Dacheng Xie

FOXM1 Promotes the Warburg Effect and Pancreatic Cancer Progression via Transactivation of LDHA Expression

Purpose: The transcription factor Forkhead box protein M1 (FOXM1) plays critical roles in cancer development and progression. However, the regulatory role and underlying mechanisms of FOXM1 in cancer metabolism are unknown. In this study, we characterized the regulation of aerobic glycolysis by FOXM1 and its impact on pancreatic cancer metabolism. Experimental Design: The effect of altered expression of FOXM1 on expression of glycolytic enzymes and tumor development and progression was examined using animal models of pancreatic cancer. Also, the underlying mechanisms of altered pancreatic cancer glycolysis were analyzed using in vitro molecular biology. The clinical relevance of aberrant metabolism caused by dysregulated FOXM1 signaling was determined using pancreatic tumor and normal pancreatic tissue specimens. Results: We found that FOXM1 did not markedly change the expression of most glycolytic enzymes except for phosphoglycerate kinase 1 (PGK-1) and lactate dehydrogenase A (LDHA). FOXM1 and LDHA were overexpressed concomitantly in pancreatic tumors and cancer cell lines. Increased expression of FOXM1 upregulated the expression of LDHA at both the mRNA and protein level and elevated LDH activity, lactate production, and glucose utilization, whereas reduced expression of FOXM1 did the opposite. Further studies demonstrated that FOXM1 bound directly to the LDHA promoter region and regulated the expression of the LDHA gene at the transcriptional level. Also, elevated FOXM1–LDHA signaling increased the pancreatic cancer cell growth and metastasis. Conclusions: Dysregulated expression and activation of FOXM1 play important roles in aerobic glycolysis and tumorigenesis in patients with pancreatic cancer via transcriptional regulation of LDHA expression. Clin Cancer Res; 20(10); 2595–606. ©2014 AACR.

Down-regulation of MicroRNA-494 via Loss of SMAD4 Increases FOXM1 and β-Catenin Signaling in Pancreatic Ductal Adenocarcinoma Cells

BACKGROUND & AIMS Dysregulation of β-catenin and the transcriptional activator FOXM1 mediate oncogenesis, but it is not clear how these proteins become dysregulated in tumors that do not typically carry mutations in adenomatous polyposis coli (APC) or β-catenin, such as pancreatic ductal adenocarcinomas (PDACs). We searched for microRNAs that regulate levels of FOXM1 in PDAC cells and samples from patients. METHODS We identified microRNAs that affect levels of FOXM1 in PDACs using bioinformatic, genetic, and pharmacologic approaches. We altered expression of the microRNA-494 (miR-494) in PDAC cell lines (AsPC-1 and PANC-1) and examined the effects on FOXM1 and β-catenin signaling and cell proliferation and colony formation. The cells were injected into immunocompromised mice and growth of xenograft tumors and liver metastases were measured. We performed immunohistochemical analyses of 10 paired PDAC and nontumor pancreatic tissue samples collected from untreated patients during surgery. RESULTS We identified miR-494 as a negative regulator of FOXM1 levels in PDAC cells, and found that levels of this microRNA were reduced in PDAC specimens, compared with nontumor tissues. Loss of response of PDAC cells to transforming growth factor β, owing to SMAD4 deficiency, reduced expression of miR-494. Transgenic expression of miR-494 in PDAC cells produced the same effects as reducing expression of FOXM1 or blocking nuclear translocation of β-catenin, reducing cell proliferation, migration, and invasion, and increasing their sensitivity to gemcitabine. Reduced expression of miR-494 correlated with PDAC metastasis and reduced survival times of patients. CONCLUSIONS Loss of SMAD4 in PDAC cells leads to reduced levels of miR-494, increased levels of FOXM1, and nuclear localization of β-catenin. miR-494 might be developed as a prognostic marker for patients with PDAC or a therapeutic target.

FOXM1c Promotes Pancreatic Cancer Epithelial-to-Mesenchymal Transition and Metastasis via Upregulation of Expression of the Urokinase Plasminogen Activator System

Purpose: The transcription factor Forkhead box M1 (FOXM1) plays important roles in the formation of several human tumors, including pancreatic cancer. However, the molecular mechanisms by which FOXM1 promotes pancreatic tumor epithelial-to-mesenchymal transition (EMT) and metastasis are unknown. Experimental Design: The effect of altered expression of FOXM1 and urokinase-type plasminogen activator receptor (uPAR) on EMT and metastasis was examined using animal models of pancreatic cancer. Also, the underlying mechanisms of altered pancreatic cancer invasion and metastasis were analyzed using in vitro molecular biology assays. Finally, the clinical relevance of dysregulated FOXM1/uPAR signaling was investigated using pancreatic tumor and normal pancreatic tissue specimens. Results: Pancreatic tumor specimens and cell lines predominantly overexpressed the FOXM1 isoform FOXM1c. FOXM1c overexpression promoted EMT in and migration, invasion, and metastasis of pancreatic cancer cells, whereas downregulation of FOXM1 expression inhibited these processes. The level of FOXM1 expression correlated directly with that of uPAR expression in pancreatic cancer cell lines and tumor specimens. Moreover, FOXM1c overexpression upregulated uPAR expression in pancreatic cancer cells, whereas inhibition of FOXM1 expression suppressed uPAR expression. Furthermore, transfection of FOXM1c into pancreatic cancer cells directly activated the uPAR promoter, whereas inhibition of FOXM1 expression by FOXM1 siRNA suppressed its activation in these cells. Finally, we identified an FOXM1-binding site in the uPAR promoter and demonstrated that FOXM1 protein bound directly to it. Deletion mutation of this site significantly attenuated uPAR promoter activity. Conclusions: Our findings demonstrated that FOXM1c contributes to pancreatic cancer development and progression by enhancing uPAR gene transcription, and thus, tumor EMT and metastasis. Clin Cancer Res; 20(6); 1477–88. ©2014 AACR.

Activation of Vitamin D Receptor Signaling Downregulates the Expression of Nuclear FOXM1 Protein and Suppresses Pancreatic Cancer Cell Stemness

Purpose: Dysregulated signaling of nuclear transcription factors vitamin D receptor (VDR) and Forkhead box M1 (FOXM1) plays important roles in transformation and tumorigenesis. In this study, we sought to determine whether VDR signaling causally affected FOXM1 signaling in and pathogenesis of pancreatic ductal adenocarcinoma (PDAC). Experimental Design: Genetic and pharmacologic approaches were used to manipulate VDR signaling. The impacts of altered VDR signaling on FOXM1 expression and function in PDAC cells were determined using molecular and biochemical methods, whereas those on PDAC cell biology and tumorigenicity were determined using in vitro and in vivo experimental systems. The clinical relevance of our findings was validated by analyzing human PDAC specimens. Results: There was a striking inverse correlation between reduced expression of VDR and increased expression of FOXM1 in human PDAC cells and tissues. Treatment of PDAC cells with 1,25-dihydroxyvitamin D3 (1,25D), its synthetic analogue EB1089 (EB), and VDR transgenics drastically inhibited FOXM1 signaling and markedly suppressed tumor stemness, growth, and metastasis. Mechanistically, 1,25D and EB repressed FOXM1 transcription and reduced the expression level of nuclear FOXM1 protein. Conclusion: Inactivation of Vitamin D/VDR signaling is a critical contributor to PDAC development and progression via elevated expression and function of FOXM1 and enhanced PDAC cell stemness, invasion, and metastasis. Clin Cancer Res; 21(4); 844–53. ©2014 AACR.

Hippo transducer TAZ promotes epithelial mesenchymal transition and supports pancreatic cancer progression.

Transcriptional co-activator with PDZ binding motif (TAZ) is a transducer of the Hippo pathway and promotes cancer development and progression. In the present study, we sought to determine the roles and underlying mechanisms of elevated expression and activation of TAZ in pancreatic cancer development and progression. The mechanistic role of TAZ and Hippo signaling in promotion of pancreatic cancer development and progression was examined using cell culture, molecular biology, and mouse models. The relevance of our experimental and mechanistic findings was validated using human pancreatic tumor specimens. We found that TAZ expression was markedly higher in pancreatic tumors than in normal pancreatic tissue. Further analysis of the correlation of TAZ expression with tissue microarray clinicopathologic parameters revealed that this expression was positively associated with tumor differentiation. Also, TAZ expression was higher in pancreatic cancer cell lines than in pancreatic ductal epithelial cells. TAZ activation in pancreatic cancer cells promoted their proliferation, migration, invasion, and epithelial-mesenchymal transition. Further mechanistic studies demonstrated that aberrant expression and activation of TAZ in pancreatic cancer cells resulted from suppression of the expression of Merlin, a positive regulator upstream of the Hippo pathway, and that the oncogenic function of TAZ in pancreatic cancer cells was mediated by TEA/ATTS domain transcription factors. Therefore, TAZ functioned as an oncogene and promoted pancreatic cancer epithelial-mesenchymal transition and progression. TAZ thus may be a target for effective therapeutic strategies for pancreatic cancer.

The Novel KLF4/MSI2 Signaling Pathway Regulates Growth and Metastasis of Pancreatic Cancer.

Purpose: Musashi 2 (MSI2) is reported to be a potential oncoprotein in cases of leukemia and several solid tumors. However, its expression, function, and regulation in pancreatic ductal adenocarcinoma (PDAC) cases have yet to be demonstrated. Therefore, in the current study, we investigated the clinical significance and biologic effects of MSI2 expression in PDAC cases and sought to delineate the clinical significance of the newly identified Krüppel-like factor 4 (KLF4)/MSI2 regulatory pathway. Experimental Design: MSI2 expression and its association with multiple clinicopathologic characteristics in human PDAC specimens were analyzed immunohistochemically. The biological functions of MSI2 regarding PDAC cell growth, migration, invasion, and metastasis were studied using gain- and loss-of-function assays both in vitro and in vivo. Regulation of MSI2 expression by KLF4 was examined in several cancer cell lines, and the underlying mechanisms were studied using molecular biologic methods. Results: MSI2 expression was markedly increased in both PDAC cell lines and human PDAC specimens, and high MSI2 expression was associated with poor prognosis for PDAC. Forced MSI2 expression promoted PDAC proliferation, migration, and invasion in vitro and growth and metastasis in vivo, whereas knockdown of MSI2 expression did the opposite. Transcriptional inhibition of MSI2 expression by KLF4 occurred in multiple PDAC cell lines as well as mouse models of PDAC. Conclusions: Lost expression of KLF4, a transcriptional repressor of MSI2 results in overexpression of MSI2 in PDACs, which may be a biomarker for accurate prognosis. A dysregulated KLF4/MSI2 signaling pathway promotes PDAC progression and metastasis. Clin Cancer Res; 23(3); 687–96. ©2016 AACR.

Merlin/NF2 Suppresses Pancreatic Tumor Growth and Metastasis by Attenuating the FOXM1-Mediated Wnt/β-Catenin Signaling

Merlin, the protein encoded by the NF2 gene, is a member of the band 4.1 family of cytoskeleton-associated proteins and functions as a tumor suppressor for many types of cancer. However, the roles and mechanism of Merlin expression in pancreatic cancer have remained unclear. In this study, we sought to determine the impact of Merlin expression on pancreatic cancer development and progression using human tissue specimens, cell lines, and animal models. Decreased expression of Merlin was pronounced in human pancreatic tumors and cancer cell lines. Functional analysis revealed that restored expression of Merlin inhibited pancreatic tumor growth and metastasis in vitro and in vivo. Furthermore, Merlin suppressed the expression of Wnt/β-catenin signaling downstream genes and the nuclear expression of β-catenin protein, and overexpression of Forkhead box M1 (FOXM1) attenuated the suppressive effect of Merlin on Wnt/β-catenin signaling. Mechanistically, Merlin decreased the stability of FOXM1 protein, which plays critical roles in nuclear translocation of β-catenin. Collectively, these findings demonstrated that Merlin critically regulated pancreatic cancer pathogenesis by suppressing FOXM1/β-catenin signaling, suggesting that targeting novel Merlin/FOXM1/β-catenin signaling is an effective therapeutic strategy for pancreatic cancer.

Krüppel-like Factor 4 Blocks Hepatocellular Carcinoma Dedifferentiation and Progression through Activation of Hepatocyte Nuclear Factor-6.

Purpose: Tumor differentiation is a behavioral index for hepatocellular carcinoma (HCC) and a prognostic factor for patients with HCC who undergo orthotopic liver transplantation (OLT). However, the molecular basis for HCC differentiation and prognostic value of the underlying molecules that regulate HCC differentiation are unclear. In this study, we defined a potential driver pathway for HCC differentiation and prognostication. Experimental Design: The regulation and function of Krüppel-like factor 4 (KLF4) and hepatocyte nuclear factor-6 (HNF-6) in HCC differentiation was evaluated using human tissues, molecular and cell biology, and animal models, and its prognostic significance was determined according to its impact on patient survival. Results: There was a direct relationship between the expression levels of KLF4 and HNF6 in HCC. Reduced KLF4 or HNF6 expression correlated with high HCC grade. Poorly differentiated HCC cells had lower expression of KLF4 or HNF6 and differentiation-associated markers than did well-differentiated cells. Elevated KLF4 of HNF6 expression induced differentiation of poorly differentiated hepatoma cells. Mechanistically, KLF4 trans-activated HNF-6 expression. Restored HNF-6 expression upregulated expression of differentiation-associated markers and inhibited HCC cell migration and invasion, whereas HNF-6 knockdown did the opposite. Loss of KLF4 expression in primary HCC correlated with reduced overall survival and shortened relapse-free survival durations after OLT. Combination of KLF4 expression and the Milan criteria improved prognostication for HCC after OLT. Conclusions: The dysregulated KLF4/HNF-6 pathway drives dedifferentition and progression of HCC, and KLF4 is a biomarker for accurate prognostication of patients with HCC treated by OLT when integrated with the Milan Criteria. Clin Cancer Res; 22(2); 502–12. ©2015 AACR.

Targeting miRNAs for Pancreatic Cancer Therapy

Pancreatic cancer (PC) is the fourth leading cause of cancer-related deaths in the United States and has a median 5-year survival rate less than 5%. Although surgery offers the best chance for a cure for pancreatic cancer, less than 20% of patients are eligible for potentially curative resection, because in most cases, the cancer has already spread locally or to distant organs at diagnosis, precluding resection. MicroRNAs (miRNAs) are small noncoding, endogenous, single-stranded RNAs that are pivotal regulators of posttranscriptional gene expression. Extensive studies of miRNAs over the past several years have revealed that the expression of miRNAs is frequently deregulated in pancreatic cancer patients and that this deregulation contributes to the pathogenesis and aggressiveness of the disease. Currently, investigators are studying the use of miRNAs as diagnostic and/or prognostic biomarkers and therapeutic tools for pancreatic cancer. Rapid discovery of many miRNA targets and their relevant pathways has contributed to the development of miRNA-based therapeutics. In particular, the transcription factor Forkhead box M1 (FOXM1) is overexpressed in the majority of cancer patients, including those with pancreatic cancer. This overexpression is implicated to have a role in tumorigenesis, progression, and metastasis. This important role of FOXM1 affirms its usefulness in therapeutic interventions for pancreatic cancer. In this review, we summarize the current knowledge and concepts concerning the involvement of miRNAs and FOXM1 in pancreatic cancer development and describe the roles of the miRNA-FOXM1 signaling pathway in pancreatic cancer initiation and progression. Additionally, we describe some of the technical challenges in the use of the miRNA-FOXM1 signaling pathway in pancreatic cancer treatment.

Loss of KLF4 and consequential downregulation of Smad7 exacerbate oncogenic TGF-β signaling in and promote progression of hepatocellular carcinoma

Hyperactivation of transforming growth factor-β (TGF-β) signaling pathway is a common feature of hepatocellular carcinoma (HCC) progression. However, the driver factors leading to enhanced TGF-β activity are not well characterized. Here, we explore the mechanisms that loss of Krüppel-like factor 4 (KLF4) exacerbates oncogenic TGF-β signaling in human HCC. The expression of KLF4 and TGF-β signaling components in primary HCC and their clinicopathologic relevance and significance was evaluated by using tissue microarray and immunohistochemistry. Cellular and molecular impacts of altered KLF4 expression and TGF-β signaling were determined using immunofluorescence, western blot, reverse-transcriptase PCR, chromatin immunoprecipitation and promoter reporter assays. Loss of KLF4 expression in primary HCC closely correlated with decreased Smad7 expression, increased p-Smad2/3 expression and independently predicts reduced overall and relapse-free survival after surgery. TGF-β signaling components were expressed in most HCC cells, and activation of TGF-β signaling promoted cell migration and invasion. Enforced KLF4 expression blocked TGF-β signal transduction and inhibited cell migration and invasion via activation of Smad7 transcription, whereas deletion of its C-terminal zinc-finger domain diminished this effect. KLF4 protein physically interacts with the Smad7 promoter. Promoter deletion and point mutation analyses revealed that a region between nucleotides –15 bp and –9 bp of the Smad7 promoter was required for the induction of Smad7 promoter activity by KLF4. Our data indicate that KLF4 suppresses oncogenic TGF-β signaling by activation of Smad7 transcription, and that loss of KLF4 expression in primary HCC may contribute to activation of oncogenic TGF-β signaling and subsequent tumor progression.