Background
Primary cilia are microtubule-based, nonmotile cell appendages protruding from most mammalian cells that were identified more than 100 years ago as vestigial organelles [
1]. Primary cilia extend from the mother centriole into the extracellular environment and function as antennas sensing and transducing signals from the external milieu into cells [
2‐
5]. Signaling through primary cilia regulates a number of biological processes, including cell polarity, migration, and differentiation, in part through the activation of primary cilia-associated components, including members of the Hedgehog, mTORC1, platelet-derived growth factor receptor (PDGFR), and Wnt signaling pathways [
2,
5]. Primary cilia also play important roles during development, with defects in the establishment of primary cilia due to mutation- or transcription-driven alterations in ciliary genes being responsible for a number of ciliopathies and disorders, such as polycystic kidney disease, situs inversus, and Bardet-Biedl syndrome. In cancer tissues, including pancreatic, prostate, and breast cancers, primary cilia are decreased in incidence compared with normal-matched tissues [
6‐
9]. Recently, a study conducted with a collection of breast cancer cell lines and tissues revealed that primary cilia are lost at a very early stage during breast cancer development, raising the possibility that they may have tumor-suppressive functions in breast cancer and possibly other cancer types [
10,
11]. Yet, in another study, researchers reported that the presence of primary cilia in cancer cells correlates with poor prognosis and lymph node metastasis in patients with pancreatic cancer [
12]. Indeed, primary cilia are still poorly characterized and studied organelles whose functions as sensing and signaling platforms in normal and transformed mammalian cells are only beginning to be explored.
Split ends (SPEN), also known as SMRT/HDAC1-associated repressor protein, is a large nuclear protein with essential roles in transcriptional regulation and chromosome X inactivation [
13‐
16]. SPEN contains four N-terminal RNA recognition motifs and a highly conserved Spen paralogue and orthologue C-terminal domain (SPOC) that is required for its transcriptional repression of nuclear hormone receptors (HRs) [
17]. Given the presumptive role of SPEN in endocrine response, our laboratory has characterized SPEN functions in estrogen receptor alpha (ERα)-positive breast cancers and established SPEN as a transcriptional corepressor of the ERα, tumor suppressor gene, and candidate predictive biomarker of tamoxifen response in hormone-dependent breast cancers [
13].
In the present study, we examined the hormone-independent transcriptional program and functions regulated by SPEN in breast cancer and found that
SPEN is significantly coexpressed with genes involved in ciliary biology. We demonstrate that SPEN positively regulates primary cilia formation and cell migration in breast cancer, possibly via the transcriptional regulation of
RFX3, a ciliogenic transcription factor [
18‐
21]. Interestingly, we found that
SPEN knockdown attenuates cell migration in breast cancer cells when accompanied with a concomitant decrease in primary cilia levels, indicating that SPEN may regulate cellular movement through primary cilia-dependent mechanisms. We also report that high
SPEN expression levels are predictive of early metastasis in patients with ERα-negative but not ERα-positive breast cancers. Altogether, our results establish SPEN as a new player in primary cilia formation and cell migration in breast cells, two functions that may collectively explain why
SPEN expression is associated with time to metastasis in patients with ERα-negative breast cancers.
Methods
Cell lines
All cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). T47D clones (CTL and SPEN) were stably transfected with control and SPEN-expressing vectors as previously described and were cultured in Gibco DMEM (Life Technologies, Carlsbad, CA, USA) supplemented with 10% FBS (Wisent Bio Products, Saint-Bruno, PQ, Canada) and G418 (500 μg/ml) under conditions specified by the manufacturer. BT20 and MDA-MB-436 cells were cultured in Eagle’s minimum essential medium and RPMI 1640 (ATCC), respectively, supplemented with 10% FBS. MCF10A cells were maintained in RPMI 1640 (ATCC) containing 10% FBS, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, and 20 ng/ml human epidermal growth factor. MCF10A-CT cells were maintained in Gibco DMEM/Ham’s F-12 Nutrient Mixture (Life Technologies) containing 5% horse serum, 10 μg/ml insulin, 0.5 μg/ml hydrocortisone, 20 ng/ml human epidermal growth factor, and 100 ng/ml cholera toxin (CT).
DNA microarray expression profiling and analysis
Expression profiling was conducted according to the manufacturer’s instructions and following the One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technologies, Santa Clara, CA, USA). Briefly, the integrity and concentration of the input RNA was evaluated with the Agilent 2011 Bioanalyzer (Agilent Technologies). Total RNA (100 ng) was reverse-transcribed into complementary RNA (cRNA), amplified, and labeled with cyanine 3 dye. The resulting labeled cRNA was purified using the RNeasy Mini Kit (QIAGEN Sciences, Germantown, MD, USA) according to the instructions of the manufacturer and hybridized to a Sure Print G3 Human Gene Expression 8 × 60 K microarray (Agilent Technologies) for 17 h at 65 °C. The array was then washed and scanned on the Agilent DNA microarray scanner at a resolution of 3 μm. Images were extracted and normalized with Feature Extraction version 9.5 software (Agilent Technologies). Expression values of three biological RNA replicates for each probe in the expression array were analyzed using GeneSpring GX software (Agilent Technologies). The microarray data from this study are available on ArrayExpress under accession numbers [E-MTAB-4974 and E-MTAB-4875].
Statistical analysis
Microarray expression data were processed with GeneSpring GX software. Data were normalized to the 75th percentile of all values on the microarrays and to the median expression levels of all samples. The normalized gene expression data were filtered on flags, and all detected and undetected flags were allowed to pass the filter and were included in the analysis. The expression profiles of genes differentially expressed by more than 1.5-fold on the basis of three biological replicates were compared using unpaired t tests.
Ingenuity Pathway Analysis
Microarray analyses were performed using the Ingenuity Pathway Analysis software (QIAGEN Bioinformatics, Redwood City, CA, USA). Genes upregulated by 2.00-fold (P < 0.05) in T47D-SPEN compared with its control (T47D-CTL) and downregulated by 2.00-fold (P < 0.05) in SPEN-silenced compared with control MCF10A cells were considered for further analyses.
Immunoprecipitation
Cells were rinsed in PBS, harvested, and lysed in buffer (250 mM NaCl, 0.5% Nonidet P-40, 5 mM ethylenediaminetetraacetic acid [EDTA], 50 mM Tris) freshly supplemented with protease inhibitors (sodium fluoride, sodium orthovanadate, phenylmethylsulfonyl fluoride, aprotinin, and leupeptin). After micro-bicinchoninic acid quantification, 0.5 mg of proteins was incubated overnight with 1 μg of anti-SPEN antibody (HPA015825; Sigma-Aldrich, St. Louis, MO, USA), RFX3 (TA505916; OriGene Technologies, Rockville, MD, USA), or immunoglobulin G (IgG) control antibody (ab46540-1; Abcam, Cambridge, MA, USA). Protein lysates were incubated for 3 h with protein A beads (45000116; GE Healthcare Bio-Sciences, Piscataway, NJ, USA), centrifuged (4000 rpm), and washed three times with 400 μl of lysis buffer. Precipitated beads were then incubated at 95 °C for 7 minutes in 60 μl of the loading buffer, followed by centrifugation at 13,000 rpm for 1 minute. SPEN-knockdown post-small interfering RNA (post-siRNA) transfection was confirmed twice for each of the four cell lines under study. Immunoprecipitation assays for the measurement of RFX3 protein expression levels were performed at least three times.
siRNA transfections
All siRNA transfections were done using Lipofectamine RNAiMAX Transfection Reagent (Life Technologies) following the manufacturer’s protocol. Briefly, 2.5 × 105 cells were plated in six-well plates, and the transfection complex (containing 9 μl of Lipofectamine RNAiMAX Transfection Reagent and 30 pmol of siRNAs) was added to cells 24 h later. After another 72 h, transfected cells were analyzed for RNA and/or protein expression by qRT-PCR and Western blotting, respectively. A fraction of transfected cells was also analyzed by fluorescence-activated cell sorting for cell migration, or it was plated in low serum-containing medium for primary cilia quantification by immunofluorescence. All experiments were done within 6 days posttransfection.
Immunofluorescence
For immunofluorescence staining of cultured cells, cells were seeded onto coverslips in six-well plates (T47D cells) or into eight-chamber cell culture slides (siRNA-treated cells) and allowed to grow for 3 days. T47D cells were cultured in hormone-depleted medium, whereas all other cell lines were grown in their respective media supplemented with 1% FBS. Cells were washed with PBS, then fixed in 4.0% paraformaldehyde in DMEM with 10% FBS for 10 minutes, and then incubated in blocking solution (1% goat serum, 0.1% Triton X-100 in PBS) for 30 minutes. Cells were then incubated in primary antibodies for 1 h at room temperature. Primary antibodies used were mouse antiacetylated α-tubulin (1:4000 dilution, clone 6-11B-1; Sigma-Aldrich) and rabbit γ-tubulin (1:1000 dilution; Sigma-Aldrich). After being washed four times for 10 minutes each with PBS, cells were incubated with secondary antibodies (donkey antimouse Alexa Fluor 594 and goat antirabbit Alexa 488; Invitrogen/Life Technologies) for 1 h at room temperature. Cells were incubated for 4 minutes with 4′,6-diaminodino-2-phenylindole (5 μg/ml; Sigma-Aldrich) and washed four times with PBS for 10 minutes. Slides were mounted with CureMount II mounting medium (Leica Biosystems, Buffalo Grove, IL, USA). Images were obtained with a × 63 or × 100 lens objective. Primary cilia were identified as such when a ciliary acetylated α-tubulin structure, a marker of the axoneme, was colocalized to γ-tubulin staining, a marker for centrosomes. No minimal length cutoff was used. Primary cilia were manually counted in at least 15 independent microscopic fields (except for the KIF3A-knockdown assay, in which 10 independent microscopic fields were examined) per slide, and the frequency of primary cilia was calculated by dividing the number of cilia by the number of counted nuclei. Primary cilia levels were quantified in at least three independent experiments for each cell line (>750 cells were counted in total per experimental condition, except for lines of fibroblasts, in which approximately 400 cells were counted because of slow growth rates).
Western blotting
Subconfluent cells were collected by trypsinization, washed in ice-cold PBS, and lysed in lysis buffer freshly supplemented with protease inhibitors. Lysates were then centrifuged for 15 minutes at 4 °C. Supernatants were subjected to Bradford quantification, and 50 μg of proteins were loaded and run by SDS-PAGE in a 10% gel for 1 h. Proteins were transferred to nitrocellulose membranes and incubated with antibodies for RFX3 (TA505916; OriGene Technologies), KIF3A (D7G3, #8507; Cell Signaling Technology, Danvers, MA, USA), sperm flagellar 2 (SPEF2, catalog number ab57761; Abcam), and α-tubulin or β-actin. All Western blot experiments were performed at least three times.
Fluorescence activated cell sorting
For cell cycle analyses, cells were detached with trypsin, washed in PBS supplemented with 5 mmol/L EDTA, suspended in a fixing solution (1 ml of PBS, 5 mmol/L EDTA for 3 ml of 100% ethanol), and incubated at −20 °C for at least 24 h. Then cells were washed with PBS/EDTA and resuspended in 1 ml of staining solution (PBS, 50 mg/ml propidium iodide, and 20 mg/ml RNase A). The analysis was performed using BD CellQuest (BD Biosciences, San Jose, CA, USA), ModFit (Verity Software House, Topsham, ME, USA), and FlowJo (FlowJo, Ashland, OR, USA) software. At least three independent experiments were performed for each assay.
Intersection probabilities
To determine the statistical significance of intersection between two lists of genes, we assessed the probability of this intersection to occur by performing 10,000 independent simulations with randomly selected lists of genes of the same size. P values were calculated using a hypergeometric test.
Migration assays
Cell migration was assessed using a Boyden chamber assay. For these experiments, 5 × 105 cells (T47D cells) or 2.5 × 105 cells (all other cell lines) were seeded onto the upper well of a Costar Transwell chamber (8 μM; Corning Life Sciences, Tewksbury, MA, USA) in serum-free medium, except for T47D cells. The latter were seeded in complete medium, which was replaced with 0% FBS-containing medium 24 h later. For all cell lines except T47D clones, cells that had migrated to the bottom side of the membrane were fixed in 70% ethanol and stained with crystal violet 24 h after plating. This was done 72 h after seeding for T47D cells. After being stained, nonmigrating cells in the upper chamber were removed using a cotton-tipped applicator. The membranes were mounted onto object slides, and six random fields per slide were counted with a × 10 or × 20 lens objective. At least three independent experiments were performed for each assay.
Discussion
Primary cilia have long been considered as organelles of little to no functional and biological relevance. However, it is now clear that primary cilia play important roles during development, with ciliary dysfunction being associated with a variety of diseases, including polydactyly, brain malformations, situs inversus (abnormal left-right patterning), and polycystic kidney disease. More recently, primary cilia were also shown to be required for appropriate branching morphogenesis during the maturation of the breast, a bilayered ductal system composed of apically oriented luminal epithelial cells encircled by contractile and basement membrane-associated basal cells [
36,
37]. Interestingly, this study uncovered that primary cilia are found on both luminal and basal cells during branching morphogenesis but disappear from luminal cells after completion of breast development. Consistently, analysis of reduction mammoplasties from 12 women revealed that primary cilia are highly prevalent in basal (median 23.6%) but not luminal (median 1.1%) breast epithelial cells [
10]. Altogether, the data reported in these studies indicate that primary cilia may be more functionally and biologically important in basal than in luminal breast cells.
In the present study, we have established a novel role for SPEN in the regulation of primary cilia formation in breast epithelial cells. Indeed, we report that SPEN silencing reduces primary cilia formation in MCF10A and Hs578T cells, two cell types known to harbor high levels of primary cilia. Although luminal breast cancer cells are typically nonciliated, it should not be excluded that SPEN may also regulate primary cilia formation in these cells, as observed in our T47D model. Indeed, SPEN reexpression in T47D cells was able to restore the primary cilia to a level that is within the range previously reported for normal luminal breast epithelial cells (median 1.1%) and breast cancer cells (0.3–3.3%) [
9]. It is possible that characteristics intrinsic to luminal cells, including their unique gene expression profile and anatomical location, are responsible for their low abundance of primary cilia. This may also provide an explanation for the very low degree of restored primary cilia expression in T47D-SPEN cells to a level that is within the intrinsic capacity range of luminal breast cell populations.
Although identified more than 100 years ago, primary cilia are cellular structures whose functions in normal and cancer cells remain elusive. Indeed, it has been proposed that primary cilia have tumor-suppressive functions because they decrease in incidence in cancer compared with normal tissues. Yet, a large body of evidence also indicates that primary cilia may have tumor-promoting effects, owing to their ability to sense and integrate signals from oncogenic pathways, including the Hedgehog, Wnt, and PDGFR transduction pathways. As suggested by others, primary cilia may have dual roles in cancer development, functioning as tumor suppressors early in cancer formation but conferring aggressiveness at later stages of the disease through the regulation of oncogenic signaling pathways [
38‐
40].
In addition to its regulation of primary cilia formation, we also report that SPEN coordinates migration in breast epithelial cells, but only in those that are ciliated. Indeed, we found a correlation between SPEN-mediated effects on migration and primary cilia abundance, suggesting that SPEN may control migration through primary cilia-dependent mechanisms. To evaluate the clinical significance of these results, we assessed whether SPEN expression levels are correlated with metastasis in breast cancer and found that high SPEN RNA expression levels are predictive of early metastasis in two independent cohorts of ERα-negative but not ERα-positive breast cancers. These clinical findings are consistent with the very low abundance of primary cilia in luminal and ERα-positive breast cancer cells in vitro. However, it is important to note that cilia expression is very low in clinical breast cancer samples and not significantly different between ERα-positive and ERα-negative breast tumors. This may be explained by the 3D nature of primary tissues and the altered structural architecture of cancer compared with normal tissues, which may render the assessment of primary cilia levels in clinical samples more difficult and imprecise than for cultured cells. Preclinical studies with mouse models may provide further clues to better understand the role of primary cilia, the incidence of which is influenced by SPEN levels, in the metastatic process in breast cancer.
Metastasis is a very complex multistep process that imposes many more barriers and challenges to cancer cells than a requirement for migration. Yet, several lines of evidence are compatible with a role for SPEN through its regulation of primary cilia abundance and migration in one or several steps in the metastatic process. For instance, Emoto et al. [
12] recently demonstrated that patients with primary cilia-positive pancreatic cancers have a higher probability of developing lymph node metastasis than those whose tumors are nonciliated. In their report, they further showed that primary cilia expression constitutes an independent poor prognostic factor of overall patient survival in pancreatic cancer (HR 3.47,
P = 0.01) [
12]. Such findings are compatible with a role for primary cilia in cancer cells dissemination to distant organ sites and may explain why
SPEN expression is linked to metastasis in patients with ERα-negative breast cancers.
Although this study was focused on addressing the role of SPEN in primary cilia formation in breast cancer, it highlights the functions of SPEN in primary ciliogenesis that may also apply to other cell types, as seen in fibroblasts, and possibly many more cancer types. With hormone-dependent and hormone-independent functions, SPEN is a complex protein whose roles with regard to the establishment of primary cilia should be further investigated, especially in skin, pancreatic, and lung cancers, which are aggressive and highly metastatic types of cancer. In general, the results presented in this study reveal new functions for SPEN in the regulation of primary cilia formation and migration in breast cells. In addition, they shed light on research avenues that deserve further consideration in the field of cancer metastasis, which is still responsible for more than 90% of cancer-related deaths.
Acknowledgements
The authors thank past and current members of the M. Basik Laboratory, particularly Simona Sala for technical assistance, as well as Isabelle Royal, Vincent Giguère, Sylvie Mader, Frédéric Charron and Volker Blank for assistance and discussion. The authors also acknowledge the help of Lilian Amrein and Christian Young from the imaging and flow cytometry facilities, respectively, of the Lady Davis Institute for Medical Research.