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SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells.

Chen YW, Hsiao PJ, Weng CC, Kuo KK, Kuo TL, Wu DC, Hung WC, Cheng KH - BMC Cancer (2014)

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan. khcheng@faculty.nsysu.edu.tw.

ABSTRACT

Background: 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.

Methods: 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.

Results: Here, we report that re-expression of SMAD4 in SMAD4- 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.

Conclusions: 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.

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A Model of phenotypic alteration involving SMAD4 loss in PDAC cells. SMAD4 wild type PDAC cells exhibits more fibroblast-like morphology, which high express Nestin, SMA, CD44 gene, with the increase of activation of p38 MAPK and TGFβ1 signaling, and acquisition of a more high expression levels for the c-Jun, c-Fos, Hes1 NF-κb transcription factor genes. In contrast, inactivation of SMAD4 in PDAC cells exhibits more well differentiate epithelial like (cobblestone) morphology and leads to an overall increase in E-cadherin, CD133, VEGF, EGFR and Sp-1 expressions with high levels of activation p-44/42 and PI3K/Akt signaling pathways.
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Figure 7: A Model of phenotypic alteration involving SMAD4 loss in PDAC cells. SMAD4 wild type PDAC cells exhibits more fibroblast-like morphology, which high express Nestin, SMA, CD44 gene, with the increase of activation of p38 MAPK and TGFβ1 signaling, and acquisition of a more high expression levels for the c-Jun, c-Fos, Hes1 NF-κb transcription factor genes. In contrast, inactivation of SMAD4 in PDAC cells exhibits more well differentiate epithelial like (cobblestone) morphology and leads to an overall increase in E-cadherin, CD133, VEGF, EGFR and Sp-1 expressions with high levels of activation p-44/42 and PI3K/Akt signaling pathways.

Mentions: Most importantly, our present study provides the first experimental evidence that inactivation of SMAD4 enhances EGFR and CD133 expression, whereas re-expression of SMAD4 suppresses EGFR and CD133 levels in PDAC cells. These results are consistent with a previous report using HPDEC cells in which the knockdown of SMAD4 expression was found to increase EGFR expression [24]. Meanwhile, the down-regulation of EGFR expression in SMAD4-proficient cells may result from the reduced expression of the transcriptional factor Sp-1 (Figure 7). Recently, the CD133 molecule has been linked to tumor malignancy and invasiveness, and overexpression of EGFR and its ligands significantly contributes to the malignant phenotype and correlates with decreased survival in pancreatic cancer patients [46-49]. Further insight is needed to evaluate the relationship between the expression levels of EGFR and the presence of CD133 in PDAC, and the association between EGFR and CD133 may represent an important mechanism in the control of SMAD4- inactivated PDAC cell proliferation and malignancy. Our data further indicated increased Nestin expression upon SMAD4 reconstitution in PDAC, a result which may be related to the restoration of the TGF-β1/SMAD signaling pathway in PDAC cells. Nestin was first identified as an important neuronal stem cell marker during central nervous system development [50,51]. The long carboxy-terminal portion of Nestin has been reported to serve as the link or cross-bridge between intermediate filaments and microtubule, helping to mediate cell migration. Recently, Matsuda and colleagues illustrated the importance of Nestin in pancreatic cancer cell migration, invasion and metastasis by selectively modulating the expression of actin and other cell adhesion molecules [52]. They proposed that Nestin expression is crucial for colonizing distant sites in metastasis and thus may be a marker of metastasis-initiated “cancer stem cells”. How SMAD4 regulates Nestin expression in PDAC is not yet clear. The Nestin promoter does harbor several potential SMAD-binding sites, two SBE-related sequence 5′-CAGACA-3′-box at position -2067 and -566. Thus, it could exert control via transcriptional regulation. More recently, we proposed that increased Nestin expression could provide a positive feedback loop to induce TGF-β1/SMAD signaling by increasing the expression of TGF-β1 and TβR1a and TβR2 receptors [20]. Nestin is also involved in regulating the Wnt effector; the CD44 gene, a known putative cancer stem cell marker involved in mediating tumor cell metastasis [53]. Thus, this study provides the first evidence linking SMAD4 status and the expression patterns of CSC markers of PDAC.


SMAD4 loss triggers the phenotypic changes of pancreatic ductal adenocarcinoma cells.

Chen YW, Hsiao PJ, Weng CC, Kuo KK, Kuo TL, Wu DC, Hung WC, Cheng KH - BMC Cancer (2014)

A Model of phenotypic alteration involving SMAD4 loss in PDAC cells. SMAD4 wild type PDAC cells exhibits more fibroblast-like morphology, which high express Nestin, SMA, CD44 gene, with the increase of activation of p38 MAPK and TGFβ1 signaling, and acquisition of a more high expression levels for the c-Jun, c-Fos, Hes1 NF-κb transcription factor genes. In contrast, inactivation of SMAD4 in PDAC cells exhibits more well differentiate epithelial like (cobblestone) morphology and leads to an overall increase in E-cadherin, CD133, VEGF, EGFR and Sp-1 expressions with high levels of activation p-44/42 and PI3K/Akt signaling pathways.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4007528&req=5

Figure 7: A Model of phenotypic alteration involving SMAD4 loss in PDAC cells. SMAD4 wild type PDAC cells exhibits more fibroblast-like morphology, which high express Nestin, SMA, CD44 gene, with the increase of activation of p38 MAPK and TGFβ1 signaling, and acquisition of a more high expression levels for the c-Jun, c-Fos, Hes1 NF-κb transcription factor genes. In contrast, inactivation of SMAD4 in PDAC cells exhibits more well differentiate epithelial like (cobblestone) morphology and leads to an overall increase in E-cadherin, CD133, VEGF, EGFR and Sp-1 expressions with high levels of activation p-44/42 and PI3K/Akt signaling pathways.
Mentions: Most importantly, our present study provides the first experimental evidence that inactivation of SMAD4 enhances EGFR and CD133 expression, whereas re-expression of SMAD4 suppresses EGFR and CD133 levels in PDAC cells. These results are consistent with a previous report using HPDEC cells in which the knockdown of SMAD4 expression was found to increase EGFR expression [24]. Meanwhile, the down-regulation of EGFR expression in SMAD4-proficient cells may result from the reduced expression of the transcriptional factor Sp-1 (Figure 7). Recently, the CD133 molecule has been linked to tumor malignancy and invasiveness, and overexpression of EGFR and its ligands significantly contributes to the malignant phenotype and correlates with decreased survival in pancreatic cancer patients [46-49]. Further insight is needed to evaluate the relationship between the expression levels of EGFR and the presence of CD133 in PDAC, and the association between EGFR and CD133 may represent an important mechanism in the control of SMAD4- inactivated PDAC cell proliferation and malignancy. Our data further indicated increased Nestin expression upon SMAD4 reconstitution in PDAC, a result which may be related to the restoration of the TGF-β1/SMAD signaling pathway in PDAC cells. Nestin was first identified as an important neuronal stem cell marker during central nervous system development [50,51]. The long carboxy-terminal portion of Nestin has been reported to serve as the link or cross-bridge between intermediate filaments and microtubule, helping to mediate cell migration. Recently, Matsuda and colleagues illustrated the importance of Nestin in pancreatic cancer cell migration, invasion and metastasis by selectively modulating the expression of actin and other cell adhesion molecules [52]. They proposed that Nestin expression is crucial for colonizing distant sites in metastasis and thus may be a marker of metastasis-initiated “cancer stem cells”. How SMAD4 regulates Nestin expression in PDAC is not yet clear. The Nestin promoter does harbor several potential SMAD-binding sites, two SBE-related sequence 5′-CAGACA-3′-box at position -2067 and -566. Thus, it could exert control via transcriptional regulation. More recently, we proposed that increased Nestin expression could provide a positive feedback loop to induce TGF-β1/SMAD signaling by increasing the expression of TGF-β1 and TβR1a and TβR2 receptors [20]. Nestin is also involved in regulating the Wnt effector; the CD44 gene, a known putative cancer stem cell marker involved in mediating tumor cell metastasis [53]. Thus, this study provides the first evidence linking SMAD4 status and the expression patterns of CSC markers of PDAC.

Bottom Line: 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.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 80424, Taiwan. khcheng@faculty.nsysu.edu.tw.

ABSTRACT

Background: 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.

Methods: 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.

Results: Here, we report that re-expression of SMAD4 in SMAD4- 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.

Conclusions: 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.

Show MeSH
Related in: MedlinePlus