The cyclin-like protein Spy1/RINGO promotes mammary transformation and is elevated in human breast cancer
- Mohammad Al Sorkhy†1,
- Rosa-Maria Ferraiuolo†2,
- Espanta Jalili2,
- Agnes Malysa2,
- Andreea R Fratiloiu3,
- Bonnie F Sloane4 and
- Lisa A Porter2Email author
© Al Sorkhy et al; licensee BioMed Central Ltd. 2012
Received: 11 August 2011
Accepted: 26 January 2012
Published: 26 January 2012
Spy1 is a novel 'cyclin-like' activator of the G1/S transition capable of enhancing cell proliferation as well as inhibiting apoptosis. Spy1 protein levels are tightly regulated during normal mammary development and forced overexpression in mammary mouse models accelerates mammary tumorigenesis.
Using human tissue samples, cell culture models and in vivo analysis we study the implications of Spy1 as a mediator of mammary transformation and breast cancer proliferation.
We demonstrate that this protein can facilitate transformation in a manner dependent upon the activation of the G2/M Cdk, Cdk1, and the subsequent inhibition of the anti-apoptotic regulator FOXO1. Importantly, we show for the first time that enhanced levels of Spy1 protein are found in a large number of human breast cancers and that knockdown of Spy1 impairs breast cancer cell proliferation.
Collectively, this work supports that Spy1 is a unique activator of Cdk1 in breast cancer cells and may represent a valuable drug target and/or a prognostic marker for subsets of breast cancers.
The Speedy/RINGO family of proteins are novel regulators of the cell cycle, capable of activating the cyclin-dependent kinases (Cdks) independent of cyclin binding and phosphorylation within the Cdk T-loop . The human Speedy/RINGO homolog, herein referred to as Spy1, is constitutively expressed in most human tissues and is essential for somatic cell cycle progression . Ectopic expression of Spy1 promotes rapid cell cycle progression through G1/S phase that is attributed, at least in part, to the activation of Cdk2 . Spy1 can prevent the inhibitory effects of the tumor suppressor p27Kip1 on Cdk2 by directly promoting p27 degradation, suggesting yet another mechanism by which Spy1 can enhance both normal and aberrant cell growth [3–5]. SAGE analysis has shown that Spy1 is expressed at elevated levels in one case of invasive ductal carcinoma of the breast . Spy1 protein levels have also been implicated as a prognostic marker in hepatic carcinogenesis and ectopic overexpression of Spy1 can accelerate mammary tumorigenesis in vivo [6–8]. Of potential importance, two independent linkage studies have resolved that the chromosomal loci at the precise location of Spy1 (2p23.2) may be a candidate site contributing toward breast cancer risk, particularly in women under the age of 50 [9, 10]. Hence, how Spy1 levels contribute to the initiation and/or progression of tumorigenesis is of high priority for understanding both normal and abnormal cell growth programs.
Spy1 protein levels are tightly regulated during the cell cycle, being transcriptionally upregulated by the oncogene c-Myc [8, 11, 12]. We have resolved three residues within the N-terminal region of the protein; T15, S22, and T33 which are essential for targeting Spy1 for ubiquitin-mediated degradation in G2/M phase of the cell cycle . Mutation of these residues generates a non-degradable form of Spy1 (Spy1-TST) which significantly enhances cell proliferation over that of wild-type Spy1 . Herein, we demonstrate that: (1) elevated levels of Spy1 is a transforming event, (2) Spy1-mediated transformation relies on the activation of Cdk1 and may mediate an inhibition of the pro-apoptotic regulator FOXO1, (3) levels of Spy1 protein are highly elevated in aggressive human breast cancers and (4) downregulation of Spy1 can significantly inhibit breast cancer cell growth. Collectively these data support that Cdk1 kinase activity is essential for Spy1-mediated transformation and may indicate a therapeutically relevant mechanism of treating tumors with elevated levels of Spy1 protein.
The mouse embryonic fibroblast cell line NIH3T3 (ATCC), human embryonic kidney cell line HEK-293 (293; ATCC) and human breast cancer line MDA-MB-231 (MDA-231; ATCC) were maintained in DMEM medium (Sigma) supplemented with 10% (vol/vol) calf serum (Sigma) for NIH3T3 cells, and fetal bovine serum (FBS; Sigma) for 293 cells. The BALB/c mouse mammary epithelial cell line HC11 (Dr. C. Shermanko) and breast cancer MCF7 cells (Dr. T. Seagroves) were maintained in RPMI 1640 medium (Sigma) containing 10% (vol/vol) fetal calf serum and supplemented with 5 μg/ml insulin (Sigma), and 10 ng/ml EGF (Gibco). Human breast MCF10A series cell lines (ATCC & Drs. B. Sloane and F. Miller) were maintained in DMEM-F12 media containing 0.5 ug/ml hydrocortisone, 10 ug/ml insulin, 20 ng/ml human EGF and 5% (vol/vol) horse serum heat inactivated. The MMTV-Myc cell line was derived from a freshly dissected mammary adenocarcinoma from a 3 month multiparous MMTV-Myc female mouse. All cell lines were maintained in a media containing 2 mM L-glutamine (G7513; Sigma), penicillin and streptomycin (15140; GIBCO), and were cultured in a 5% CO2 environment. Cells not received from ATCC were tested for tissue/species specific genes and characteristic receptor status via Q-RT-PCR. These tests, as well as testing for mycoplasma, bacteria, fungi contamination and cytogenetic characterization are performed on cells obtained from ATCC.
Plasmid and mutagenesis
Creation of the Myc-Spy1-PCS3 vector was described previously . Spy1-TST was created using QuickChange PCR Multi-Site-Directed Mutagenesis (Stratagene) of Spy1-PCS3 in 3 sequential steps to generate alanine mutations at positions T15, S22 and T33. Successful cloning was determined by DNA sequencing (Robarts Sequencing Facility; UWO). Plasmids for FLAG-FOXO1 (#9036), HA-Cdk1 (#1888), HA-Cdk1-DN (#1889), pLKO-scrambled control (#8453), the luciferase reporter construct 3xIRS (#13511) and lentiviral constructs: pMD.G (#12259), pMDLg/pRE (#12251) and pRSV-Rev (#12253) were all obtained from Addgene. pLKO Spy1 was cloned to express the short hairpin previously demonstrated to specifically knockdown Spy1 in place of the scrambled sequence in pLKO above . pSUPER (Oligoengine) containing a scrambled siRNA and pSUPER with siSpy1 are previously described . Generous gifts; FOXO1-A3/S249A (Dr. H. Huang; University of Minnesota), luciferase control plasmids (Dr. B. Vogelstein; Johns Hopkins University) and Ras-V12 (Dr. S. Lowe; Cold Spring Harbor).
Primary antibodies were as follows: Spy1 (NB100-2521; Novus), Myc (9E10 and C19; Santa Cruz), HA (Y11 and F7; Santa Cruz), Cdk1 (ab31687; Abcam), TOTO-3 (T-3600; Molecular Probes), IgG (SC66186; Santa Cruz), Cdk2 (SC-6428; Santa Cruz), Flag (F3040; Sigma) and actin (MAB1501R; Chemicon).
Cells were transfected using polyethylenimine (PEI) branched reagent (408727; Sigma). In brief, for experiments using a fixed amount of DNA per construct 10 μg of DNA was mixed with 50 μL of 150 mM NaCl and 3 μL of 10 mg/ml PEI for 10 min then added to a 10 cm tissue culture plate. For transfections requiring more DNA as specified, the relative amounts of NaCl and PEI were scaled up accordingly. Media was changed after 8 h for NIH3T3s and MCF7s and remained overnight for the 293 cells. For viral preparation: media was changed after 8 h transfection of packaging lines and virus harvested 24 h later. Virus was sterilized using a 0.45 μm filter and concentrated using ultracentrifugation for 3 h at 25 K rpm at 4°C. Viral titre was determined using HC11 cells. Using a titre of 107/ml and an MOI of 10, MDA-231 cells were infected ~70-80% confluency in serum free and antibiotic free media containing polybrene (25 μg/mL). Media was replaced 6 h post-infection. Proliferation assays were conducted by counting live and dead cell populations using trypan blue exclusion and using a haemocytometer; counts were also verified using a TC10 automated cell counter (Biorad).
Cell synchronization and flow cytometry
Cells were synchronized using double thymidine block; cells were cultured in a media containing 2 mM thymidine for 16 h, released to normal media for 8 h, followed by a 14 h block in 2 mM thymidine and then released in 70 ng nocodazole. NIH3T3 cells were synchronized by being cultured in a 2% serum containing media for 24 h, followed by release in standard culture media.
Flow cytometry analysis; cells were carefully collected at indicated times (taking care to include even floating cell populations), washed twice in PBS, and then either used immediately or fixed for future analysis. Fixed cells were resuspended at 2 × 106 cells in 1 ml of PBS, fixed by the dropwise addition of an equal amount of ethanol, and frozen at -80°C. Within 1 week, fixed cells were pelleted, washed, and resuspended in 300 μl of PBS. Samples of resuspended fixed cells or fresh cells were treated with 1 μl of 10 mg/ml stock of DNase free RNase (Sigma) and 50 μl of 500 mg/ml propidium iodide solution. Cells shown were first gated for size using forward scatter and side scatter parameters. A minimum of 300,000 cells were analyzed per treatment using a Beckman Coulter FC500.
Immunoblotting (IB) and immunoprecipitation (IP)
Cells were lysed in 0.1% NP-40 lysis buffer (5 ml 10% NP-40, 10 ml 1 M Tris pH 7.5, 5 ml 0.5 M EDTA, 10 ml 5 M NaCl up to 500 ml RO water) containing protease inhibitors (10 μg/ml PMSF, 60 μg/ml aprotinin, 10 μg/ml leupeptin) for 30 min on ice. Bradford Reagent was used to determine the protein concentration (Sigma). 20-30 μg protein were subjected to electrophoresis on denaturing SDS polyacrylamide gels and transferred to PVDF-Plus 0.45U transfer membranes (Osmonics Inc.) for 2 h at 30 V using a wet transfer method. Blots were blocked for 2 h in TBST containing 3% non-fat dry milk (blocker) at RT, primary antibodies were reconstituted in blocker and incubated overnight at 4°C, secondary antibodies were used at a 1:10,000 dilution in blocker for 1 h at RT. Blots were washed three times with TBST following incubation with both the primary and secondary antibodies. Washes were 6 min each following the primary antibody and 10 min each following the secondary antibody. Blots were visualized using Chemilumiminescent Peroxidase Substrate (Pierce) and quantified on an Alpha Innotech HD2 (Fisher) using AlphaEase FC software.
For IP, equal amounts of protein were incubated with primary antisera as indicated overnight at 4°C, followed by the addition of 10 ul protein A-Sepharose (Sigma) and incubated at 4°C with gentle rotation for an additional 2 h. These complexes were then washed 3× with 0.1% NP-40 lysis buffer and resolved by 10% SDS-PAGE.
Tissue microarray (TMA) analysis
Paraffin embedded TMA slides (cat # BR721 and BR962; US Biomax) consisting of 165 tissue cores in total were deparaffinized and rehydrated in decreasing percentages of ethanol according to the manufacturer's instructions. Antigen retrieval was performed at 95°C in 0.01 M sodium citrate buffer, pH 6.0. Slides were washed in 1× PBS, permeabilized in 1 × PBS/0.2% Triton X-100 at RT followed by 3 washes with 1 × PBS. Sections were blocked in goat serum for 1 h at 37'C, followed by incubation in primary antibody for 2 h at 37°C. Slides were washed twice for 10 min in PBS-1% normal goat serum. Secondary antibodies were applied for 1 h at 37°C, washed 3× for 10 min each in 1 × PBS and incubated 30 min with a nuclear counterstain for TOTO-3. The fluorescent signal was detected and quantified by ScanArray Express (Perkin Elmer Inc.). The Spy1 signal intensity was normalized to nuclear stain signal.
Soft agar assay
Two layers of agar/media mixture were plated into 60 mm culture dishes. Briefly, 0.6 g of Noble agar (UBS) was suspended in 100 ml DI water to yield a 0.6% bottom agar solution. This mixture was poured into two 50 ml tubes, sealed and placed at 40°C for 40 min. Simultaneously; DMEM media enriched with 20% FBS was also incubated at 40°C. The bottom agar layer contained 1.5 ml of 0.6% agar solution and 1.5 ml of media solution was poured and plated. When solidified, a top agar layer was prepared containing a 3% agar solution. Cells (~2.5 × 105) were harvested, counted and added to the top agar + media mix and then promptly layered onto the bottom agar layer. Plates were set for 1 h under sterile conditions and then incubated at 37°C in 5% CO2 for 12-14 days. Triplicate transfections for each experiment were observed using light microscopy and pictures were taken using an Apha Innotech HD2 camera. Colonies were counted manually using light microscopy.
Low passage cells were grown in 10 cm plates, transfected as above and grown to confluence. Media with 2% calf serum was gently changed every other day for up to two weeks. Plates were stained with 0.5% crystal violet, photographed using an Apha Innotech HD2 camera and the number of colonies were counted manually.
Fat pad transplants
Mice were maintained following the guidelines of the Canadian Council on Animal Care under the ethical approval of the Animal Care Committee, University of Windsor (AUPP #06-19). Fat pad transplant assays were conducted using BALB/c mice, which are syngenic for the HC11 cell line as previously described . In brief, 5 × 105 cells were injected into the cleared fat pad of 4th inguinal mammary glands of 22 day old mice and allowed to grow for 1 to 8 week. Tumor presence was monitored weekly by palpitation of the gland. Animals were sacrificed humanely at the specified time points and glands dissected for analysis. Tumor volume was calculated as length (mm) × width (mm) × height (mm) using manual calipers.
Cells were transfected, cultured in 10% FBS and lysed in 0.1% NP-40 lysis buffer. 16 h post-transfection IP was carried out as described above and precipitates were washed four times prior to the addition of 50 μl of kinase buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl2, 1 mM DTT, 20 mM EGTA, 50 mM ATP, 10 μCi of [γ-32P]ATP) and 74 μg/ml H1 histone (Boehringer Mannheim). Reactions were incubated for 10 min at 30°C, sample buffer was added to stop the reaction and 50 μl of each sample were analyzed by 10% SDS-PAGE. Incorporated phosphorylation was visualized using a Cyclone storage phosphor system and quantified using OptiQuant software (Perkin Elmer). IPs were subsequently probed on the same membrane.
Cells were transfected with the appropriate luciferase reporter construct, harvested 24 h post-transfection and 50 ul of cell suspension was mixed with 50 ul of Bright-glo reagent (E2620; Promega). Luminescence spectra of the samples were measured using a plate reader (Wallac Victor 1420).
Results are presented as the mean ± standard error. Statistical significance was assessed through either a student's t-test (analysis of two means) or analysis of variance (ANOVA) followed by a post-hoc Tukey test (for more than two means with equal variances) or post-hoc Bonferroni (for more than two means without equal variances). StatSoft's STATISTICA software was used for all analysis.
Results and Discussion
To test this hypothesis, soft agar assays were conducted using similar protein levels of either Spy1-WT or Spy1-TST, along with activated Ras (Ras-V12) as a positive control and empty pCS3 as a negative control (Figure 1C). Colonies were formed in the presence of Ras-V12 as well as Spy1-TST, but no colonies were present in the negative control and few colonies were detected for Spy1-WT (Figure 1C). Quantification over three separate experiments demonstrated that Spy1-TST yielded 4 times more colonies than the Spy1-WT counterpart (Figure 1D). Densitometry analysis of protein levels of transfected Spy1 at the time of seeding, normalized using Actin levels, show less than a 2 fold increase in the overall Spy1 protein levels with Spy1-TST (Figure 1E). The modest increase in protein accumulation with Spy1-TST is not surprising given that we are utilizing asynchronous cells and there are multiple mechanisms for regulating Spy1 protein levels [11, 12]. Functionally, this supports that the transforming properties of Spy1-TST are not explained merely by the accumulation of overall amounts of protein.
Spy1 protein is known to regulate cell cycle progression at least in part through the direct binding to Cdks . In somatic cells the primary partner for Spy1, and an essential regulator of Spy1-mediated proliferation, appears to be the G1/S Cdk, Cdk2 . Spy1 is capable of also binding and activating Cdk1 when overexpressed . To determine the role of Cdk proteins on Spy1-mediated transformation we utilized a mutant form of Spy1-TST where the aspartic acid residue at position 90 is mutated to a nonpolar alanine group (Spy1-TST/D90A), a modification previously demonstrated to significantly reduce Spy1-Cdk binding . We demonstrate that this mutation also abrogates the ability of Spy1-TST to interact with Cdk1 (Figure 4A). The Spy1-TST/D90A mutation reduced colony formation in soft agar approximately 5-fold compared to Spy1-TST (Figure 4B & C). To further investigate the relative contribution of each kinase individually to this effect, soft agar assays were performed using Spy1-TST in the presence of the dominant negative form of either Cdk1 (Cdk1-DN) or Cdk2 (Cdk2-DN) (Figure 4D). Interestingly, Cdk1-DN reduced colony formation by ~60% with high statistical significance over 3 separate trials while Cdk2-DN demonstrated reduced colony numbers but this result was not statistically significant. This was not due to inefficient function of the constructs as both DN constructs effectively reduced the kinase activity of their relevant Cdk (Additional file 1: Figure S1). Collectively this data supports that the oncogenic function of Spy1 is dependent, at least in part, on the binding and activation of Cdk1.
Collectively, this work supports that critical levels of Spy1 protein trigger transformation dependent upon the activation of the G2/M cyclin-dependent kinase, Cdk1. We further show that this mechanism is sensitive to inhibition by the apoptotic regulator FOXO1. We have demonstrated for the first time that levels of Spy1 are elevated in all human breast cancer samples tested and that knockdown of Spy1 can reduce breast cancer cell growth and may represent a novel target for breast cancer therapy.
We thank Drs. H. Huang, B. Vogelstein, E. Harlow, W. Sellers, S. Lowe., K.Guan, C. Shermanko, T. Seagroves and F. Miller for providing plasmids and cell lines. The Breast Cancer Society of Canada for an equipment grant. Our appreciation to J. Tubman and J. Maimaiti for critical evaluation of this manuscript and technical support. MAS and R-MF acknowledge scholarship support from NSERC and the CIHR. LAP gratefully acknowledges salary support from the CIHR New Investigator Program and Assumption University. This study is supported by operating funds from the Canadian Cancer Society (CCS)/Canadian Breast Cancer Research Alliance (CBCRA) #020513.
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