cell carcinoma. Clin Cancer Res. 2006, 12: 5136-5141. 10.1158/1078-0432.CCR-05-1650.View ArticlePubMed
- Shang X, Burlingame SM, Okcu MF, Ge N, Russell HV, Egler RA, David RD, Vasudevan SA, Yang J, Nuchtern JG: Aurora A is a negative prognostic factor and a new therapeutic target in human neuroblastoma. Mol Cancer Ther. 2009, 8: 2461-2469. 10.1158/1535-7163.MCT-08-0857.PubMed CentralView ArticlePubMed
- Jeng YM, Peng SY, Lin CY, Hsu HC: Overexpression and amplification of Aurora-A in hepatocellular carcinoma. Clin Cancer Res. 2004, 10: 2065-2071. 10.1158/1078-0432.CCR-1057-03.View ArticlePubMed
- Pugacheva EN, Jablonski SA, Hartman TR, Henske EP, Golemis EA: HEF1-dependent aurora A activation induces disassembly of the primary cilium. Cell. 2007, 129: 1351-1363. 10.1016/j.cell.2007.04.035.PubMed CentralView ArticlePubMed
- Auersperg N, Maines-Bandiera SL, Dyck HG: Ovarian carcinogenesis and the biology of ovarian surface epithelium. J Cell Physiol. 1997, 173: 261-265. 10.1002/(SICI)1097-4652(199711)173:2<261::AID-JCP32>3.0.CO;2-G.View ArticlePubMed
- Teilmann SC, Christensen ST: Localization of the angiopoietin receptors Tie-1 and Tie-2 on the primary cilia in the female reproductive organs. Cell Biol Int. 2005, 29: 340-346. 10.1016/j.cellbi.2005.03.006.View ArticlePubMed
- Auersperg N, Siemens CH, Myrdal SE: Human ovarian surface epithelium in primary culture. In Vitro. 1984, 20: 743-755. 10.1007/BF02618290.View ArticlePubMed
- Auersperg N, Maines-Bandiera SL, Dyck HG, Kruk PA: Characterization of cultured human ovarian surface epithelial cells: phenotypic plasticity and premalignant changes. Lab Invest. 1994, 71: 510-518.PubMed
- Wong AS, Maines-Bandiera SL, Rosen B, Wheelock MJ, Johnson KR, Leung PC, Roskelley CD, Auersperg N: Constitutive and conditional cadherin expression in cultured human ovarian surface epithelium: influence of family history of ovarian cancer. Int J Cancer. 1999, 81: 180-188. 10.1002/(SICI)1097-0215(19990412)81:2<180::AID-IJC3>3.0.CO;2-7.View ArticlePubMed
- Kruk PA, Maines-Bandiera SL, Auersperg N: A simplified method to culture human ovarian surface epithelium. Lab Invest. 1990, 63: 132-136.PubMed
- Fogh J, Trempe G: New human tumor cell lines. Human Tumor Cells in Vitro. Edited by: Fogh J. 1975, Plenum Press, New York, 115-159.View Article
- Hamilton TC, Young RC, McKoy WM, Grotzinger KR, Green JA, Chu EW, Whang-Peng J, Rogan AM, Green WR, Ozols RF: Characterization of a human ovarian carcinoma cell line (NIH:OVCAR-3) with androgen and estrogen receptors. Cancer Res. 1983, 43: 5379-5389.PubMed
- Buick RN, Pullano R, Trent JM: Comparative properties of five human ovarian adenocarcinoma cell lines. Cancer Res. 1985, 45: 3668-3676.PubMed
- Dyck HG, Hamilton TC, Godwin AK, Lynch HT, Maines-Bandiera S, Auersperg N: Autonomy of the epithelial phenotype in human ovarian surface epithelium: changes with neoplastic progression and with a family history of ovarian cancer. Int J Cancer. 1996, 69: 429-436. 10.1002/(SICI)1097-0215(19961220)69:6<429::AID-IJC1>3.0.CO;2-6.View ArticlePubMed
- Ong A, Maines-Bandiera SL, Roskelley CD, Auersperg N: An ovarian adenocarcinoma line derived from SV40/E-cadherin-transfected normal human ovarian surface epithelium. Int J Cancer. 2000, 85: 430-437. 10.1002/(SICI)1097-0215(20000201)85:3<430::AID-IJC21>3.0.CO;2-Q.View ArticlePubMed
- Pedersen LB, Veland IR, Schroder JM, Christensen ST: Assembly of primary cilia. Dev Dyn. 2008, 237: 1993-2006. 10.1002/dvdy.21521.View ArticlePubMed
- Clement CA, Kristensen SG, Mollgard K, Pazour GJ, Yoder BK, Larsen LA, Christensen ST: The primary cilium coordinates early cardiogenesis and hedgehog signaling in cardiomyocyte differentiation. J Cell Sci. 2009, 122: 3070-3082. 10.1242/jcs.049676.PubMed CentralView ArticlePubMed
- Follit JA, Tuft RA, Fogarty KE, Pazour GJ: The intraflagellar transport protein IFT20 is associated with the Golgi complex and is required for cilia assembly. Mol Biol Cell. 2006, 17: 3781-3792. 10.1091/mbc.E06-02-0133.PubMed CentralView ArticlePubMed
- Pazour GJ, Dickert BL, Vucica Y, Seeley ES, Rosenbaum JL, Witman GB, Cole DG: Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J Cell Biol. 2000, 151: 709-718. 10.1083/jcb.151.3.709.PubMed CentralView ArticlePubMed
- Haycraft CJ, Banizs B, Aydin-Son Y, Zhang Q, Michaud EJ, Yoder BK: Gli2 and Gli3 localize to cilia and require the intraflagellar transport protein polaris for processing and function. PLoS Genet. 2005, 1: e53-10.1371/journal.pgen.0010053.PubMed CentralView ArticlePubMed
- Pan Y, Bai CB, Joyner AL, Wang B: Sonic hedgehog signaling regulates Gli2 transcriptional activity by suppressing its processing and degradation. Mol Cell Biol. 2006, 26: 3365-3377. 10.1128/MCB.26.9.3365-3377.2006.PubMed CentralView ArticlePubMed
- Sasaki H, Nishizaki Y, Hui C, Nakafuku M, Kondoh H: Regulation of Gli2 and Gli3 activities by an amino-terminal repression domain: implication of Gli2 and Gli3 as primary mediators of Shh signaling. Development. 1999, 126: 3915-3924.PubMed
- Lih CJ, Cohen SN, Wang C, Lin-Chao S: The platelet-derived growth factor alpha-receptor is encoded by a growth-arrest-specific (gas) gene. Proc Natl Acad Sci U S A. 1996, 93: 4617-4622. 10.1073/pnas.93.10.4617.PubMed CentralView ArticlePubMed
- Schneider L, Clement CA, Teilmann SC, Pazour GJ, Hoffmann EK, Satir P, Christensen ST: PDGFRalphaalpha signaling is regulated through the primary cilium in fibroblasts. Curr Biol. 2005, 15: 1861-1866. 10.1016/j.cub.2005.09.012.View ArticlePubMed
- Schneider L, Cammer M, Lehman J, Nielsen SK, Guerra CF, Veland IR, Stock C, Hoffmann EK, Yoder BK, Schwab A, Satir P, Christensen ST: Directional cell migration and chemotaxis in wound healing response to PDGF-AA are coordinated by the primary cilium in fibroblasts. Cell Physiol Biochem. 2010, 25: 279-292. 10.1159/000276562.PubMed CentralView ArticlePubMed
- Pan J, Wang Q, Snell WJ: An aurora kinase is essential for flagellar disassembly in Chlamydomonas. Dev Cell. 2004, 6: 445-451. 10.1016/S1534-5807(04)00064-4.View ArticlePubMed
- Pugacheva EN, Golemis EA: The focal adhesion scaffolding protein HEF1 regulates activation of the aurora-A and Nek2 kinases at the centrosome. Nat Cell Biol. 2005, 7: 937-946. 10.1038/ncb1309.PubMed CentralView ArticlePubMed
- Kwon YE, Kim YS, Oh YM, Seol JH: Nuclear localization of Chfr is crucial for its checkpoint function. Mol Cells. 2009, 27: 359-363. 10.1007/s10059-009-0046-7.View ArticlePubMed
- Oh YM, Yoo SJ, Seol JH: Deubiquitination of Chfr, a checkpoint protein, by USP7/HAUSP regulates its stability and activity. Biochem Biophys Res Commun. 2007, 357: 615-619. 10.1016/j.bbrc.2007.03.193.View ArticlePubMed
- Daniels MJ, Marson A, Venkitaraman AR: PML bodies control the nuclear dynamics and function of the CHFR mitotic checkpoint protein. Nat Struct Mol Biol. 2004, 11: 1114-1121. 10.1038/nsmb837.View ArticlePubMed
- Castiel A, Danieli MM, David A, Moshkovitz S, Aplan PD, Kirsch IR, Brandeis M, Krämer A, Izraeli S: The Stil protein regulates centrosome integrity and mitosis through suppression of Chfr. J Cell Sci. 2011, 124: 532-10.1242/jcs.079731.PubMed CentralView ArticlePubMed
- Burgess A, Labbe JC, Vigneron S, Bonneaud N, Strub JM, Van Dorsselaer A, Lorca T, Castro A: Chfr interacts and colocalizes with TCTP to the mitotic spindle. Oncogene. 2008, 27: 5554-5566. 10.1038/onc.2008.167.View ArticlePubMed
- Xu J, Li H, Wang B, Xu Y, Yang J, Zhang X, Harten SK, Shukla D, Maxwell PH, Pei D, Esteban MA: VHL inactivation induces HEF1 and aurora kinase A. J Am Soc Nephrol. 2010, 21: 2041-2046. 10.1681/ASN.2010040345.PubMed CentralView ArticlePubMed
- Nielsen SK, Mollgard K, Clement CA, Veland IR, Awan A, Yoder BK, Novak I, Christensen ST: Characterization of primary cilia and Hedgehog signaling during development of the human pancreas and in human pancreatic duct cancer cell lines. Dev Dyn. 2008, 237: 2039-2052. 10.1002/dvdy.21610.View ArticlePubMed
- Privette LM, Gonzalez ME, Ding L, Kleer CG, Petty EM: Altered expression of the early mitotic checkpoint protein, CHFR, in breast cancers: implications for tumor suppression. Cancer Res. 2007, 67: 6064-6074. 10.1158/0008-5472.CAN-06-4109.View ArticlePubMed
- Toyota M, Sasaki Y, Satoh A, Ogi K, Kikuchi T, Suzuki H, Mita H, Tanaka N, Itoh F, Issa JP, Jair KW, Schuebel KE, Imai K, Tokino T: Epigenetic inactivation of CHFR in human tumors. Proc Natl Acad Sci U S A. 2003, 100: 7818-7823. 10.1073/pnas.1337066100.PubMed CentralView ArticlePubMed
- Scolnick DM, Halazonetis TD: Chfr defines a mitotic stress checkpoint that delays entry into metaphase. Nature. 2000, 406: 430-435. 10.1038/35019108.View ArticlePubMed
- Hubbert C, Guardiola A, Shao R, Kawaguchi Y, Ito A, Nixon A, Yoshida M, Wang XF, Yao TP: HDAC6 is a microtubule-associated deacetylase. Nature. 2002, 417: 455-458. 10.1038/417455a.View ArticlePubMed
- Haggarty SJ, Koeller KM, Wong JC, Grozinger CM, Schreiber SL: Domain-selective small-molecule inhibitor of histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation. Proc Natl Acad Sci U S A. 2003, 100: 4389-4394. 10.1073/pnas.0430973100.PubMed CentralView ArticlePubMed
- Matsuyama A, Shimazu T, Sumida Y, Saito A, Yoshimatsu Y, Seigneurin-Berny D, Osada H, Komatsu Y, Nishino N, Khochbin S, Horinouchi S, Yoshida M: In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. EMBO J. 2002, 21: 6820-6831. 10.1093/emboj/cdf682.PubMed CentralView ArticlePubMed
- Sharma N, Kosan ZA, Stallworth JE, Berbari NF, Yoder BK: Soluble levels of cytosolic tubulin regulate ciliary length control. Mol Biol Cell. 2011, 22: 806-816. 10.1091/mbc.E10-03-0269.PubMed CentralView ArticlePubMed
- Zhang Y, Kwon SH, Yamaguchi T, Cubizolles F, Rousseaux S, Kneissel M, Cao C, Li N, Cheng HL, Chua K, Lombard D, Mizeracki A, Matthias G, Alt FW, Khochbin S, Matthias P: Mice lacking histone deacetylase 6 have hyperacetylated tubulin but are viable and develop normally. Mol Cell Biol. 2008, 28: 1688-1701. 10.1128/MCB.01154-06.PubMed CentralView ArticlePubMed
- Schrøder JM, Larsen J, Komarova Y, Akhmanova A, Thorsteinsson RI, Grigoriev I, Manguso R, Christensen ST, Pedersen SF, Geimer S, Pedersen LB: EB1 and EB3 promote cilia biogenesis by several centrosome-related mechanisms. J Cell Sci. 2011, 124: 2539-2551. 10.1242/jcs.085852.PubMed CentralView ArticlePubMed
- Christensen ST, Guerra C, Wada Y, Valentin T, Angeletti RH, Satir P, Hamasaki T: A regulatory light chain of ciliary outer arm dynein in Tetrahymena thermophila. J Biol Chem. 2001, 276: 20048-20054. 10.1074/jbc.M008412200.View ArticlePubMed
- Rompolas P, Pedersen LB, Patel-King RS, King SM: Chlamydomonas FAP133 is a dynein intermediate chain associated with the retrograde intraflagellar transport motor. J Cell Sci. 2007, 120: 3653-3665. 10.1242/jcs.012773.View ArticlePubMed
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.