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SMAD4 Loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells
- Yu-Wen Chen†1,
- Pi-Jung Hsiao†2,
- Ching-Chieh Weng1,
- Kung-Kai Kuo3,
- Tzu-Lei Kuo1,
- Deng-Chyang Wu4, 5,
- Wen-Chun Hung6 and
- Kuang-Hung Cheng1Email author
© Chen et al.; licensee BioMed Central Ltd. 2014
Received: 14 September 2013
Accepted: 28 February 2014
Published: 14 March 2014
SMAD4 is a gastrointestinal malignancy-specific tumor suppressor gene found mutated in one third of colorectal cancer specimens and half of pancreatic tumors. SMAD4 inactivation by allelic deletion or intragenic mutation mainly occurs in the late stage of human pancreatic ductal adenocarcinoma (PDAC). Various studies have proposed potential SMAD4-mediated anti-tumor effects in human malignancy; however, the relevance of SMAD4 in the PDAC molecular phenotype has not yet been fully characterized.
The AsPC-1, CFPAC-1 and PANC-1 human PDAC cell lines were used. The restoration or knockdown of SMAD4 expression in PDAC cells were confirmed by western blotting, luciferase reporter and immunofluorescence assays. In vitro cell proliferation, xenograft, wound healing, quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), Western blotting, and immunohistochemistry analysis were conducted using PDAC cells in which SMAD4 was either overexpressed or knocked down.
Here, we report that re-expression of SMAD4 in SMAD4-null PDAC cells does not affect tumor cell growth in vitro or in vivo, but significantly enhances cells migration in vitro. SMAD4 restoration transcriptionally activates the TGF-β1/Nestin pathway and induces expression of several transcriptional factors. In contrast, SMAD4 loss in PDAC leads to increased expression of E-cadherin, vascular endothelial growth factor (VEGF), epidermal growth factor receptor (EGFR) and CD133. Furthermore, SMAD4 loss causes alterations to multiple kinase pathways (particularly the phosphorylated ERK/p38/Akt pathways), and increases chemoresistance in vitro. Finally, PDAC cells with intact SMAD4 are more sensitive to TGF-β1 inhibitor treatment to reduced cell migration; PDAC cells lacking SMAD4 showed decreased cell motility in response to EGFR inhibitor treatment.
This study revealed the molecular basis for SMAD4-dependent differences in PDAC with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-β or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects.
Pancreatic cancer is one of the most insidious forms of human cancer whose incidence nearly equals its death rate. Histologically, ductal adenocarcinomas of the pancreas (PDAC) account for > 90% of all exocrine pancreatic cancers. PDAC remains the eighth leading cause of cancer death worldwide, with the lowest 5-year survival rate of any gastrointestinal cancer. Several features conspire to make PDAC a formidable clinical issue: poor early detection, the advanced nature of most tumors at the time of diagnosis, and lack of specific or effective therapy. In contrast to other major cancers, decades of clinical trials have failed to provide appreciable survival and less toxicity benefit for PDAC . For example, FOLFIRINOX and nab-Paclitaxel for treatment of advanced pancreatic cancer have shown to be effective for overall survival, progression-free survival, and response rate, but was associated with increased toxicity and serious side effects [2–4]. Indeed, this continual cycle of clinical trial for PDAC therapy followed by failure has led some to conclude that there is insufficient knowledge of the mechanisms underlying this particular type of lethal disease [5, 6].
A number of studies of PDAC have elucidated a detailed profile of genetic alterations associated with PDAC initiation and progression — including the activation KRAS and loss of INK4A, p53, and SMAD4 — providing clues for investigation of the molecular and biochemical basis for this malignancy [7, 8]. SMAD4 is recognized as an intracellular common mediator for the TGF-β superfamily signaling pathways, including TGF-β1, activin, and BMP signaling, responsible for embryonic patterning, differentiation and a variety of homeostatic processes [9, 10]. During the initiation phase of carcinogenesis, most malignant epithelial tumors develop resistance to TGF-β/SMAD-mediated growth inhibition. However, excessive levels of TGF-β1 are associated with malignant tumor progression in many cancers, suggesting that inactivation of the SMAD proteins could be an important event in this process . With respect to cellular growth control, the effects of TGF-β are highly dependent on the cell type and cell context, which exert alternating growth-promoting and growth-inhibitory effects in different cell types and at different stages of tumorigenesis. Several independent studies indicate that deletions or intragenic mutations of the SMAD4 gene are present in more than 50% of human PDACs, but are rare in other malignancies such as lung or breast cancer [12–16]. Hence, SMAD4 is a distinguishing molecular feature of two major types of PDAC. Although many lines of evidence indicate that SMAD4 status in PDAC is associated with specific histopathological phenotypes, the detailed molecular basis of SMAD4-dependent phenotypic changes in cancer biology has yet to be determined.
Although many lines of evidence indicate that inactivation of SMAD4 in PDAC is generally restricted to high grade Pancreatic intraepithelial neoplasia (PanIN) and PDAC, implying a specific role for SMAD4 in malignant progression, the specific anti-tumorigenic impact of SMAD4 loss has not been fully characterized [8, 17]. Notably, studies of human cell lines have given inconsistent results of how SMAD4 status influences TGF-β responsiveness and of other tumor biological properties, leading to conflicting conclusions on the impact of SMAD4 defects on PDAC prognosis [18, 19]. Overall, these studies suggest that TGF-β/SMAD4 signaling may have pleiotropic and context-dependent roles during PDAC progression. These features add significant complexity to attempts to design therapeutic strategies to deregulate the SMAD4 pathway. In this study, we used SMAD4-proficient and -deficient human PDAC cell lines AsPC-1, CFPAC-1, and PANC-1 to compare the molecular profiles of SMAD4-positive and -negative PDAC cells; assess their relationship to SMAD4 status; and further demonstrate the ability of SMAD4 to modulate cell proliferation, affect cell motility, regulate the epithelial-mesenchymal-transition (EMT) process, activate kinase pathways, change expression of cancer stem-like cell (CSC) markers and affect sensitivity to chemodrugs in PDAC. The objective of the present study was therefore to dissect the molecular circuits that contribute to the inactivation of SMAD4 in different phenotypes of PDAC.
Cell culture, RNA isolation, and cDNA synthesis and inhibitors treatments
The HEK293T and human PDAC cell lines were obtained from sources described previously [8, 20]. Treatments with TGF-β1 (5 ng/ml), cisplatin, paciltaxol, gemcitabine, SB231542 and gefitinib were performed according to previously-described procedures [20, 21]. The RNA isolation and cDNA synthesis from the cell lines were also conducted according to previously-described protocols [20, 22].
Plasmid and retroviral construction
A full length cDNA clone for the SMAD4 gene was originally obtained from the Bert Vogelstein laboratory and subcloned in pBabe-puro plasmid (Addgene, Cambridge, MA) to create a pBabe-SMAD4-puro vector . In brief, for SMAD4 gene restoration, pBabe-puro plasmid was digested with restriction enzyme BamHI and Hind ΙΙΙ to obtain the full length of SMAD4 cDNA, then ligated into BamHI/XhoI-digested pBabe-puro backbone vector. The insert fragment of SMAD4 cDNA was subcloned into the pBABE-puro backbone by using T4 ligase (NEB) subjected to Klenow enzyme reaction and ligated. All plasmids were verified by DNA sequencing (Genome International Biomedical Co., Ltd., New Taipei City, Taiwan).
Retroviral production and infection of target cells
Retrovirus was generated by co-transfection of pBabe-puro empty vector or pBabe- puro-SMAD4 with pVSV-G (envelope) and pVSV-GP (packaging) plasmids in 293 T cells. Target cells were infected overnight with 4 ml of virus-containing medium in the presence of 10 μg/ml polybrene. The following day, cells were cultured in fresh medium and allowed to grow for another 24 hrs. After this medium was replaced with fresh regular medium, cells were selected with 2 μg/ml puromycin for 2 weeks. Positive stable clones were then characterized and utilized in further assays.
Lentivirus production and shRNA for gene knockdown
All plasmids required for shRNA lentivirus production were purchased from the National RNAi Core Facility, Academia Sinica, Taipei, Taiwan. The pLKO.1-shRNA vector used for knockdown of SMAD4 was TRCN000010323 (SMAD4), and the scrambled lentiviral control vector was pLKO_TRC025. Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA) was used for lentiviral production in 293 T cells with a packaging construct (pCMV-ΔR8.91), an envelope construct (pMD.G) and different shRNA constructs as previously described .
Western blotting was performed as described previously [20, 21]. The following antibodies were used in this study: anti-SMAD4 (sc-7154 or sc-7966), anti-E-cadherin (sc-8426), anti-vimentin (sc-7557), anti-CD133 (sc-8304), anti-CD44 (sc-18849), anti-Sp1(sc-14027), anti-c-Jun (sc-1694), anti-Fos (sc-52), anti-Fast-1 (sc-377358), anti-Hes1 (sc-25392), anti-GAPDH (sc-32233; Santa Cruz Biotechnology, Inc.), anti-p-Akt (#4060), anti-Akt (#4691), anti-p-p44/42 (#9101),anti-p44/42 (#4695), anti-Pten (#9272), anti-NF-κB (#4764S), anti- EGFR (#4267), anti-p-EGFR tyr 992 (#2235), anti-p-EGFR tyr 1068 (#3777), anti-Smad2/3 (#5339), anti-p-Smad2/3 (#3101), anti-p-c-Jun (#2361; Cell Signaling Technology, Inc.), anti-Nestin (N5413), mouse anti-β-actin (Sigma- Aldrich Co.), anti-CD133/1 (AC133, Miltenyi Biotec.) and anti-TGF-β1 (ab9758, Abcam, Plc.).
Quantitative reverse transcription polymerase Chain reaction (RT-qPCR) analysis
Total RNA prepared from samples was used for cDNA synthesis. PCR amplification and results of the delta computed tomography (CT) measurements were described previously [20, 22]. The primers sequence used in thi stsudy were as follows: GAPDH primer sequences: forward 5′-GAAGGTGAAGGTCGGAGTCA-3′. Reverse 5′-AATGAAGGGGTCATTGAT GG-3′. SMAD4 primer pair: Forward 5′-CGCTTTTGTTTGGGTC AACT-3′. Reverse: 5′-CCCAAACATCACCTTCACCT-3. CD133 primer pair: Forward 5′-CCCCAGGAAATTT GAGGAAC-3′. Reverse 5′- TC CAACAATCCATTCCCTGT-3′. E-cadherin primer pair: Forward 5′-ATTGCAAATTCCTGCCATTC-3′. Reverse 5′-CTCTTCTCCGCCTCCTTCTT-3′. N-cadherin primer pair: Forward 5′-CCTTGTGCTGATGTTTGTGG-3′. Reverse 5′-TGGATGGGTCTTTCATCCAT-3′. vimentin primer pair: Forward 5′-GGGAGAAATTGC AGGAGGAG-3′. Reverse 5′-ATTCCACTTTGCGTTCAAGG-3′. CD44 primer pair: Forward 5′-AG ACACCATGCATGGTGCACC-3′. Reverse 5′-TAACAGCATCAGGAGTG-3′. EGFR primer pair: Forward 5′-TCAGCCACCCATATGTACCA-3′. Reverse: 5′-CATTC TTTCATCCCCCTGAA-3′. VEGF primer pair: Forward 5′-CCCACTGAGGAGTCC AACAT-3′. Reverse: 5′-T GCATTCACATTTGTTGTGC-3′. The PCR reactions were repeated three times from three independent experiments.
Transient transfections and luciferase reporter assays
Transient transfections and SBE4 (four repeats of SMAD binding element), CD133 and Nestin luciferase reporter assays were performed as described previously .
Cell proliferation assay
Cell proliferation assay was performed as previously described [20, 22]. Briefly, 5X 103 cells were seeded in 96-well plates, and incubated overnight. The cells were treated with or without drugs, and incubated for 1 to 3 days. 5 mg/ml MTT (thiazolyl blue tetrazolium bromide) (Americo Chemical Co) 25 μl in 500 μl medium was then added, and incubated for another 2 hours for reaction. The medium was removed, and crystal was completely dissolved with 200 μl DMSO (Sigma). The OD570 reading was then detected with a BioTek ELISA reader (Molecular Device, Sunnyvale, CA).
In vitrocell migration/invasion assays
For wound healing cell migration assay, cells were pretreated with 0.02% (0.2 mg/mL) mitomycin C for 2 hours, and wounded by removing a 300–500 μm-wide strip of cells across the well with a standard 200 μL yellow tip. Wounded monolayers were washed twice with 1xPBS to remove nonadherent cells. The cells were cultured in low FBS media and incubated for pre-determined times to monitor wound closing. Wound closure was recorded by phase-contrast microscopy according to previously published protocols [20, 22]. For transwell migration assays, 5 × 104 cells were plated in the top chamber with a non-coated filter membrane (6-well insert, pore size 8 μm; BD Biosciences, San Jose, CA) in low serum medium. The bottom medium was supplemented with 10% FBS. Cells were incubated for 24 hours. Cells that did not migrate through the pores were removed by cotton swab. Cells on the lower surface of the membrane were stained with crystal violet before photography. The crystal violet was dissolved in 10% acetic acid and absorbance was measured by using the BioTek enzyme-linked immunosorbent assay (ELISA) reader OD570 (Level BioTek Instruments, Inc., Winooski, VT) for quantitative analysis .
Mice and injections
To study in vivo tumorigenicity, pathogen-free female C.B17/lcr- SCID mice, eight weeks old, were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). Technology from Charles River Laboratories (Wilmington, MA, USA) was used for breeding in the animal center at the Department of Medical Research, Kaohsiung Medical University (KMU) Hospital. Mice were housed at the Experimental Animal Center, KMU under specific pathogen-free (SPF) conditions under protocols approved by the KMU IACUC institutional guidelines for the care and use of experimental animals were followed. Mice were injected subcutaneously in the left and right flank with 1 × 106 cells in 0.1 ml of medium. After two months, tumor volumes, overall health and total body weights of the mice were assessed as previously described . Each experimental group contained > 4 mice.
Mouse surgery, necropsy, histopathology and immunohistochemistry
Tissue samples were fixed in 10% buffered formalin for 12 h, washed with PBS and transferred to 70% ethanol, embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E). Immunohistochemical analysis of SMAD4, EGFR, E-cadherin, CD133 and Nestin were performed as described previously [8, 20].
Data are presented as mean ± standard error of the mean. The continuous data were statistically analyzed using Student’s t-test and categorical data were subjected to Chi-square test. All statistical calculations were performed using SAS for Windows version 12.2 (SAS, Inc., Cary, NC). A p value of less than 0.05 was considered significant .
Generated stable SMAD4 over-expression and knockdown of human PDAC cells
Further, we determined that the intact TGF-β signal pathway was fully restored in AsPC-1 and CFPAC-1 stable SMAD4 reconstituted cells by using a SBE4 luciferase reporter assay, and by detecting the levels of SMAD2 phosphorylation after TGF-β1 treatment in AsPC-1 cells after SMAD4 restoration (Figure 1B and C). We also observed that TGF-β1 treatment leads to nuclear translocation of SMAD4 in SMAD4-re-expressing AsPC-1 cells by immunofluorescence analysis (Figure 1D). Meanwhile, we utilized a shRNA-mediated RNA interference approach to knockdown the expression of SMAD4 in the PANC-1 cell line. Results of Western blots from the PANC-1 shSMAD4 cells showed a significant reduction of SMAD4 protein levels compared to mock control cells (Figure 1A). We also confirmed the reduced TGF-β1 signaling by phospho-SMAD2 western blot analysis and SBE4-luciferase activity assay in PANC-1 shSMAD4 cells when compared with control cells. (Figure 1B and C).
SMAD4 restoration does not affect their proliferation in vitro and in vivo, but increases PDAC cells migration in vitro
To further investigate the effect of SMAD4 expression on the migratory potential of AsPC-1, CFPAC-1 and PANC-1 cells in vitro, in vitro wound healing assays were employed in SMAD4-proficient and -deficient CFPAC-1 and AsPC-1 cells. Monolayers of cells were pretreated with mytomycin-C for 2 hrs before being scratched with a pipette tip, and then cultured in the regular culture condition containing 5% fetal bovine serum (FBS). After overnight incubation, our results indicated that SMAD4 restoration significantly enhanced the ability in vitro of CFPAC-1 and AsPC-1 cells to migrate as compared to control cells (Figure 2C). In addition, knockdown of SMAD4 by shRNA significantly decreased the in vitro migratory potential of PANC-1 cells (P < 0.05; Figure 2C). Further, our results with in vitro invasion assay using a transwell chemotaxis invasion approach in AsPC-1 and PANC-1 cells also showed that SMAD4 enhanced the invasive ability of PDAC cells in vitro (P < 0.05; Figure 2D and Additional file 1: Figure S1).
SMAD4 modulates EMT and regulates CSC-associated gene expression
Re-expression of SMAD4 reduces EGFR and VEGF expression and repression phosphorylation in the Akt and ERK signaling pathways, but enhances the p38 MAP kinase pathway
SMAD4 defect confers chemoresistance and leads to augmented EGFR-mediated cancer cell motility in PDAC
SMAD4, also known as deleted in pancreatic carcinoma, locus 4 (DPC4), was first identified on the basis of frequent homozygous deletions and mutations affecting 18q21.1 in the pancreatic tumor, and was found to be involved in the TGF-β1 signaling pathway [11, 30]. Germline mutations in SMAD4/DPC4 have also been identified in certain types of juvenile polyposis [31, 32]. Hahn and colleagues reported that about 90 percent of pancreatic carcinomas show allelic loss at chromosome 18q21.1, and further studies have confirmed that the SMAD4/DPC4 gene, localized to 18q21, was the target for 50% of the PDAC that exhibited 18q deletion . During carcinogenesis, TGF-β1 may act in an autocrine and/or paracrine fashion to exert a biphasic effect on cancer progression. Early in tumor formation, TGF-β1 functions to suppress cell cycle progression and block tumor growth. In contrast, cancer cells later adapt to develop a resistance to TGF-β1-mediated growth inhibition by increasing expression of TGF-β1 antagonist, mutating the TGF-β1 receptor or inactivating the SMAD4 gene. Subsequently, TGF-β1 ceases to function in tumor suppression and switches to the converse role of enhancing tumor metastasis by promoting tumor cells’ epithelial-mesenchymal transition (EMT) or inducing the angiogenic phenotype [33, 34]. TGF-β1 is known to transduce signaling cascades through SMAD-dependent, as well as SMAD-independent, non-canonical pathways. A number of studies have reported that TGF-β1 can activate non-canonical SMAD-independent pathways through Ras/Erk (p44/42), PI3K/Akt, JNK or TAK1/p38 kinase [35, 36]. However, the overall effect of Erk, Akt or p38 MAPK activation by TGF-β and the biological consequences are poorly characterized. Upon SMAD4 inactivation or deletion, TGF-β1 may preferentially signal through a SMAD-independent pathway, instead of the canonical SMAD-dependent pathway, leading to the phenotypic changes seen in tumor cells.
The study reported by Dai et al.  revealed that he antitumor activity of SMAD4 induces G1 arrest and apoptosis through the nuclear translocation of SMAD4 in MDAMB468 breast cancer cells, revealing the anti-tumor proliferation mediation of SMAD4-dependent signaling. Although most attention has focused on the cell cycle arrest mediated by TGF-β1/SMAD4 signaling, the other tumor suppressive effects of SMAD4 in preventing late stage tumor progression are still not fully understood. Until recently, our group and others have found SMAD4 involved in suppression of metastasis, angiogenesis and chemo-resistance in many different types of cancers [21, 38]. For example, Schwarte- Waldhoff and his colleagues reported that the restoration of SMAD4 in SW480 colon cells reduced expression levels of the endogenous urokinase-type plasminogen activator and plasminogen-activator-inhibitor-1 (PAI-1) genes, involved in the degradation of extracellular matrix proteins and the control of tumor cell migration and invasion . In 2000, they further demonstrated that SMAD4 re-expression in the human PDAC cell line Hs766T suppresses angiogenesis through down-regulation of VEGF and up-regulation of throbospondin-1 (TSP-1), a potent endogenous angiogenesis inhibitor . Recently, our research group also reported that SMAD4 suppresses the development of malignant phenotypes of human colorectal cancer through interacting with HIF1α to suppress VEGF and MMP expression under hypoxic conditions . Although these studies provide promising evidence of the role of SMAD4 as a tumor suppressor gene, our mechanistic understanding of SMAD4 is still in its infancy.
In the present study, using human PDAC cell lines, we first examined the overall effects of the restoration and knockdown SMAD4 expression in human PDAC cells. Specifically, we found that all PDAC cells exhibit increased cell migration in vitro after SMAD4 re-expression, although PDAC cell growth was not significantly affected after SMAD4 reconstitution. In addition, we observed that SMAD4 deficiency in human PDAC cells induces E-cadherin expression and such cells exhibit epithelial morphology, a result consistent with our previous report with SMAD4-conditional knockout mice demonstrating that genetically engineered mouse (GEM) models of Pdx Kras Smad4L/L Ink/ArfL/+ mice develop more well-differentiated lesions with glandular structures of PDAC tumors than SMAD4 wild type Pdx Kras Ink/ArfL/+ mice . Here, we also demonstrated an increase in the noncanonical or non-SMAD TGF-β pathways, including the MEK/ERK and PI3K/Akt signaling pathways, in SMAD4-negative PDAC cells compared to SMAD4-positive PDAC cells. Intriguingly, we also observed the down-regulated PTEN gene expression in SMAD4-deficient PDAC cells, an effect which may be partly due to the mediation of the inhibitory effects of NF-κB activation . Previous studies have shown that TGF-β-activated kinase 1 (TAK1) is implicated in p38 MAPK activation in response to TGF-β1 in several cell systems . In addition, TGF-β-induced EMT was blocked by inhibiting the activation of p38 MAPK in mouse mammary epithelial cells, and p38 MAPK inhibitors blocked TGF-β1-stimulated migration of non-tumor and tumor cells, which suggest that p38 MAPK may act in parallel or in cooperation with a SMAD-dependent pathway in chemotactic responses to TGF-β1 [42, 43]. In this study, we also observed an increased activation of the p38 MAPK pathway in the presence of SMAD4 in PDAC. In addition, our result revealed that restoration of SMAD4 induces the increased activation of p38 MAPK signaling, which may in turn enhance the expression of c-Jun, c-fos or Fast-1 transcriptional factors in PDAC [44, 45].
We also demonstrated that reconstitution of SMAD4 in PDAC cells resulted in an increase in apoptotic death after treatment with cisplatin, gemcitabine, or paclitaxel when compared with SMAD4-deficient PDAC cells. This result is in agreement with our previously published work in the colorectal cancer model, which found that SMAD4 loss increased resistance to the chemotherapeutic agent 5′-fluorouracil . Many more recent studies have shown that TGFβ1 and EGFR inhibitors are promising for the treatment of pancreatic cancer [54–56]. Like many chemotherapeutic agents, the effectiveness of EGFR inhibitors have been approved by Food and Drug Administration for use in several tumor cases, alone and in combination with gemcitabine for pancreatic cancer [57, 58]. In the present study, we concluded that treatment of SMAD4-proficient PDAC cells with TGF-β1 inhibitor resulted in a profound reduction in cell migration in vitro. In contrast, treatment with EGFR inhibitor remarkably inhibited cell migration in SMAD4-deficient PDAC cells, implying that the SMAD4 defect results in a gain to the EGFR signaling pathway during PDAC development.
The present study revealed the molecular basis for SMAD4-dependent and -independent differences in PDAC tumor biology with the aim of identifying the subset of patients likely to respond to therapies targeting the TGF-β or EGFR signaling pathways (Figure 7) The use of model system illustrated here may help to identify additional nodes of therapeutic intervention in PDAC patients devoid of SMAD4.
This study was supported in part by the following grants: NSC 101-2314-B-110-001-MY2 and 101-2628-B-110-001-MY2 from the National Science Council, Taiwan ROC (to K.H. Cheng), a grant from National Sun Yat-Sen University- Kaohsiung Medical University Joint Research Center (to K.H. Cheng and D.C. Wu).
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