Objectives Pancreatic cancer (PC) is highly aggressive with multiple oncogenic mutations. The efficacy of current chemotherapy is poor, and new therapeutic targets are needed. The forkhead box (FOX) proteins are multidirectional transcriptional factors strongly implicated in malignancies. Their expression is consistently suppressed by several oncogenic pathways such as PI3K/AKT signaling activated in PC. A recent study showed that class IIa histone deacetylases (HDAC) can act as a transcriptional suppressor. In this study, we hypothesized that HDAC class IIa inhibition would upregulate FOXO3a expression, thereby inducing its transcription-dependent antitumor effects. Methods We confirmed the change of FOXO3a expression and the effect of the cell growth inhibition by HDAC class IIa inhibition in AsPC-1 cells. Because FOXO3a is subject to ubiquitylation-mediated proteasome degradation, we examined the synergistic activation of FOXO3a by HDAC class IIa selective inhibitor TMP269 combined with proteasome inhibitor carfilzomib. Results We observed that TMP269 induced FOXO3a expression in a dose-dependent manner and inhibited cell growth in AsPC-1 cells. G1/S arrest was observed. FOXO3a expression was further increased and cell growth inhibition was dramatically enhanced by TMP269 combined with carfilzomib. Conclusions Dual inhibition of class IIa HDACs and proteasome could be a promising new strategy for modifying FOXO3a activity against PC.
Background and Aim: Reliable predictors for hepatocellular carcinoma (HCC) are urgently needed. The psoas muscle index (PMI) is a simple and rapid method for evaluating muscle atrophy. Furthermore, the neutrophil/lymphocyte ratio (NLR) is a prognostic factor that is easy to calculate in everyday clinical practice. We aimed to investigate the value of the PMI and NLR as prognostic factors for patients receiving nonsurgical HCC therapy, hepatic arterial infusion chemotherapy (HAIC), transcatheter arterial chemoembolization (TACE), or molecular targeted drugs such as sorafenib (SOR) and lenvatinib (LEN). Methods: We enrolled 87 patients with HCC who were treated with HAIC, TACE, SOR, or LEN. The primary endpoint was overall survival (OS) with variable PMI or NLR status. For Barcelona Clinic Liver Cancer (BCLC)-B patients, useful prognostic factors were examined by comparing the OS between stratified groups. Prognostic factors including PMI and NLR were evaluated by univariate and multivariate analysis.Results: Analysis of HAIC or TACE (HAIC/TACE) and SOR or LEN (SOR/LEN) patients showed significant differences in OS between low and high PMI. In patients treated with TACE, there was a significant difference in OS between low and high NLR. For BCLC-B and low PMI, the prognosis was significantly worse for SOR/LEN than for TACE, although there was no difference for high PMI, suggesting that PMI may be useful for treatment selection. In addition, the prognostic formula composed of PMI, NLR, and up-to-seven criteria developed in the present study may be useful. Conclusion: PMI and NLR are considered to be independent prognostic factors for HCC.
Background. It has been reported that iron absorption from gastrointestinal tract was enhanced in a subset of patients with myelodysplastic syndrome (MDS) exhibiting ineffective erythropoiesis. Iron absorption was achieved via an iron transporter ferroportin which was downregulated by hepcidin. Recently, three erythroid regulators such as growth differentiation factor 15 (GDF15), twisted gastrulation protein homolog 1 (TWSG1) and erythroferrone (ERFE) which down regulated hepatic hepcidin production has been identified. However, it has been not yet clarified which molecules could contribute to the increased iron absorption in patients with MDS. Materials and Methods. In the present study, we examined the expression level of GDF15, TWSG1 and ERFE mRNA during ex vivo erythroid differentiation from CD34+ bone marrow (BM) cells in the presence of 4 U/mL erythropoietin (EPO), 100 U/mL interleukin-3, 10 ng/mL stem cell factor, 20 ng/mL insulin-like growth factor (IGF)-1 and 500 micro g/mL iron-saturated transferrin. We further analyzed the expression level of GDF15 and ERFE in BM mononuclear cells (MNCs) derived from BM derived from MDS patients and lymphoma patients without BM involvement as a control by using quantitive RT-PCR. The expression of EPO-R was analyzed by flow cytometry. The CD34+ MDS cells were seeded on fibronectin substratum in 5 mL of a serum-free medium supplemented with 50 ng/mL human thrombopoietin (TPO), 10 ng/mL human SCF, 50 ng/mL human Fms-related tyrosin kinase 3 ligand (FLT3LG) and 100 ng/mL human delta like protein 4 (DLL4) with or without 4 U/mL EPO. For analysis of CD34+ cells, a GEO dataset (GSE58831) was downloaded as a matrix by GEOquery package (Bioconductor). The numerical data of the matrix were normalized by quantile normalization using limma package. Clinical and sequencing data were downloaded from supplementary materials. Those were combined with a GEO dataset (GSE58831) before analysis. Results. The level of ERFE mRNA was dramatically increased during erythroid differentiation from control CD34+ cells in response to EPO in vitro (Figure 1) although increase of the level of GDF15 and TWSG1 was marginal. Moreover, the level of ERFE mRNA in BM MNCs derived from MDS patients was significantly higher than that from control. Furthermore, the expression level of ERFE mRNA correlated with the percentage of CD34+ cells, but not percentage of erythroblasts derived from MDS patients. Using GEO data sets (GSE58831), the level of ERFE mRNA in CD34+ cells derived from MDS patients was significantly elevated as compared with that from healthy volunteers. Importantly, flow cytometric analysis indicated that CD34+ MDS cells highly expressed EPO receptors and the level of ERFE mRNA in CD34+ cells in a subset of MDS patients was enhanced after exposure of EPO ex vivo. In addition, the level of ERFE mRNA positively correlated with the level of EPO-R in CD34+ MDS cells (GSE58831). Conclusion. These results indicated that CD34+/EPO-R+ double positive MDS cells is one of the major sources of ERFN. The level of ERFE in CD34+ MDS cells may be associated with abnormal iron metabolism in MDS patients. These finding may be important for understanding the abnormal iron metabolism and predicting the efficacy of EPO administration. Disclosures No relevant conflicts of interest to declare.
Pancreatic cancer is highly chemo-resistant associated with oncogenic mutations such as KRAS and/or p53. The effect of conventional chemotherapy is not sufficient and new target and strategy is urgently needed. The forkhead box (Fox) proteins are multidirectional transcriptional factors strongly implicated in malignancies. Although Fox O (FoxO) protein, and particularly FoxO3a, works as negative regulator of cell proliferation by repressing cyclin proteins and inducing cell cycle inhibitors such as p21Waf1/Cip1, its expression is consistently suppressed by several oncogenic pathways including phosphatidylinositol-3 kinase (PI3K) / AKT pathway, constitutively activated in pancreatic cancer. Thus, upregulating FoxO3a activity could be a promising target of pancreatic cancer treatment without impact of underlying oncogenic mutations. Class IIa Histone deacetylase (HDAC) is a subgroup of HDAC. Though HDAC inhibitors (HDACi) have been extensively investigated as a cancer target, its action mechanism is considered due to histone modification by class I. Biological significance of class IIa HDACs which have minimal histone deacetylation activity have not been elucidated yet. Recent studies show class IIa HDACs act as a transcriptional regulator including FoxO3a. In this study, we investigate the biologic impact of HDAC class IIa inhibition on FoxO3a and anti-tumor effect against pancreatic cancer cell line using selective class IIa HDACi TMP269. TMP269 treatment showed increased FoxO3a expression in a dose dependent manner with immunoblotting and modest cell growth inhibition effect at 57.5 μM of IC50 dose for 48-hour treatment against AsPC-1 in MTT. G1/S arrest was observed with cell cycle assay. Upregulated p21Waf1/Cip1 and downregulated CDK2 and 4/6 and cyclin D1 and D2 expressions were further observed, consistent with inducing G1/S arrest and transcriptionally activated FoxO3a. Importantly, upregulated p21Waf1/Cip1 was observed in AsPC-1 p53 null cell line, suggesting independent with p53 pathway. These findings suggest upregulated FoxO3a induced by HDAC class IIa inhibition activated its transcription and resulted in cell growth inhibition. Because PI3K/AKT leads FoxO3a to the ubiquitylation-mediated proteasome degradation, we examined irreversible proteasome inhibitor carfilzomib (CFZ) combined with TMP269, aiming synergistic FoxO3a upregulating. As expected, FoxO3a expression was further increased in TMP269 combined with CFZ compared with TMP269 or CFZ alone. Following the activated FoxO3a, p21Waf1/Cip1 expression was upregulated and cell growth inhibition was dramatically enhanced. In conclusion, HDAC class IIa inhibition modified FoxO3a transcriptional activation and upregulating FoxO3 by dual inhibition of HDAC class IIa and proteasome is promising target against pancreas cancer. Citation Format: Makoto Usami, Shohei Kikuchi, Kohichi Takada, Yusuke Sugama, Yohei Arihara, Naotaka Hayasaka, Hajime Nakamura, Yuki Ikeda, Yusuke Kamihara, Masahiro Hirakawa, Makoto Yoshida, Masayoshi Kobune, Koji Miyanishi, Junji Kato. FoxO3a activation by HDAC class IIa inhibition induces cell cycle arrest in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2112. doi:10.1158/1538-7445.AM2017-2112
Background. It has been shown that bone marrow (BM) microenvironment and physiological hematopoiesis were disturbed during development of acute myelogenous leukemia (AML). However, little is known the molecular mechanism involved in this process. We have recently demonstrated that high number of extracellular vesicles (EVs) including exosomes exist in BM interstitial fluid. Myeloid neoplasm derived EVs carry miRNAs and transmitted into mesenchymal stem cells (MSCs) (Highlights of ASH 2015, GSE64029). A subset of extracellular vesicular miRNAs including miR-7977 was increased in BM cavity and regulate the expression level of mRNA stabilizer poly(rC) binding protein 1 (PCBP1) and modulated the function of hematopoietic supporting capacity of MSCs. On the other hand, a relatively large subset of extra vesicular miRNAs was actually decreased in BM cavity of AML as compared with that of healthy volunteer. However, the clinical significance of these decreased miRNAs is unresolved. In the present study, miRNA pathway analysis was conducted to elucidate the role of decreased miRNAs in BM environment of AML. Methods. To harvest EVs from 2 x 105primary AML CD34+ cells, normal BM CD34+ cells and leukemic cell lines including TF-1 and Kasumi-1, cells were cultured in serum-free StemPro®-34 medium with a cytokine cocktail on plates coated with fibronectin fragments. EV miRNA from the supernatant of CD34+ hematopoietic and leukemic cells were prepared. Microarray analysis of the miRNA profiles was done with the human miRNA Oligo chip (Human_miRNA_V20) and the 3D-Gene® miRNA labeling kit. For analysis of transcriptome in CD34+/CD38- normal and AML cells, data sets (GSE24395) was downloaded as a matrix by GEOquery package (Bioconductor, R commander version 3.2.2). The data sets (GSE24395) and our data sets (GSE64029) were normalized by rma method using limma package before analysis. Clustering analysis were performed with amap package to find a subset of miRNA decreased. For miRNA pathway analysis, DIANA-mirPath version 3 was utilized. Results. Thirty EV miRNAs derived from AML were decreased as compared with those from normal CD34+ cells. Especially, miR-92a-3p, miR-125a-3p, miR-4448, miR-4484 and miR-4270 were remarkably reduced. The miRNA pathway analysis indicated that a KEGG pathway (hsa04520), adherens junction strongly correlated with these decreased miRNAs (P=0.00000025). The miRNAs including miR-4448, miR-4484 and miR-4270 could be associated with downstream molecules of adherens junction including beta-catenin, IGF-1R, WAVE, Vinculin, TGF-beta and Smad4. Importantly, we found that CD34+CD38- AML stem cells highly expressed JAM family molecules (JAM2 and JAM3) associated with adherens junction (GSE24395). These results suggested that these miRNAs regulate the pathway of adherens junction in BM microenvironment. In addition, adherens junction could be enhanced concomitant with reduction of miRNAs released from AML. Conclusion. EV miRNAs derived from AML are involved in the regulation of the pathway of adherens junction in BM microenvironment. Adherens junction is known to contribute to quiescence, apoptosis and drug resistance via HIPPO signaling pathway, EV miRNAs may have an important role on the development and drug resistance of AML. Disclosures No relevant conflicts of interest to declare.
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