Pituitary tumor-transforming gene (PTTG), the index mammalian securin, is abundantly expressed in several tumors and regulates tumor growth and progression. Molecular mechanisms elucidating PTTG regulation and actions remain elusive. Here, we provide evidence that PTTG acts as a signal transducer and activator of transcription factor 3 (STAT3) target gene. Total STAT3 and Tyr705 phosphorylated STAT3 were concordantly expressed with PTTG in human colorectal tumors (n=97 and n=95, respectively, P<0.001). STAT3 specifically bound the human PTTG promoter and induced PTTG transcriptional activity (twofold) as assessed by chromatin immunoprecipitation and luciferase reporter assays. STAT3 transfection increased PTTG mRNA and protein abundance twofold in HCT116 human colon cancer cells, and induction was further enhanced (threefold) by constitutively active STAT3 (STAT3-C), whereas strongly abrogated by dominant-negative STAT3 (STAT3-DN). Attenuating PTTG expression by siRNA in STAT3 HCT116 stable transfectants suppressed cell growth and colony formation in vitro, and PTTG cell knockout also constrained activated STAT3-induced explanted murine tumor growth in vivo. STAT3 increased HCT116 cell migration and invasion up to fivefold, whereas cell mobility was abolished by STAT3-DN (>85%). Impairing PTTG expression by siRNA also strongly suppressed STAT3-faciliated cell migration and invasion by up to 90%. Knocking out PTTG in STAT3-C HCT116 stable transfectants strongly decreased tumor metastases in nude mice, indicating the requirement of PTTG for STAT3-promoted metastasis. These results elucidate a mechanism for tumor cell PTTG regulation, whereby STAT3 induces PTTG expression to facilitate tumor growth and metastasis, and further support the rationale for targeting PTTG to abrogate colorectal cancer growth.
Pituitary tumor-transforming gene (PTTG) and signal transducer and activator of transcription factor 3 (STAT3) are both involved in tumor transformation and metastatic progression.1, 2 PTTG, the vertebrate securin,3 mediates sister chromatid separation during mitosis.4 PTTG also facilitates cell-cycle progression, maintains chromosomal stability,5 responds to DNA damage6, 7 and mediates cell transformation in vitro and tumor formation in vivo.4, 8 PTTG is highly expressed in several human tumors, including colorectal,9 pituitary,10, 11 thyroid,12 breast13 and esophageal cancers.14 PTTG overexpression correlates with tumor invasiveness, differentiation, recurrence and prognosis,15, 16 and PTTG has been identified as a key signature gene associated with tumor metastases.17 PTTG abundance promotes lymph node metastases in esophageal carcinomas through modulating metastasis-related factors.18 PTTG exerts oncogenic functions by disrupting genetic stability, altering oncoprotein expression and regulating growth factors. Overexpressed PTTG results in abnormal mitosis and chromosomal instability.19 PTTG activates c-Myc20 and cyclin D3 (CCND3)21 to facilitate cell proliferation and also increases basic fibroblast growth factor and vascular endothelial growth factor expression to induce angiogenesis,22 and induces interleukin (IL)-8 to function in metastasis.23 Factors inducing PTTG expression include estrogen, insulin, basic fibroblast growth factor and epidermal growth factor.2 PTTG is also regulated by beta-catenin/transcription factor14 and Rb/E2F1 pathways,24 which both have important roles in tumorigenesis. Nevertheless, proximal regulatory mechanisms enabling tumor PTTG abundance remain elusive. In this study, we present evidence supporting PTTG as a STAT3 target.
STAT3 acting as a transcription factor, responds to cytokines, growth factors and hormones by Janus kinase phosphorylation, followed by dimerization and nuclear translocation, to regulate target gene transcription.25 STAT3 regulates cell proliferation, survival and differentiation and is also involved in tumorigenesis, angiogenesis, metastasis and tumor-promoting inflammation.26 Constitutive STAT3 activation results in NIH3T3 cell transformation and tumor formation in nude mice.27 Overexpression and/or constitutive tumor STAT3 activation are associated with poor prognosis and occur in a wide variety of cancers, including colorectal,28 leukemia, myelomas, melanomas, breast, prostate, pancreatic, ovarian and head and neck cancers.1 Inhibition of constitutive STAT3 activation, by inhibiting tyrosine kinase signaling, is associated with cell growth suppression and induction of cell death.29, 30, 31 Several genes involved in tumorigenesis have been identified as STAT3 targets, including c-Myc,32 cyclin D1,33 vascular endothelial growth factor34 and matrix metalloproteinase-2 (MMP-2).35
In this study, we present evidence that PTTG, behaving as a STAT3 target gene, acts to mediate STAT3-induced cell transformation and motility. Concordant colorectal cancer STAT3 and PTTG overexpression is accompanied by STAT3 binding to the PTTG promoter and induction of PTTG. Attenuating PTTG inhibited colon cancer cell growth and colony formation in vitro, and constrained STAT3-induced tumor growth in vivo. Furthermore, PTTG inhibition by siRNA attenuated STAT3-promoted cell motility in vitro and strongly decreased STAT3-facilitated tumor metastasis in vivo. These results elucidate a mechanism for PTTG regulation, whereby STAT3 induces PTTG to promote cell transformation and motility, supporting the PTTG role in tumorigenesis and providing a rationale for PTTG targeting to abrogate colorectal cancer growth.
Total STAT3 and Tyr705 phosphorylated STAT3 are concordantly expressed with PTTG
As both STAT3 and PTTG are upregulated in several cancers, we assessed confocal immunofluorescence patterns of STAT3 and PTTG in 97 colorectal tumors and 9 normal tissues. Normal tissue STAT3 and PTTG immunoreactivity was weak or undetectable, whereas STAT3 and PTTG expression was abundant in tumor specimens (Figure 1A). Upregulated immunoreactive STAT3 and PTTG were detected in 82% (80 of 97) and 74% (72 of 97) of colorectal tumors, respectively (Table 1). Sixty-six tumors with strong STAT3 expression were also immunoreactive for PTTG, and 11 cases with unchanged STAT3 levels did not exhibit increased PTTG immunoreactivity. Taken together, 79% of tumor samples exhibited a concordant pattern of STAT3 and PTTG expression (P<0.001, Pearson’s χ2-test).
As STAT3 is activated by phosphorylation at Tyr705, we co-stained Tyr705 phospho-STAT3 and PTTG in 95 colorectal tumors and 9 normal tissues. Discrete nuclear phospho-STAT3 and PTTG staining was detected in a small percentage of normal cells; by contrast, most tumor tissues strongly stained with a high percentage of positive cells (Figure 1B). Upregulated phospho-STAT3 and PTTG expression were observed in 64% and 73% of tumor tissues, respectively. Remarkably, 71 of 95 tumors (77%), including 53 cases showing both overexpressed (56%) and 18 cases showing both unchanged (19%), exhibited concordant expression of phospho-STAT3 and PTTG (P<0.001, Pearson’s χ2-test) (Table 2).
IL-6 induces STAT3 phospharylation and PTTG expression, while STAT3 inhibitor attenuates STAT3 phosphorylation and PTTG expression
As STAT3 is activated by IL-6, we serum-starved HCT116 cells and subsequently treated with IL-6 for 16 h. IL-6 dose-dependently (0–50 ng/ml) increased STAT3 Try705 phosphorylation and PTTG expression as measured by western blotting. Total STAT3 expression did not change as obviously as did phospho-STAT3 (Figure 2a). IL-6 also increased PTTG mRNA expression as assessed by real-time PCR (Figure 2a). Moreover, a specific STAT3 inhibitor S3I-201 inhibited STAT3 phosphorylation and also attenuated PTTG expression in HCT116 (Figure 2b) and SW620 (Figure 2c) colon tumor cells. When cells were serum-starved and subsequently treated with IL-6 and S3I-201 for 16 h, IL-6 induction of phospho-STAT3 and PTTG were dose-dependently attenuated (Figure 2d).
STAT3 binds human PTTG promoter and activates PTTG transcription
We screened the human PTTG promoter with Genomatix MatInspector and detected several STAT-binding sites (Figure 3a). Accordingly, we performed chromatin immunoprecipitation (ChIP) to identify STAT3 binding to the PTTG promoter. Equal amounts of sonicated HCT116 chromatin DNA were incubated with immunoglobulin G control or STAT3 antibody, respectively. Protein G bead-captured chromatin DNA was amplified as template, and five pairs of PTTG promoter primers used for real-time PCR (Figure 3a), with primer 1 being closest to the ATG translation initiation site and primer 5 furthest.36 Human c-Fos promoter primers were used as positive controls and α-satellite repeat primers as negative controls. Anti-STAT3-immunoprecipitated DNA with the enriched STAT locus was strongly amplified by primers 4 and 5, indicating specific STAT3 binding to the PTTG promoter around these two primer regions (Figure 3b). Similar results were obtained by using Tyr705 phospho-STAT3 antibody (not shown).
To ascertain whether STAT3 regulates PTTG transcription, we used two different PTTG promoter plasmids, −2642/−1 and −1717/−1 cloned in pGL3-Basic vector,36 to measure promoter activity by a dual luciferase reporter assay in response to STAT3. The −2642/−1 construct contains multiple STAT-binding motifs as shown in Figure 3a, whereas the shorter −1717/−1 construct is devoid of the STAT-binding sites that exhibited strong binding affinity in ChIP assays. We also cloned two different STAT3 expression plasmids: wild-type STAT3 and constitutively active STAT3 (STAT3-C)27 in pIRES2-ZsGreen1 vector. Figure 3c shows that STAT3 induced PTTG promoter transcriptional activity. Co-transfection of the PTTG promoter (−2642/−1) with STAT3 or STAT3-C plasmid resulted in ∼twofold induction of promoter activity compared with empty vector pIRES2-ZsGreen1. The shorter PTTG promoter (−1717/−1), which lacks STAT-binding sites close to ChIP primers 4 and 5 (Figure 3a), exhibited modest responses to STAT3 and STAT3-C (Figure 3c). This result implies that STAT3-binding motifs located between −2642 and −1717 bp are required for STAT3-induced PTTG transcriptional activity.
Furthermore, we treated PTTG promoter-transfected HCT116 cells with STAT3 inhibitor S3I-201 and measured luciferase activity. As shown in Figure 3d, transcriptional activity of two PTTG promoters (−2642/−1 and −1717/−1) were both suppressed (up to 66% at 100 μM) by S3I-201, indicating that STAT3 is required in PTTG transactivation. To ensure specificity, we performed the same experiment using another STAT3 inhibitor, Stattic, with similar results (data not shown).
STAT3 induces PTTG mRNA and protein expression
As STAT3 binds and activates the PTTG promoter, we tested STAT3 regulation of endogenous PTTG expression in HCT116 stable transfectants. We cloned a dominant-negative STAT3 (STAT3-DN) in pIRES2-ZsGreen1 vector, by replacing tyrosine 705 to phenylalanine. We, respectively, transfected empty vector pIRES2-ZsGreen1, STAT3, STAT3-C and STAT3-DN in HCT116 cells and selected stable transfectants, all of which expressed green fluorescent protein ZsGreen, and sorted before experiments to ensure expression. PTTG mRNA expression was measured by real-time PCR using β-actin as an internal control. As shown in Figure 3e, overexpressed STAT3 doubled PTTG mRNA levels. This induction was further enhanced by STAT3-C (∼2.5-fold) and was specifically attenuated by STAT3-DN. Cell lysates analyzed by western blotting verified that PTTG protein was altered similarly to mRNA (Figure 3e). Upregulated STAT3 induced PTTG protein expression 1.8-fold and was further enhanced by STAT3-C to threefold. Transfection of STAT3-DN abolished STAT3-induced PTTG protein expression.
Attenuating PTTG expression decreases STAT3 stable cell growth and transformation
As PTTG promotes cell proliferation and mediates cell transformation, we examined whether inhibiting PTTG would constrain STAT3-facilitated cell growth and transformation. We transfected PTTG siRNA and negative controls in STAT3-overexpressing HCT116 stable cells, and measured cell proliferation using WST-1 cell proliferation reagent. Compared with negative siRNA controls, PTTG siRNA transfected cells exhibited ∼30% reduction of WST-1 absorbance after 72 h (Figure 4a), demonstrating that attenuating PTTG expression constrains stable HCT116 transfectant growth.
We then performed soft agar colony-formation assays to determine PTTG involvement in STAT3-promoted in vitro transformation. Compared with pIRES2-ZsGreen1 controls, STAT3 overexpression enhanced colony-forming ability of HCT116 stable transfectants (1.6-fold), whereas STAT3-DN overexpression did not exhibit a difference (Figure 4b). Furthermore, suppressing PTTG in STAT3 stable cells by siRNA strongly abrogated STAT3-facilitated colony formation (∼50%; Figure 4b). A similar result was shown in STAT3-C stably expressed HCT116 cells (∼38% reduction), further supporting the role of PTTG in STAT3-facilitated anchorage-independent HCT116 colony formation.
PTTG knock out in STAT3-C stable cells suppresses xenografted tumor growth
To assess whether blocking PTTG expression constrains tumor growth in vivo, we subcutaneously injected STAT3-C overexpressed HCT116 PTTG+/+ or HCT116 PTTG−/− stable cells in nude mice and examined xenografted tumor growth. Compared with STAT3-C-overexpressed PTTG+/+ controls, knocking out PTTG in STAT3-C stable cells strongly suppressed tumor growth. As shown in Figure 4c, tumor volumes were higher in STAT3-C HCT116 PTTG+/+ than STAT3-C HCT116 PTTG−/−, starting from day 9 (218±17.0 vs 92±8.4 mm3, P<0.001). On day 12, the average volume of STAT3-C PTTG+/+ tumor was 366±49.3 vs 122±12.2 mm3 for STAT3-C PTTG−/− tumors (67% inhibition, P<0.001). On day 16, average STAT3-C PTTG+/+ tumor volume was 718±89.1 vs 204±29.4 mm3 for STAT3-C PTTG−/− tumors (82% reduction, P<0.001). Seventeen days after tumor cell implantation, mice were killed and tumors harvested. The average weight of STAT3-C PTTG+/+ tumors was 484±67.8 mg, while STAT3-C PTTG−/− tumors weighed 89±14.2 mg (82% reduction, P<0.001). These results indicate that attenuating PTTG expression inhibits tumor growth in vivo.
Impairing PTTG expression in STAT3-C stable tranfectants suppresses CCND3 and KRAS expression
We further studied mechanisms underlying PTTG involvement in STAT3-induced tumor development. By using RT2 Profiler PCR arrays (SABiosciences), we examined alterations of gene expression affected by STAT3–PTTG pathway (data not shown) and further focused on several candidates, including CCND3, KRAS and CDKN1A (cyclin-dependent kinase inhibitor 1A). Induced STAT3 or STAT3-C expression in HCT116 cells elevated Cyclin D3 and KRAS expression, as measured by western blotting (Figure 5a). By contrast, STAT3-DN overexpression attenuated inductions of Cyclin D3 and KRAS. STAT3 bands were visualized to verify stable exogenous gene expression. Furthermore, we impaired PTTG expression by siRNA in STAT3-C stable cells and showed Cyclin D3 and KRAS attenuation. Western blot of PTTG confirmed PTTG siRNA suppression (Figure 5b). In addition, attenuating PTTG in STAT3-C stable transfectants elevated expression of p21, a cyclin-dependent kinase inhibitor (Figure 5b). We also measured these gene expressions by real-time PCR, and similar results were obtained. As shown in Figure 5c, inhibiting PTTG expression in STAT3-C stable cells decreased mRNA levels of CCND3 and KRAS, by up to 90%. By contrast, expression of CDKN1A was moderately elevated after PTTG siRNA (∼1.5-fold) (Figure 5c). These results indicate that attenuating PTTG constrained the expression of pro-proliferative factors CCND3 and KRAS.
PTTG is involved in STAT3-facilitated cell motility by regulating MMPs expression
Using pIRES2-ZsGreen1 HCT116 stable transfectants as controls, we used STAT3, STAT3-C, STAT3-DN HCT116 stable transfectants to measure in vitro cell migration and invasion. Elevated STAT3 expression or activation increased cell migration and invasion abilities (Figures 6a and b). Overexpression of wild-type STAT3 increased cell migration 2.6-fold and enhanced invasion twofold (Figures 6a and b). Both migration and invasion were further enhanced by activated-STAT3 (STAT3-C) up to fivefold compared with pIRES2-ZsGreen1 controls, whereas cell mobility was strongly abrogated by STAT3-DN (>88%; Figures 6a and b). Moreover, we transfected PTTG siRNA and negative controls to STAT3 or STAT3-C stable transfectants, respectively. Attenuating PTTG expression by siRNA strongly suppressed STAT3- or STAT3-C-induced cell migration and invasion by up to 90% (Figures 6a and b). Impairing PTTG expression in STAT3-C stable transfectants reduced MMP-3 (70% reduction), MMP-9 (30% reduction), MMP-10 (30% reduction) and MMP-13 (40% reduction) expressions as assessed by real-time PCR (Figure 6c), indicating that inhibiting MMPs may underlie the constraint of STAT3-facilitated cell mobility.
PTTG is required for STAT3-promoted tumor metastasis in vivo
To investigate PTTG roles in STAT3-promoted in vivo metastases, we implanted stable STAT3-C-overexpressing HCT116 PTTG+/+ or HCT116 PTTG−/− cells into the spleen of nude mice. Mice were killed 5 weeks later, when weight loss was observed. As shown in Figure 6d, in the STAT3-C HCT116 PTTG+/+ implanted group (n=14), all 14 mice (100%) developed liver tumors (2–30 metastatic loci) and 5 of 14 mice (36%) harbored splenic tumors. In addition, two mice exhibited a peritoneal tumor, two showed intestine tumors and one had ascites. In the STAT3-C HCT116 PTTG−/− control group (n=15), only one mouse (6.6%) exhibited small liver tumor foci, while 14 did not exhibit visible metastatic tumor. The incidence of tumor metastasis in the STAT3-C HCT116 PTTG+/+ group (100%) was higher than controls (6.6%; P<0.01). These results support our in vitro cell motility findings and indicate that PTTG is required for STAT3-promoted tumor metastasis in vivo.
Persistent activation or frequent overexpression of STAT3 and PTTG have been reported in several solid tumor types. Both STAT3 and PTTG have been implicated in multiple steps of cancer development and progression, but mechanisms elucidating their coordinated functions were not known. In the present study, we provide evidence that PTTG acts as a STAT3 target gene. Increased expression of STAT3/phosphor-STAT3 and PTTG were concordantly detected in human colorectal cancers (Figure 1). In support of this observation, STAT3 is shown to bind the human PTTG promoter, and activate PTTG transcription, while STAT3 induced PTTG mRNA and protein expression. Moreover, a STAT3-DN mutant, by abolishing Tyr705 phosphorylation, specifically attenuated PTTG induction (Figure 3). This novel regulatory mechanism elucidating induction of intracellular PTTG expression expands our understanding of tumor PTTG abundance and also extends our knowledge of STAT3 actions in tumor growth.
STAT3, which responds to several cytokines and growth factors, is a mediator of cell proliferation and also has important roles in cellular transformation and tumor formation.1, 26 As a STAT3 downstream gene, oncogenic PTTG is involved in G2/M mitosis and enables cell proliferation and transformation. Transfecting PTTG siRNA into STAT3 or STAT3-C stable transfectants attenuated cell growth and transformation in vitro (Figure 4). Furthermore, using HCT116 cells devoid of PTTG, depletion of PTTG constrained xenografted tumor growth in vivo. As compared with STAT3-C HCT116 PTTG+/+ controls, xenografted tumors generated from STAT3-C HCT116 PTTG−/− cells exhibited 82% reduction of tumor weight. These results illustrating PTTG actions exemplify PTTG as a potential therapeutic target to abrogate tumor growth.
In addition, mechanisms underlying PTTG actions for STAT3-mediated tumor growth include STAT3-increased CCND3 and KRAS expression. Attenuating PTTG expression by siRNA constrained expression of these pro-proliferative factors and also increased tumor-suppressor p21 expression. Several direct STAT3 targets are involved in proliferation and cell-cycle progression, such as CCND133 and c-Myc.32 Moreover, CCND3,21 as well as c-Myc,20 are regulated by PTTG. In this study, we confirmed previous reports, wherein CCND3 was induced by STAT3 and suppressed by PTTG siRNA. Importantly, we observed that KRAS was regulated by STAT3–PTTG signaling in these colorectal cancer cells. KRAS is frequently mutated in several cancers,37 and mutant KRAS upregulates fibroblast genes involved in proliferation and angiogenesis.38 Our findings indicate that PTTG siRNA constrains cell transformation and tumor formation in STAT3 HCT116 stable transfectants, associated with decreased CCND3 and KRAS expression.
STAT3 may be required for cancer cell migration and invasion,39, 40, 41, 42 while overexpressed PTTG correlates with metastasis and poor survival in several cancers, and is a key signature gene predicting metastasis.17 Here we show that both migratory and invasive activities of HCT116 cells were induced by STAT3 overexpression and further enhanced by persistent STAT3 activation, whereas cell mobility was abrogated by STAT3-DN. Moreover, siRNA-mediated inhibition of endogenous PTTG expression suppressed STAT3-induced cell motility. Remarkably, PTTG depletion in STAT3-C HCT116 cells strongly abrogated tumor metastasis in vivo, suggesting that PTTG is required for metastasis, and attenuating PTTG expression could be a novel approach to constrain STAT3-facilitated tumor progression.
MMPs, a family of zinc-dependent endopeptidases, contribute to tumor invasion and angiogenesis.43 High levels of distinct MMPs are associated with metastasis, and several cancer MMPs have been reported as STAT3 target genes,35, 44, 45 while PTTG also increases MMP-2 expression.46 Our results here suggest a role for PTTG in STAT3-facilitated tumor invasion, whereby STAT3 regulates PTTG expression to induce MMP expression and promote cell motility.
In summary, we provide evidence that PTTG acts as a STAT3 target gene. STAT3 induces PTTG expression and promotes cell transformation and motility. As attenuated PTTG constrained tumor development and progression, these findings expand our knowledge on regulatory mechanisms underlying tumor PTTG abundance and indicate PTTG as a potential therapeutic target to abrogate colorectal cancer growth.
Materials and methods
Immunofluorescence and confocal microscopy
Tissue arrays of human colorectal cancer specimens were obtained from US Biomax (Cat #BC051110, Rockville, MD, USA), and each pathologically confirmed. Slides were deparaffinized, hydrated, antigen retrieved and blocked as described.24 Slides were hybridized with antibodies against STAT3 (1:600, Cell Signaling #9139, Danvers, MA, USA), phospho-STAT3 (1:300, Cell Signaling #9131) or PTTG (1:250, Abcam DCS-280, Cambridge, MA, USA) at 4 °C overnight. Alexa Fluor antibodies (Molecular Probes, Carlsbad, CA, USA) were used as secondary antibody. Slides were mounted with ProLong Gold Antifade Reagent with DAPI (4,6-diamidino-2-phenylindole; Life Technologies, Grand Island, NY, USA), and nuclei dyed by DAPI with blue fluorescence.
Samples were imaged with a Leica TCS/SP spectral confocal scanner (Leica Microsystems, Mannheim, Germany) in dual-emission mode to separate autofluorescence from specific staining. For STAT3 and PTTG staining, a spectral window from 500 to 550 nm-wavelength-detected Alexa 488 emission appeared green. A second window from 560 to 620 nm-detected-autofluorescence contribution colored red. The two images were merged, so autofluorescence appears yellow, and true signals appear green. For double staining, PTTG stained with Alexa 488 were colored in green, and phospho-STAT3 stained with Alexa 568 were imaged with a 540-nm HeNe laser and colored in red.
Evaluation and statistical analysis
Immunofluorescence slides were independently examined by two blinded observers. Specimens staining for STAT3, Tyr705 phospho-STAT3 or PTTG were calculated as a percentage of positively stained cells. The average score of nine normal colon tissues was used as basal control and compared with all tumors (scaled as 40% for STAT3, 19% for phospho-STAT3 and 4% for PTTG). STAT3 expression in tumor tissues was evaluated as: (1) no change, if score is ⩽60%; and (2) overexpression, if score is >60%. Tumor phospho-STAT3 expression was evaluated as: (1) no change, if score is ⩽30%; and (2) overexpression, if score is >30%. Tumor PTTG expression was evaluated as: (1) no change, if score is ⩽10%; and (2) overexpression, if score is >10%. STAT3, phospho-STAT3 and PTTG expression were correlated using Pearson’s Chi-Square Test with statistical software SPSS10.0 (IBM Software, Armonk, NY, USA) and P<0.05 was considered significant.
Cell culture, transfection and stable cell selection
HCT116 cells were obtained from the American Tissue Culture Collection (ATCC, Manassas, VA, USA). HCT116 PTTG+/+ and HCT116 PTTG−/− cells were kindly provided by Dr Bert Vogelstein, Johns Hopkins University (Baltimore, MD, USA). Generation of HCT116 PTTG+/+ and PTTG−/− cells are described.5 Cell transfection was performed in 70–80% confluent cells using Lipofectamine 2000 (Life Technologies) according to the manufacturer’s protocol. G418 (0.5 mg/ml) was added for 14 days to select stable cells. Stable HCT116 transfectants expressed green fluorescent protein ZsGreen. A mass population of green stable transfectants was sorted by MoFlo Cell Sorter (Beckman Coulter, Miami, FL, USA) to ensure expression before in vitro and in vivo experiments.
Ten million cells were cross-linked and lysed using ChIP-IT express kit (Active Motif, Carlsbad, CA, USA). Chromatin was sonicated to 200–800 bp length fragments with eight rounds of 10-s pulses using 25% power. Normalized inputs of sheared chromatin DNA were incubated with 4 μg negative control immunoglobulin G, STAT3 (Cell Signaling #9139) or phospho-STAT3 antibody (Cell Signaling #9131) overnight at 4 °C. Real-time PCR reactions were amplified using precipitated immunocomplexes as template, and the PTTG promoter primers described.24 Human c-Fos promoter primers were used as positive controls (Cell Signaling #4663) and α-satellite repeat primers (Cell Signaling #4486) as negative controls.
Plasmids and siRNA
PTTG promoters, −2642/−1 and −1717/−1, were cloned into pGL3-Basic luciferase reporter vector (Promega, Madison, WI, USA) as described.36 Wild-type STAT3 expression plasmid was amplified from MHS1011-76652 clone (Open Biosystems, Lafayette, CO, USA) using TaKaRa LA Taq (Clontech, Mountain View, CA, USA) and cloned into the pIRES2-ZsGreen1 vector (Clontech). The following primers were used: forward: 5′-IndexTermCCGCTCGAGACCATGGCCCAATGGAATCAGCTACAGCA-3′; and reverse: 5′-IndexTermTCCCCGCGGTCACATGGGGGAGGTAGCGCACTCCGA-3′. The STAT3-C construct27 was made by site-directed mutagenesis (QuikChange II Site-Directed Mutagenesis Kit, Agilent Technologies, Santa Clara, CA, USA) using primers 5′-IndexTermGGGCTATAAGATCATGGATTGTACCTGCATCCTGGTGTCTCCACTGG-3′ and 5′-IndexTermCCAGTGGAGACACCAGGATGCAGGTACAATCCATGATCTTATAGCCC-3′. The STAT3-DN construct was mutated by primers 5′-IndexTermCCAGGTAGCGCTGCCCCATTCCTGAAGACC-3′ and 5′-IndexTermGGTCTTCAGGAATGGGGCAGCGCTACCTGG-3′, to replace tyrosine at position 705 to phenylalanine. Plasmids were sequenced by Sequetech (Mountain View, CA, USA). Pre-designed PTTG siRNA (ID#4390824) and negative control siRNA (Cat#AM4611) were obtained from Ambion (Grand Island, NY, USA).
Cells were split into 24-well plates, and each well co-transfected with 200 ng luciferase vector pGL3-Basic as control, PTTG promoter −2642/−1 or −1717/−1, together with 800 ng pIRES2-ZsGreen1 as control, STAT3 or STAT3-C plasmids. pRL-Tk (Promega) encoding Renilla luciferase was used as an internal control (5 ng/well) to assess transfection efficiency. For STAT3 inhibitors S3I-201 and Stattic (EMD Millipore, Billerica, MA, USA) treatment, cells were transfected with 1000 ng pGL3-Basic or PTTG promoter (−2642/−1 or −1717/−1) and subsequently treated with STAT3 inhibitors. After 24 h, whole-cell lysate was collected for reporter detection by the Dual Luciferase Reporter System (Promega). Reactions were measured using an Orion Microplate Luminometer (Berthold Detection System, Tanja Vicentic, Germany). Transfections were performed in triplicate and repeated three times to assure reproducibility.
RNA extraction and real-time PCR
Total RNA was isolated using TRIZOL Reagent (Life Technologies). Two micrograms of total RNA were used to synthesize cDNA with SuperScript II Reverse Transcriptase (Life Technologies). Real-time PCR was amplified in 20 μl reaction mixtures (100 ng template, 0.5 μM of each primer, 10 μl 2 × SYBR GREEN Master Mix (Life Technologies)) using the following parameters: 95 °C for 1 min, followed by 40 cycles of 95 °C for 20 s, 60 °C for 40 s. β-actin was used as internal control. Real-time PCR primers were designed as follows. PTTG Forward: 5′-IndexTermTGATCCTTGACGAGGAGAGAG-3′; reverse: 5′-IndexTermGGTGGCAATTCAACATCCAGG-3′. KRAS forward: 5′-IndexTermTTGAACTAGCAATGCCTGTG-3′; reverse: 5′-IndexTermACCAATTAGAAGGTCTCAACTG-3′. CCND3 forward: 5′-IndexTermCAGATGTCACAGCCATAC-3′; reverse: 5′-IndexTermGATGGGTAGGACCAGATC-3′. CDKN1A forward: 5′-IndexTermGCTCTACATCTTCTGCCTTAGTC-3′; reverse: 5′-IndexTermACCTCTCATTCAACCGCCTAG-3′. MMP-3 forward: 5′-IndexTermGGTCTCTTTCACTCAGCCAACAC-3′; reverse: 5′-IndexTermCAGGCGGAACCGAGTCAGG-3′. MMP-13 forward: 5′-IndexTermCCTTGATGCCATTACCAGTCTC-3′; reverse: 5′-IndexTermTCAATACGGTTGGGAAGTTCTG-3′. β-actin forward: 5′-IndexTermCATGTACGTTGCTATCCAGGC-3′; and reverse: 5′-IndexTermCTCCTTAATGTCACGCACGAT-3′. MMP-9 and MMP-10 primers were purchased from SABiosciences (#PPH00152E and #PPH00896B, Valencia, CA, USA).
Western blots were performed as described.24 Primary antibodies used were: STAT3: 1:1000, Cell Signaling #9139; phospho-STAT3: 1:1000, Cell Signaling #9131; PTTG: 1:500, Abcam DCS-280; CCND3: 1:500, Santa Cruz sc-182 (Dallas, TX, USA); cK Ras: 1:1000, Abcam ab84573; β-actin: 1:20000, Millipore.
Cell proliferation and soft agar colony-formation assay
STAT3 HCT116 stable transfectants were separately transfected with PTTG siRNA or negative control. After 24 h, cells (104 cells/well in 100 μl medium) were plated in flat-bottomed 96-well plates. Each group (PTTG siRNA or negative control) consisted of eight parallel wells. After 72 h, premixed WST-1 cell proliferation reagent (Clontech) was added (1:10) and incubated for 4 h at 37 °C in a humidified atmosphere maintained at 5% CO2, after which absorbance was measured at 450 nm.
STAT3 or STAT3-C HCT116 stable transfectants were separately transfected with PTTG siRNA or negative control. One thousand cells were plated in the top layer containing 0.3% agarose, and the bottom support layer comprised 0.6% agarose. Cells were stained 10 days later with 0.2% iodonitrotetrazolium chloride (Life Technologies) and photographed.
In vitro migration and invasion assay
Cell migration and invasion assays were performed in 6.5 mm Transwells (Corning #3422, Corning, NY, USA). Cells (2 × 105) were suspended in 100 μl serum-free medium, added to the upper chamber and the lower chamber was filled with complete medium with 10% serum. Cells were migrated at 37 °C for 48 h. After removing non-migrated cells, membranes were fixed in methanol and stained with 0.05% crystal violet. Migrated cells were photographed and quantified in five random fields per membrane. Each sample was assayed in triplicate. A similar system, with Matrigel-coated membranes, was used for assessing invasion.
Subcutaneously xenografted tumor growth
Animal protocols were approved by the Cedars-Sinai Institutional Animal Care and Use Committee. Forty female nude mice (5–6-week old, obtained from Jackson Laboratory, Bar Harbor, ME, USA) were randomly separated into two groups. One group was injected subcutaneously with STAT3-C HCT116 PTTG+/+ stable transfectants and the second group was injected with STAT3-C HCT116 PTTG−/− stable transfectants (106 cells/100 μl Matrigel per animal) into the left lumbar area to generate tumors. Tumor size was measured by caliper measurements twice a week when tumors became visible, and volume calculated with the formula: (length × width2)/2. Seventeen days after cell injection, animals were euthanized and excised tumors weighed.
Experimental metastasis in vivo
Animal protocols were approved by the Cedars-Sinai Institutional Animal Care and Use Committee. STAT3-C HCT116 PTTG+/+ or STAT3-C HCT116 PTTG−/− stable cells (106 cells/50 μl phosphate-buffered saline per animal) were injected into the spleen of 6-week-old female nude mice. Each group included 14–15 animals. Mice were weighed once a week and metastasis development monitored. Five weeks later, mice were killed when weight loss or any adverse effect was observed. Liver, spleen and other organs were visibly observed for tumor metastasis. Tumors were harvested and metastatic loci counted.
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We are grateful to Dr Bert Vogelstein at Johns Hopkins University (Baltimore, MD, USA) for kindly providing HCT116 PTTG+/+ and HCT116 PTTG−/− cells; and thank Patricia Lin at the Cedars-Sinai Flow Cytometry Core. This work was supported by National Institutes of Health Grant CA75979 (to SM) and the Doris Factor Molecular Endocrinology Laboratory.
The authors declare no conflict of interest.
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