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

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-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-ß1/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-ß1 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-ß or EGFR signaling pathways and of identifying potential therapeutic interventions for PDAC patients with SMAD4 defects.

ARID1A loss in pancreas leads to islet developmental defect and metabolic disturbance.

ARID1A is a tumor suppressor gene mutated in 7-10% of pancreatic cancer patients. However, its function in pancreas development and endocrine regulation is unclear. We generated mice that lack Arid1a expression in the pancreas. Our results showed that deletion of the Arid1a gene in mice caused a reduction in islet numbers and insulin production, both of which are associated with diabetes mellitus (DM) phenotype. RNA sequencing of isolated islets confirmed DM gene signature and decrease of developmental lineage genes. We identified neurogenin3, a transcription factor that controls endocrine fate specification, is a direct target of Aird1a. Gene set enrichment analysis indicated the enhancement of histone deacetylase (HDAC) pathway after Arid1a depletion and a clinically approved HDAC inhibitor showed therapeutic benefit by suppressing disease onset. Our data suggest that Arid1a is required for the development of pancreatic islets by regulating Ngn3+-mediated transcriptional program and is important in maintaining endocrine function.

Deciphering The Potential Role of Hox Genes in Pancreatic Cancer.

The Hox gene family plays an important role in organogenesis and animal development. Currently, 39 Hox genes that are clustered in four chromosome regions have been identified in humans. Emerging evidence suggests that Hox genes are involved in the development of the pancreas. However, the expression of Hox genes in pancreatic tumor tissues has been investigated in only a few studies. In addition, whether specific Hox genes can promote or suppress cancer metastasis is not clear. In this article, we first review the recent progress in studies on the role of Hox genes in pancreatic cancer. By comparing the expression profiles of pancreatic cancer cells isolated from genetically engineered mice established in our laboratory with three different proliferative and metastatic abilities, we identified novel Hox genes that exhibited tumor-promoting activity in pancreatic cancer. Finally, a potential oncogenic mechanism of the Hox genes was hypothesized.

ß-catenin-activated autocrine PDGF/Src signaling is a therapeutic target in pancreatic cancer.

K-ras mutation and p53 loss are the most prevalent genetic alterations in pancreatic cancer. In addition to these two alterations, pancreatic tumors frequently contain a third genetic defect. Mutations in the WNT/ß-catenin signaling molecules occur in 15-20% of pancreatic cancer patients and co-exist with K-ras mutation and p53 loss. However, the contribution of the WNT/ß-catenin pathway in pancreatic tumorigenesis is still unclear. Methods: We generated Pdx1-CreKrasG12Dp53L/+APCL/+ (KPA) mice and compared their phenotypes with Pdx1-CreKrasG12Dp53L/+ (KPC) mice. The signaling pathways specifically activated in the KPA mice were investigated and the therapeutic effect by targeting the activated pathways was evaluated. We finally validated our findings in human blood and tumor samples. Results: Survival of the KPA mice was shorter than that of the KPC mice. The KPA cancer cells are highly invasive and exhibit distorted morphology in organoid culture with extensive invadopodia formation and elevated matrix metalloproteinase (MMP) activity. The platelet-derived growth factor (PDGF) pathway is upregulated in the KPA cancer cells, and PDGF production induced by ß-catenin triggers constitutive activation of the Src kinase via the PDGF receptor in the cells. Serum PDGF concentration of the KPA mice is much higher than that of the normal and KPC mice. The Src inhibitor dasatinib effectively inhibits tumor growth and metastasis of the KPA cancer cells. Patient's serum PDGF level is significantly correlated with the expression of PDGF and phosphor-Src in tumors and elevated PDGF/phosphor-Src level in tumors predicts increased recurrence and poor survival. Moreover, mutations of the WNT/ß-catenin signaling molecules are higher in patients with elevated PDGF/phosphor-Src level. Conclusion: ß-catenin activation, coupled with K-ras mutation and p53 loss, activates an autocrine PDGF/Src signaling in pancreatic cancer and defines a subset of patients who might be sensitive to Src inhibition. In addition, serum PDGF level could be a reliable biomarker for patient selection in clinic.

Utilization of liquid chromatography mass spectrometry analyses to identify LKB1-APC interaction in modulating Wnt/ß-catenin pathway of lung cancer cells.

UNLABELLED: STK11/LKB1, a serine/threonine protein kinase and tumor suppressor, is a key upstream kinase of adenine monophosphate-activated protein kinase, which is a kinase involved in controlling cell polarity and maintaining cellular energy homeostasis. LKB1 is mutated in a significant number of Peutz-Jeghers syndrome (PJS) cases and sporadic cancers, and is most frequently mutated in lung adenocarcinomas; however, little is known about how LKB1 is involved in lung cancer progression. In this study, immunoprecipitation-HPLC tandem mass spectrometry (IP-LC-MS/MS) was performed to identify novel proteins interacting with LKB1 in lung cancer. Interestingly, many LKB1-interacting proteins acquired from the LC-MS/MS approach were mapped, using MetaCore pathway analysis, to the cystic fibrosis transmembrane conductance regulator activation pathway. Moreover, it was determined that LKB1 directly interacts with APC, and this LKB1-APC interaction was further confirmed by reverse immunoprecipitation assays, but GSK3ß was dispensable for the association of LKB1 and APC. Importantly, LKB1 binds to APC to suppress the Wnt/ß-catenin signaling pathway, which is known to be involved in cell proliferation and migration. Subsequent analysis of the downstream targets of the Wnt/TCF pathway led to the identification of several Wnt-regulated genes, such as CD44, COX-2, survivin, and c-Myc, whose expression levels are downregulated by LKB1. In summary, these results demonstrate that LKB1 regulates the Wnt pathway through a direct interaction with APC to suppress the tumorigenic/metastatic potential of lung tumors. IMPLICATIONS: LKB1 status influences the molecular circuitry (Wnt/ß-catenin pathway), cellular biology, and may serve as a potential therapeutic node in genetically defined subsets of lung cancer.

Stem cell marker nestin is critical for TGF-ß1-mediated tumor progression in pancreatic cancer.

The stem cell marker nestin is an intermediate filament protein that plays an important role in cell integrity, migration, and differentiation. Nestin expression occurs in approximately one third of pancreatic ductal adenocarcinoma (PDAC), and its expression strongly correlates with tumor staging and metastasis. Little is known about the mechanisms by which nestin influences PDAC progression. Here, nestin overexpression in PDAC cells increased cell motility and drove phenotypic changes associated with the epithelial-mesenchymal transition (EMT) in vitro; conversely, knockdown of endogenous nestin expression reduced the migration rate and reverted cells to a more epithelial phenotype. Mouse xenograft studies showed that knockdown of nestin significantly reduced tumor incidence and volume. Nestin protein expression was associated with Smad4 status in PDAC cells; hence, nestin expression might be regulated by the TGF-ß1/Smad4 pathway in PDAC. We examined nestin expression after TGF-ß1 treatment in human pancreatic cancer PANC-1 and PANC-1 shSmad4 cells. The TGF-ß1/Smad4 pathway induced nestin protein expression in PDAC cells in a Smad4-dependent manner. Moreover, increased nestin expression caused a positive feedback regulator of the TGF-ß1 signaling system. In addition, hypoxia was shown to induce nestin expression in PDAC cells, and the hypoxia-induced expression of nestin is mediated by the TGF-ß1/Smad4 pathway. Finally, the antimicrotubule inhibitors, cytochalasin D and withaferin A, exhibited anti-nestin activity; these inhibitors might be potential antimetastatic drugs. Our findings uncovered a novel role of nestin in regulating TGF-ß1-induced EMT. Anti-nestin therapeutics may serve as a potential treatment for PDAC metastasis.

The activation of MEK/ERK signaling pathway by bone morphogenetic protein 4 to increase hepatocellular carcinoma cell proliferation and migration.

Hepatocellular carcinoma (HCC) is one of the most common visceral malignancies worldwide, with a very high incidence and poor prognosis. Bone morphogenesis protein 4 (BMP4), which belongs to the TGF-ß superfamily of proteins, is a multifunctional cytokine, which exerts its biologic effects through SMAD- and non-SMAD-dependent pathways, and is also known to be involved in human carcinogenesis. However, the effects of the BMP4 signaling in liver carcinogenesis are not yet clearly defined. Here, we first show that BMP4 and its receptor, BMPR1A, are overexpressed in a majority of primary HCCs and that it promotes the growth and migration of HCC cell lines in vitro. We also establish that BMP4 can induce HCC cyclin-dependent kinase (CDK)1 and cyclin B1 upregulation to accelerate cell-cycle progression. Our study indicates that the induction of HCC cell proliferation is independent of the SMAD signaling pathway, as Smad4 knockdown of HCC cell lines still leads to the upregulation of CDK1 and cyclin B1 expression after BMP4 treatment. Using mitogen-activated protein/extracellular signal-regulated kinase (MEK) selective inhibitors, the induction of CDK1, cyclin B1 mRNA and protein were shown to be dependent on the activation of MEK/extracellular signal-regulated kinase (ERK) signaling. In vivo xenograft studies confirmed that the BMPR1A-knockdown cells were significantly less tumorigenic than the control groups. Our findings show that the upregulation of BMP4 and BMPR1A in HCC promotes the proliferation and metastasis of HCC cells and that CDK1 and cyclin B1 are important SMAD-independent molecular targets in BMP4 signaling pathways, during the HCC tumorigenesis. It is proposed that BMP4 signaling pathways may have potential as new therapeutic targets in HCC treatment.