A novel function for p21Cip1 and acetyltransferase p/CAF as critical transcriptional regulators of TGFβ-mediated breast cancer cell migration and invasion
© Dai et al.; licensee BioMed Central Ltd. 2012
Received: 29 May 2012
Accepted: 24 August 2012
Published: 20 September 2012
Tumor cell migration and invasion are critical initiation steps in the process of breast cancer metastasis, the primary cause of breast cancer morbidity and death. Here we investigated the role of p21Cip1 (p21), a member of the core cell cycle machinery, in transforming growth factor-beta (TGFβ)-mediated breast cancer cell migration and invasion.
A mammary fat pad xenograft mouse model was used to assess the mammary tumor growth and local invasion. The triple negative human breast cancer cell lines MDA-MB231 and its sub-progenies SCP2 and SCP25, SUM159PT, SUM149PT, SUM229PE and SUM1315MO2 were treated with 5 ng/ml TGFβ and the protein expression levels were measured by Western blot. Cell migration and invasion were examined using the scratch/wound healing and Transwell assay. TGFβ transcriptional activity was measured by a TGFβ/Smad reporter construct (CAGA12-luc) using luciferase assay. q-PCR was used for assessing TGFβ downstream target genes. The interactions among p21, p/CAF and Smad3 were performed by co-immunoprecipitation. In addition, Smad3 on DNA binding ability was measured by DNA immunoprecipitation using biotinylated Smad binding element DNA probes. Finally, the association among active TGFβ/Smad signaling, p21 and p/CAF with lymph node metastasis was examined by immunohistochemistry in tissue microarray containing 50 invasive ductal breast tumors, 25 of which are lymph node positive.
We found p21 expression to correlate with poor overall and distant metastasis free survival in breast cancer patients. Furthermore, using xenograft animal models and in vitro studies, we found p21 to be essential for tumor cell invasion. The invasive effects of p21 were found to correlate with Smad3, and p/CAF interaction downstream of TGFβ. p21 and p/CAF regulates TGFβ-mediated transcription of pro-metastatic genes by controlling Smad3 acetylation, DNA binding and transcriptional activity. In addition, we found that active TGFβ/Smad signaling correlates with high p21 and p/CAF expression levels and lymph node involvement using tissue microarrays from breast cancer patients.
Together these results highlight an important role for p21 and p/CAF in promoting breast cancer cell migration and invasion at the transcriptional level and may open new avenues for breast cancer therapy.
p21 was originally identified as a cell cycle regulator through inhibition of different cyclin/cyclin-dependent kinase complexes . p21 is a member of the Cip/Kip family of cell cycle inhibitors, which also includes p27Kip1 and p57Kip2 [2–4]. In addition to its role in cell cycle control, p21 is involved in the regulation of cellular senescence, gene transcription, apoptosis and actin cytoskeleton [5–7]. The role of p21 in breast cancer development and progression has not been fully investigated. While p21 is involved in cell cycle control and is a downstream target of the tumor suppressor p53, it does not fulfill the classic definition of a tumor suppressor. Germline or somatic mutations in the p21 gene are not common in human cancers . Furthermore, in vivo studies using p21 knockout mice showed that, while loss of p21 expression efficiently blocked the ability of the cells to undergo G1 arrest following DNA damage, these animals developed normally . Intriguingly, p21 is often overexpressed in aggressive tumors, including carcinomas of the pancreas, breast, prostate, ovary and cervix [10–13]. Together these observations suggest that the role played by p21 in cancer is more complex than initially thought and that, in addition to its well-known cell cycle regulatory effect, it may have uncharacterized roles in promoting carcinogenesis.
Tumor cell migration and invasion are critical steps in the metastatic process and are regulated by numerous tumor-secreted factors which modify the tumor microenvironment by acting on stromal recruitment and extracellular matrix (ECM) degradation, resulting in tumor cell migration and invasion . Among these tumor-secreted factors, TGFβ has been shown to play a pivotal role in promoting tumor metastasis . The TGFβ family regulates asymmetric cell division and cell fate determination during embryogenesis and exerts profound effects on reproductive functions, immune responses, cell growth, bone formation, tissue remodeling and repair throughout adult life . The effects of TGFβ in breast cancer are complex. TGFβ is thought to play a dual role in breast cancer progression, acting as a tumor suppressor in normal and early carcinoma, and as a pro-metastatic factor in aggressive carcinoma . The growth inhibitory effects of TGFβ are known to be mediated through transcriptional repression of the c-myc gene  and induction of the cell cycle inhibitors p15Ink4b (p15) and p21, leading to G1 arrest [19, 20]. During tumor progression, however, the loss of TGFβ growth-inhibitory effects is frequently due to defects in c-myc and p15 regulation by TGFβ . Meanwhile, other TGFβ responses prevail, unrelated to growth inhibition and favoring tumor progression and metastasis [21–25]. Indeed, TGFβ induces degradation of the ECM, inhibits cell adhesion and stimulates cell migration and invasion, thereby promoting tumor metastasis [21–23, 25]. Moreover, during cancer progression, tumor cells secrete increasing quantities of TGFβ, which in turn alter the stroma environment, leading to stimulation of tumor angiogenesis and causing local and systemic immunosuppression, thus further contributing to tumor progression and metastasis [21–23, 25]. Together these studies highlight an important role for TGFβ in advanced breast cancer. However, the function for p21 downstream of TGFβ has not been described in breast cancer.
In this study, we found that high p21 expression correlates with poor survival in breast cancer patients. The expression of p21 is required to promote tumor cell migration and invasion in vitro and local invasion in vivo. Furthermore, p21 expression is tightly regulated by TGFβ/Smad3 signaling in a panel of human basal-like triple negative breast cancer cell lines. We found p21 to physically interact with Smad3 and the histone acetyltransferase p/CAF in response to TGFβ and identified p21 and p/CAF as key regulators of TGFβ-mediated breast cancer cell migration and invasion. We also showed that p21 and p/CAF regulate TGFβ transcriptional activity on multiple tumor-promoting target genes by controlling Smad3 acetylation and Smad3 occupancy on its DNA binding elements. Immunohistochemical analysis of tissue arrays from breast cancer patients revealed a significant correlation between active TGFβ/Smad3 signaling and high expression levels of both p21 and p/CAF in lymph node-positive invasive ductal carcinomas. Together, our findings identified p21 and p/CAF as critical regulators of cell migration and invasion downstream of TGFβ/Smad3 pathway in advanced breast cancer.
Cell culture and transfection
Human breast carcinoma MDA-MB231, SCP2 and SCP25 cells (provided by Dr. Joan Massagué) and HEK293 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS) and 2 mM L-glutamine at 37°C in 5% CO2. SUM149PT, SUM159PT and SUM229PE (provided by Dr. Stephen P Ethier) were grown in F-12 HAM'S nutrient mixture (HyClone Laboratories, Inc. Logan, Utah, USA) supplemented with 5% FBS, 5 µg/ml insulin (Sigma-Aldrich, St. Louis, MO, USA), 1 µg/ml hydrocortisone (Sigma) at 37°C in 5% CO2. SUM1315MO2 were grown in F-12 HAM'S nutrient mixture (HyClone) supplemented with 5% FBS, 5 µg/ml insulin (Sigma), 10 ng/ml epidermal growth factor (EGF) (Sigma) at 37°C in 5% CO2.
Cells were transfected with different p21, p/CAF, Smad2 and Smad3 siRNAs (Sigma), 6× myc- Smad2, myc-Smad3, p/CAF (Addgene plasmid 8941)  and Flag-tagged human p21 cDNAs (Addgene plasmid 16240)  using Lipofectamine™ 2000 reagent (Invitrogen, Carlsbad, CA, USA ), according to the manufacturer's protocol. MDA and SCPs cells were serum-starved for 24 hrs and stimulated or not with 5 ng/ml TGFβ1 (PeproTech, Rocky Hill, NJ, USA) in DMEM supplemented with 2 mM L-glutamine. For stable cell line generation, SCP2 cells were transfected with p21 shRNA (Santa Cruz Biotechnology, Santa Cruz, CA, USA) and pools of stable cells were selected with 10 ng/ml puromycin (Invitrogen). SUM159PT cells were serum-starved for 24 hrs in the absence of insulin and hydrocortisone before TGFβ1 stimulation.
Western blot analysis and immunoprecipitation
Cells were lysed in cold extraction buffer (10 mM Tris-HCl, pH 7.5, 5 mM EDTA, 150 mM NaCl, 30 mM sodium pyrophosphate, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 1% Triton X-100) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin hydrochloride, 10 µg/ml aprotinin and 10 µg/ml pepstatin A). The lysates were then centrifuged at 14,000 rpm for 15 minutes at 4°C. Protein content was measured using BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). Equal protein was analyzed by Western blot using mouse anti-p21 (F5), mouse anti-c-myc, mouse anti-p15, rabbit anti-Smad2/3 (1:1,000 dilution, Santa Cruz Biotechnology), phospho-cofilin and cofilin antibodies (1:1,000 dilution, Millipore, Billerica, MA, USA), and followed by secondary antibodies goat anti-mouse or rabbit. Immunoprecipitations were performed overnight at 4°C using antibodies against p300/CBP (Santa Cruz Biotechnology), p/CAF (Abcam®, Cambridge, MA, USA) and p21. Protein G-Sepharose (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) was added for 1 hr at 4°C, and washed four times with cold lysis buffer. The immunocomplexes were boiled with 2× sodium dodecyl sulfate (SDS) Laemmli sample buffer for five minutes and subjected to immunoblotting.
Histone proteins extraction
Total histone proteins were extracted as previously described . Briefly, 80% confluent of SCP2 cells from a 100-mm tissue culture plate were serum-starved for 24 hrs and stimulated with or without 5 ng/ml TGFβ or 1 µM trichostatin A (TSA). SCP2 cells were harvested and resuspended in cold hypotonic lysis buffer containing 10 mM Tris-HCl, pH 8.0, 1 mM KCl, 1.5 mM MgCl2, 1 mM DTT, protease inhibitors, 1 µM TSA and 10 mM sodium butyrate. Cell lysates were rotated at 4°C for 30 minutes and then centrifuged at 10,000 g, 4°C, for 10 minutes. The supernatants were discarded and nuclei pellets were resuspended in 400 µl of 0.4 N H2SO4 and incubated overnight on a rotator at 4°C. Samples were centrifuged at 16,000 g for 10 minutes and supernatants containing histones were transferred into a fresh tube. A total of 132 µl trichloroacetic acid was added drop by drop to the histone solution, inverted several times and then incubated on ice for 30 minutes. The histone precipitates were centrifuged at 16,000 g for 10 minutes and pellets were washed twice with ice-cold acetone and the histone pellets were air dried for 20 minutes. Total histone proteins were subjected to Western blot analysis using an acetylated lysine antibody (Millipore).
DNA affinity precipitation assay
SCP2 cells transiently transfected with the indicated siRNAs were stimulated with TGFβ for 30 minutes. Cell lysate were extracted in cold lysis buffer containing 10 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 30 mM sodium pyrophosphate, 1 mM sodium orthovanadate and protease inhibitors as described above. A total of 5 µg Poly (dI-dC) competitor (Sigma) was incubated with 1 mg of total cell lysate for 30 minutes at 4°C. A total of 500 pmol of double-stranded oligonucleotides (IDT) was added and incubated with cell lysates for two hours at 4°C. Streptavidin-agarose beads (65 µl; Sigma) were added, incubated overnight at 4°C and then washed three times with cold lysis buffer. The streptavidin-agarose beads containing biotinylated oligonucleotides and protein complex were boiled with 2× SDS Laemmli sample buffer for five minutes and subjected to immunoblotting. The sequences of biotin labeled double-strand oligonucleotides (IDT) were previously described . For Smad binding element oligonucleotide (4× SBE), Sense: biotin-5'-CAGACAGTCAGACAGTCAGACAGTCAGACAGT-3', antisense: 5'-ACTGTCTGACTGTCTGACTGTCTGACTGTCTG-3'. For control oligonucleotide, sense: biotin-5'-GCCCAGGCGCACCTGCTCCGATATCAATATCCGGC-3', anti-sense, 5'- GCCGGATATTGATATCGGAGCAGGTGCGCCTGGGC-3'.
SCP2 cells were transiently co-transfected with 50 nM Scr siRNA, 50 nM p21 siRNA or 0.5 µg flag-tagged p21 cDNA in combination with 0.3 µg SBE reporter construct (CAGA12-luc) and 0.1 µg pCMV-β-gal. Transfected cells were then stimulated with or without 5 ng/ml TGFβ for 16 hrs. Luciferase activity of CAGA12-luc was measured (EG & G Berthold luminometer, Berthold Technologies, Bad Wildbad, Baden-Württemberg, Germany) and normalized to β-galactosidase activity.
Total RNA was extracted using TRIzol reagents (Invitrogen). Reverse transcription of total RNA using random primers was carried out using M-MLV reverse transcriptase (Invitrogen) as per the manufacturer's instructions. Real-time PCRs were carried out using SsoFast™EvaGreen® Supermix (Bio-Rad, Hercules, CA, USA) in a Rotor Gene 6000 PCR detection system (MBI Lab Equipment, Montreal Biotech Inc. Kirkland, PQ, Canada). PCR conditions were as follows: 95°C for 30 s, 40 cycles (95°C for 5 s and 60°C for 20 s). The primer sequences were as follows: IL8 forward primer, GCAGAGGCCACCTGGATTGTGC; reverse primer, TGGCATGTTGCAGGCTCCTCAGAA; IL6 forward primer, CTCCCCTCCAGGAGCCCAGC; reverse primer, GCAGGGAAGGCAGCAGGCAA; PLAU forward primer, GCCCTGGTTTGCGGCCATCT; reverse primer, CGCACACCTGCCCTCCTTGG; MMP9 forward primer, TGGACACGCACGACGTCTTCC; reverse primer, TAGGTCACGTAGCCCACTTGGTCC; PTGS2 forward primer, AGCTTTCACCAACGGGCTGGG; reverse primer, AAGACCTCCTGCCCCACAGCAA; TGFBI forward primer, CGGCTGCTGCTGAAAGCCGACCA; reverse primer, GGTCGGGGCCAAAAGCGTGT; p21 forward primer, TGTCCGCGAGGATGCGTGTTC; reverse primer, GCAGCCCGCCATTAGCGCAT; GAPDH forward primer, GCCTCAAGATCATCAGCAATGCCT; reverse primer, TGTGGTCATGAGTCCTTCCACGAT.
Thiazolyl blue tetrazolium bromide (MTT) assay
A total of 100 µl of cell suspension (1× 105 cells/ml in DMEM supplemented with 1% FBS) was stimulated or not in the presence or absence of 5 ng/ml TGFβ and cultured in 96-well plates for two days. After two days, 25 µl 5 mg/ml MTT solution (Sigma) was added to each well and incubated for two hours. A total of 200 µl of dimethyl sulfoxide (DMSO) was added to each well and mixed well. The absorbance at 570 nm was measured on a plate reader.
Cell cycle analysis
SCP2 cells were stimulated with TGFβ for 0, 2, 6 and 24 hrs. Cells were then fixed with 70% ethanol overnight, treated with 20 µg/ml RNase (Sigma), and stained with 0.5 mg/ml propidium iodide (Santa Cruz Biotechnology). DNA content was determined using a FACScan flow cytometry analyzer.
Kinetic cell migration assay
Cells were transfected with different siRNAs and plated in Essen ImageLock 96-well plates (Essen Bioscience, Ann Arbor, Michigan, USA) at 50,000 cells per well. The use of ImageLock 96-well plates ensures that images/videos of the wound are automatically taken at the exact same location by the IncuCyte™software (Essen Bioscience). Cells were then serum-starved for six hours and confluent cell layers were scratched using the Essen Wound maker to generate approximately 800 µm width wounds. After wounding, cells were washed two times with PBS and stimulated in the presence or the absence of 5 ng/ml of TGFβ. ImageLock 96-well plates were then placed into IncuCyte (Essen Bioscience) and imaged every hour for 24 hrs. The data were analyzed by three integrated metrics: wound width, wound confluence or relative wound density automatically measured by the IncuCyte software.
Matrigel invasion assay
For the Transwell assays, 30 µl of growth factor reduced (GFR) Matrigel (BD Biosciences, diluted 1:3 in pre-chilled H2O) was coated onto each insert of 24-Tranwell invasion plate (8-µm pore size; BD Biosciences) and incubated for two hours in the cell culture incubator. SCP2 or SUM159PT (6 × 104 cells/insert) were seeded on Transwell Insert coated GFR-Matrigel and cells in the upper chamber were stimulated or not with 5 ng/ml TGFβ for 24 hrs. For SCP2 cells, bottom chambers contained 10% FBS in DMEM medium. For SUM159PT cells, bottom chambers were added to F-12 HAM'S medium with 5% FBS. After 24 hrs, cells from the upper chamber were removed by cotton swab and cells invaded through GFR-Matrigel were fixed with 3.7% formaldehyde for 10 minutes and then stained with 0.2% crystal violet for 20 minutes. Images of the invading cells were photographed using an inverted 4× or 10× microscope and total cell numbers were counted and quantified by Image J software (National Institute of Health, Bethesda, Maryland, USA).
Cells were grown on coverslips at 50% confluence, stimulated or not with TGFβ overnight. Cells were then fixed with 3.7% formaldehyde for 10 minutes and permeabilized in 0.1% Triton X-100 for 3 minutes, washed with PBS and blocked for 1 hr in 2% BSA. Cells were then incubated with anti-p21 antibody for one hour, washed with PBS and incubated with the secondary antibody Alexa Fluor®568 goat anti-rabbit IgG (1:800 dilution; Invitrogen) for one hour. Stained coverslips were mounted with SlowFade® Gold antifade reagent with DAPI (Invitrogen). Confocal analysis was performed using a Zeiss LSM 510 Meta Axiovert confocal microscope (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany) using 63× objective.
Immunohistochemistry, scoring and statistical analysis
Tissue sections (5 µm) from breast carcinoma microarray slides (BCR961 and T088, Biomax) were deparaffinized and rehydrated. The patient characteristics are in Table S1 (Additional file 1). The slides were then placed in 10 mM citrate buffer (pH 6.0) and boiled at 95°C for 15 minutes. The primary antibodies used for immunohistochemistry staining were AE1/AE3 (Thermo Scientific), p21 (c-19, Santa Cruz Biotechnology), p/CAF (ab12188, Abcam), phospho-Smad3 (Cell Signaling). HRP Polymer & DAB Plus Chromogen (Thermo Scientific) was used for detection of p21, p/CAF and phospho-Smad3. The slides were then counter stained with hematoxylin (Vector Laboratories, Burlingame, CA, USA) and dehydrated and mounted for microscopic examination. All images were scanned by ScanScope digital scanners (Aperio, Vista, CA, USA). All samples were reviewed and scored by a pathologist. The staining for p21, p/CAF and phospho-Smad3 was scored from 0 to 4 as follows: 0, no staining; 1, <25% tumor cells stained weakly; 2, 25 to 50% tumor cells stained moderately; 3, >50% tumor cells stained moderately; 4, >50% tumor cells stained strongly.
Correlations between phospho-Smad3, p/CAF and p21 were examined by the Pearson correlation test using SPSS 19 software (IBM, Armonk, NY, USA). Associations between these protein expressions and lymph node status were assessed by Fisher's exact test. P-value (two-sided) <0.05 was considered statistically significant.
Mammary fat pad and intratibia injections of nude mice
Four- to six-week old female Balb/c nude mice were obtained from Charles River (Charles River Laboratories International, Wilmington, MA, USA) and used as a model for primary mammary tumor formation and local invasion. The animal study was approved by the ethics committee and all the experimental animal protocols were in accordance with the McGill University Animal Care. Following the administration of an anesthetic cocktail of ketamine (50 mg/kg), xylazine (5 mg/kg) and acepromazine (1 mg/kg) injected intramuscularly into the mice, parental and shRNA p21 SCP2 cells were inoculated at 5× 105 cells per mouse in 100 μl of saline (20% Matrigel) with a 30-gauge needle into the mammary pad. The tumor size was measured once a week using a caliper. Tumor volume was determined according to the formula: tumor volume = shorter diameter2 × longer diameter/2. Sets of mice (eight per group) were sacrificed at eight weeks post-injection to examine invasiveness of the primary tumor. At the end of these studies, mammary tumors with surrounding fat pad and tissues were fixed in 10% neutral-buffered formalin for one day. Sections of mammary tumor were embedded in Tissue-Tek O.C.T. (VWR International, Radnor, PA, USA) compound and 9 µm thick sections were stained with hematoxylin and eosin. Images of the tumors were photographed by light microscope using 10× and 20× objectives.
For intratibia injections, parental and shRNA p21 SCP2 cells (2.0× 106) were injected intramuscularly into the left tibia of two group mice (eight per group). The mice were monitored weekly for tumor burden. Digital radiography of the hind limbs of all animals was used to monitor the development of skeletal lesions at four, six and eight weeks post-injection in a MX-20 cabinet X-ray system (Faxitron Bioptics, Tucson, Arizona, USA). On Week 8, radiographs of anesthetized mice were taken and the osteolytic lesion area was analyzed as previously described . The score of lesion area was measured as 0, no lesions; 1, minor lesions; 2, small lesions; 3, significant lesions with minor break of margins (1% to 10% of bone surface damaged); 4, significant lesions with major break in peripheral lesions (>10% of bone surface damaged).
Student's t-test was used and differences between groups were considered significant at *P < 0.05.
p21 expression correlates with poor survival in breast cancer patients
Silencing p21 prevents breast tumor local invasion in vivo and cancer cell migration and invasion in vitro
As shown in Figure S2A (Additional file 3), none of the animals in which parental or p21-depleted SCP2 cells (eight per group) were injected into the mammary fat pad developed any bone lesions after two months, the date at which mice had to be sacrificed due to the tumor size. This timing may have been insufficient for tumor cells to grow into visible distant lesions in the mouse . Thus, to investigate whether p21 is involved in the later stage of breast cancer progression, we examined its involvement in the development of bone osteolytic lesions using an intratibia injection model of parental and p21-deficient SCP2 cells in female Balb/c nude mice. By by-passing the early steps of metastasis, this experimental model allows for the assessment of tumor cell metastasis and survival in the bone marrow . As shown in Figure S2B, C (Additional file 3), following X-ray examination of the bones, both groups of mice developed secondary tumors that caused severe osteolytic bone lesions, suggesting that p21 does not affect the later stages of bone metastasis. Collectively, these results indicate that while p21 is required for breast cancer cells to acquire an invasive phenotype, its effect is restricted to the earlier stages of tumor metastasis, namely induction of local cell invasion from the tumor to the surrounding tissues.
TGFβ induces p21 expression in migratory and invasive human breast cancer cells
p21 expression is required for TGFβ-mediated cell migration
To then investigate whether p21 is required for TGFβ-induced cell migration, we knocked down p21 expression using two specific siRNAs in SCP2 cells and assessed the effect of TGFβ on cell migration dynamics by the scratch/wound healing assay. As shown in Figure 4C, TGFβ induced p21 expression in both mock and scrambled (Scr) siRNA transfected cells, while this effect was blocked in cells transfected with either p21 siRNAs, confirming the specificity and efficacy of our p21 siRNAs. Importantly, we found that while TGFβ potently induced cell migration in mock and Scr siRNA transfected SCP2 cells, this effect was completely blocked in cells in which p21 expression was depleted (Figure 4D, E). The effect of p21 siRNAs on TGFβ-induced cell migration was similar to that observed when cells were transfected with a siRNA against Smad3, used here as a positive control (Figure 4D, E). We also confirmed that these effects on cell migration were not secondary to changes in cell growth, as silencing of p21 expression had no effect on cell growth and proliferation (Figure S4C). These results demonstrate that TGFβ-mediated migration of human breast cancer cells is dependent on TGFβ-induced p21 expression.
p21 expression is required for TGFβ-mediated cell invasion
p21 interacts with Smad3 and modulates TGFβ-induced transcriptional activity and downstream genes involved in cell invasion
We next performed gene profiling experiments in parental and p21-deficient SCP2 cells, using transiently transfected p21 siRNA as well as stably transfected p21 shRNA. Our arbitrary cutoff was set up at a minimum of two-fold induction. This led us to identify multiple p21-dependent TGFβ target genes, among which were selected those known to be associated with the tumor metastasis process. This shortlist included five candidate target genes: interleukin 6 (IL6), chemokine (IL8), prostaglandin-endoperoxide synthase 2 (PTGS2), plasminogen activator (PLAU) and matrix metalloproteinase (MMP9) . To confirm that these genes were TGFβ downstream targets, SCP2 and SUM159 cells were stimulated or not with TGFβ and mRNA levels for these target genes were analyzed by quantitative real-time PCR (q-PCR). As shown in Figure 6D, E, TGFβ significantly increased the mRNA levels of IL6, IL8, PTGS2, PLAU and MMP9 in a time-dependent manner in both cell lines.
To address the contribution of these identified p21-dependent TGFβ target genes (IL6, IL8, PLAU, MMP9 and PTGS2) in regulating cell invasion, we silenced their gene expression using specific siRNAs. As shown in Figure 7C, D, inhibition of all five target genes impaired TGFβ-induced cell invasion, to a different extent. While depletion of IL6, PLAU and MMP9 drastically antagonized the TGFβ response, inhibition of PTGS2 and IL8 showed a moderate inhibitory effect. Moreover, examination of the siRNA effect on basal cell invasion indicated that IL6 and PLAU did not affect basal invasion, suggesting that they may be specifically required for the TGFβ pro-invasive response. On the other hand, inhibition of MMP9, PTGS2 and IL8 clearly affected basal cell invasion suggesting that these target genes have a broader effect on cell invasion, not limited to the TGFβ signaling pathway. Together, these results indicate that even though all five genes are important for TGFβ signaling leading to cell invasion, IL6, PLAU and MMP9 exert more predominant roles.
p21/p/CAF regulates TGFβ transcriptional activity and Smad3 DNA binding
A previous report indicated that p/CAF directly binds to Smad3 . As we have shown that TGFβ induces complex formation between Smad3 and p21 (Figure 6A), we investigated whether endogenous p/CAF is also required for Smad3 association with p21. For this, HEK293 cells were co-transfected with myc-Smad2, myc-Smad3 and flag-p21 with or without p/CAF siRNA to block expression of endogenous p/CAF. As shown in Figure 8B, TGFβ induced complex formation between p21 and Smad3, independently of Smad2. Interestingly, depletion of p/CAF completely prevented this interaction, indicating that endogenous p/CAF is required for Smad3 interaction with p21.
To investigate whether p/CAF is necessary for the regulation of p21-dependent TGFβ downstream target genes, SUM159 cells were transiently transfected with flag-tagged p21 in the presence or the absence of two different p/CAF siRNAs. The gene expression of p/CAF (also known as K(lysine) acetyltransferase 2B, KAT2B) was measured to verify the efficiency of p/CAF knockdown by q-PCR (Figure 8C). Overexpression of p21 potentiated induction of IL6, IL8 and PTGS2 mRNA by TGFβ. However, these effects were significantly blocked when p/CAF gene expression was silenced, indicating that p/CAF is required for p21-dependent gene expression of the TGFβ targets (Figure 8D). The requirement of p/CAF downstream of TGFβ was further investigated using the Transwell Matrigel assay. As shown in Figure 8E, knocking down p/CAF gene expression significantly impaired TGFβ-induced cell invasion. Efficiency of the siRNA was verified by Western blotting (Figure 8F).
Because acetyltransferase p/CAF regulates gene transcription by acetylating histones and transcription factors , we then assessed whether TGFβ could induce global changes in histone acetylation in breast cancer cells. For this, total histone proteins were extracted from SCP2 cells, treated or not with TGFβ and subjected to immunoblotting using an acetylated lysine antibody. As shown in Figure S8 (Additional file 9), TGFβ had no effect on global histone acetylation while TSA, a histone deacetylase inhibitor, showed a marked increase in the acetylation levels. This suggested that the functional relevance of the p/CAF recruitment to the p21/Smad complex may be more directed towards acetylation of specific targets rather than global histone modifications. To address this, we examined whether p/CAF could acetylate p21 and/or the Smads. Interestingly, we found that p/CAF is capable of interacting with Smad2 and Smad3, leading to an increased acetylation of both Smad proteins (Figure 8G). Moreover, the acetylation is specific to Smad2 and Smad3, as p21 did not show any increased acetylation by p/CAF (data not shown). Smad3 acetylation has been suggested to be required for its DNA binding activity . Thus, this led us to investigate whether p/CAF could associate with DNA-bound Smad3, by DNA immunoprecipitation (DNA IP) using biotinylated control and biotinylated Smad binding element (4× CAGA) DNA probes. As shown in the Figure 8H, we found TGFβ to specifically induce binding of both Smad3 and p/CAF to DNA. Furthermore, we found that gene silencing of p/CAF and p21, using siRNAs, prevented Smad3 binding to the SBE (Figure 8I), suggesting that both p21 and p/CAF are required for Smad3 DNA binding and Smad3-mediated transcriptional activity. Having shown that p21 is indeed required for Smad3-mediated transcriptional activity (Figure 6C), we then assessed the effect of knocking down p/CAF on Smad3 transcriptional activity using the CAGA12-luc reporter construct. As shown in Figure 8J, the results clearly indicate that p/CAF is required for TGFβ-induced Smad3 transcriptional activity.
Collectively, these data indicate that p21 and p/CAF regulate TGFβ transcriptional activity by controlling Smad3 occupancy on its DNA binding elements. TGFβ induces a complex formation between Smad3, p21 and p/CAF, further leading to Smad3 acetylation by p/CAF. Furthermore, both p21 and p/CAF are required for Smad3 DNA binding and Smad3-mediated transcriptional activity, highlighting a novel mechanism by which the p21/p/CAF/Smad3 complex contribute to the activation of TGFβ target gene transcription.
High expression of p/CAF/p21/p-Smad3 is associated with lymph node positivity
While p21 was initially characterized as an important cell cycle inhibitor, recent studies suggest that cytoplasmic p21 has anti-apoptotic and actin cytoskeleton regulatory functions [27, 43, 55]. The accumulation of cytoplasmic p21 is associated with Ras and HER2/neu activated tumorigenic transformation [32, 44]. Moreover, overexpression of p21 is associated with poor prognosis of many types of cancer. However, the function of p21 in breast cancer has not been established. In our study, we assessed p21 levels with clinical outcomes in breast cancer patients. High p21 expression correlates with poor overall survival and distant metastasis free survival. Furthermore, using an in vivo model of mammary fat pad transplantation of metastatic human breast cancer cells in mice, we showed that while silencing p21 gene expression did not affect the primary tumor formation, it potently prevented primary tumor cells to invade into surrounding tissues. Together, our results provide evidence of a tumor-promoting role for p21 in primary tumor local invasion.
Previous studies have indicated that during breast cancer progression, TGFβ cytostatic responses are lost while pro-migratory and pro-invasive effects are maintained . Here, we found that all invasive breast cancer cell lines tested were resistant to growth inhibition by TGFβ and that while TGFβ did not induce any change in p15 or c-myc expression levels, it strongly up-regulated p21 expression arguing that in advanced breast cancer p21 functions independently of cell cycle regulation. This is in contrast to the effect observed in human immortalized the keratinocyte cell line HaCaT, where TGFβ-mediated p21 gene expression leads to cell cycle arrest . Indeed, we found that the induction of p21 in invasive breast cancer cells is required for the pro-migratory and pro-invasive effects of TGFβ. In accordance with these results, depletion of p21 did not modulate primary tumor growth in vivo but strongly blocked tumor invasion capacity. These findings together support the notion of a direct oncogenic role for p21 in breast cancer progression.
We further report that the TGFβ-mediated increase in p21 expression is Smad-dependent and Smad3 specific. This is interesting in light of previous reports indicating that overexpression of a dominant negative form of Smad3 reduced the ability of cancer cells to metastasize  and that Smad3, but not Smad2, promotes breast cancer metastasis in mice . Furthermore, while Smad2 mutations in cancer have been described, no mutations in Smad3 or p21 have yet been reported. Together these data suggest that in breast cancer Smad3 pro-invasive functions are mediated by p21 and that targeting p21 may prove useful to improve the clinical course of metastatic patients.
Finally, we investigated the clinical relevance of TGFβ-mediated p21/p/CAF pathway in breast cancer. The prognosis of breast carcinomas is related to various clinical and pathological parameters. Axillary lymph node metastasis is one of the most important prognostic parameters in the absence of distant metastasis. There is a sharp difference in survival rate between patients with positive and negative lymph nodes. In our studies, we found a significant association of active TGFβ/Smad3 signaling, p21 and p/CAF expression with lymph node positivity, making them potential useful prognosis markers for lymph node metastasis.
In this study, we described a pro-invasive function for the cell cycle regulator p21 in human breast cancer. High expression of p21 positively correlated with poor overall and distant metastasis-free survival outcomes in breast cancer patients. We identified p21 as a novel downstream regulator of TGFβ-mediated breast cancer cell migration and invasion. We found p21 to interact with Smad3 and the acetyltransferase p/CAF and to regulate the Smad transcriptional activity, as well as gene transcription of several TGFβ-induced pro-metastatic genes. These results highlight an important role for p21/p/CAF in TGFβ-induced breast cancer cell migration and invasion at the transcriptional level.
bovine serum albumin
Dulbecco's modified Eagle's medium
distant metastasis free survival
epidermal growth factor
fetal bovine serum
growth factor reduced
matrix metalloproteinase 9
thiazolyl blue tetrazolium bromide
prostaglandin-endoperoxide synthase 2
sodium dodecyl sulphate
TGFβ type I receptor
transforming growth factor-beta
This work was supported by a Canadian Institutes for Health Research (CIHR) grant (fund code 230670 to JJL). Ms. M. Dai is supported by a Fonds de la recherche en santé du Québec (FRSQ) Scholarship Award. JJ Lebrun is the recipient of a McGill Sir William Dawson Research Chair.
We thank Dr. Joan Massagué for kindly providing us the MDA, SCP2 and SCP25 cell lines and Dr. Stephen P Ethier for kindly providing us the SUM1315MO2, SUM149PT, SUM229PE and SUM159PT cell lines. We also thank Ms Juliana Korah for critical reading of the manuscript.
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