Takeo Shimasaki

Deregulated GSK3β Sustains Gastrointestinal Cancer Cells Survival by Modulating Human Telomerase Reverse Transcriptase and Telomerase

Purpose: Glycogen synthase kinase-3 (GSK3) regulates multiple cell signaling pathways and has been implicated in glucose intolerance, neurodegenerative disorders, and inflammation. We investigated the expression, activity, and putative pathologic role of GSK3 in gastrointestinal, pancreatic, and liver cancers. Experimental Design: Colon, stomach, pancreatic, and liver cancer cell lines; nonneoplastic HEK293 cells; and matched pairs of normal and tumor tissues of stomach and colon cancer patients were examined for GSK3 expression and its phosphorylation at serine 9 (inactive form) and tyrosine 216 (active form) by Western immunoblotting and for GSK3 activity by in vitro kinase assay. The effects of small-molecule GSK3 inhibitors and of RNA interference on cell survival, proliferation, and apoptosis were examined in vitro and on human colon cancer cell xenografts in athymic mice. The effects of GSK3 inhibition on human telomerase reverse transcriptase (hTERT) expression and telomerase activity were compared between colon cancer and HEK293 cells. Results: Cancer cell lines and most cancer tissues showed increased GSK3 expression and increased tyrosine 216 phosphorylation and activity but decreased serine 9 phosphorylation compared with HEK293 cells and nonneoplastic tissues. Inhibition of GSK3 resulted in attenuated cell survival and proliferation and increased apoptosis in most cancer cell lines and in HT-29 xenografts in rodents but not in HEK293 cells. GSK3 inhibition in colon cancer cells was associated with decreased hTERT expression and telomerase activity. Conclusion: The results indicate that deregulated GSK3 sustains gastrointestinal cancer cells survival through modulation of hTERT and telomerase. (Clin Cancer Res 2009;15(22):68109)

An Emerging Strategy for Cancer Treatment Targeting Aberrant Glycogen Synthase Kinase 3β

Improvement in the outcome of cancer patients who are refractory to currently available treatments relies on the development of target-directed therapies. One group of molecular targets with potential clinical relevance is a set of protein tyrosine kinases encoded mostly by proto-oncogenes and that are frequently deregulated in cancer. Glycogen synthase kinase 3beta (GSK3beta), a serine/threonine protein kinase, has emerged as a therapeutic target for common chronic diseases including type 2 diabetes mellitus, neurodegenerative disorders, inflammation and osteoporosis. This is based on its currently known functions and primary pathologic causalities. GSK3beta has well characterized roles in the regulation of gene transcription and in oncogenic signaling. We have shown that deregulated GSK3beta promotes gastrointestinal, pancreatic and liver cancers and glioblastomas. Furthermore, we have demonstrated that inhibition of GSK3beta attenuates cell survival and proliferation, induces cell senescence and apoptosis and sensitizes tumor cells to chemotherapeutic agents and ionizing radiation. This has led us to propose GSK3beta as a potential therapeutic target in cancer. The anti-tumor effects of GSK3beta inhibition are mediated by changes in the expression and phosphorylation of molecules critical to the regulation of cell cycling, proliferation and apoptosis and underlie the pathological role for GSK3beta in cancer. Investigation of the mechanisms responsible for deregulation of GSK3beta and the consequent downstream pathologic effects in cancer cells has shed light on the molecular pathways leading to tumorigenesis. This will allow exploration of novel therapeutic strategies for cancer that target aberrant GSK3beta.

Aberrant Glycogen Synthase Kinase 3β Is Involved in Pancreatic Cancer Cell Invasion and Resistance to Therapy

Background and Purpose The major obstacles to treatment of pancreatic cancer are the highly invasive capacity and resistance to chemo- and radiotherapy. Glycogen synthase kinase 3β (GSK3β) regulates multiple cellular pathways and is implicated in various diseases including cancer. Here we investigate a pathological role for GSK3β in the invasive and treatment resistant phenotype of pancreatic cancer. Methods Pancreatic cancer cells were examined for GSK3β expression, phosphorylation and activity using Western blotting and in vitro kinase assay. The effects of GSK3β inhibition on cancer cell survival, proliferation, invasive ability and susceptibility to gemcitabine and radiation were examined following treatment with a pharmacological inhibitor or by RNA interference. Effects of GSK3β inhibition on cancer cell xenografts were also examined. Results Pancreatic cancer cells showed higher expression and activity of GSK3β than non-neoplastic cells, which were associated with changes in its differential phosphorylation. Inhibition of GSK3β significantly reduced the proliferation and survival of cancer cells, sensitized them to gemcitabine and ionizing radiation, and attenuated their migration and invasion. These effects were associated with decreases in cyclin D1 expression and Rb phosphorylation. Inhibition of GSK3β also altered the subcellular localization of Rac1 and F-actin and the cellular microarchitecture, including lamellipodia. Coincident with these changes were the reduced secretion of matrix metalloproteinase-2 (MMP-2) and decreased phosphorylation of focal adhesion kinase (FAK). The effects of GSK3β inhibition on tumor invasion, susceptibility to gemcitabine, MMP-2 expression and FAK phosphorylation were observed in tumor xenografts. Conclusion The targeting of GSK3β represents an effective strategy to overcome the dual challenges of invasiveness and treatment resistance in pancreatic cancer.

Scanning electron microscopy with an ionic liquid reveals the loss of mitotic protrusions of cells during the epithelial-mesenchymal transition.

Epithelial–mesenchymal transition (EMT) is a key event in cancer metastasis and is characterized by increase in cell motility, increase in expression of mesenchymal cell markers, loss of proteins from cell‐to‐cell junction complexes, and changes in cell morphology. Here, the morphological effects of a representative EMT inducer, transforming growth factor (TGF)‐β1, were investigated in human lung adenocarcinoma (A549) cells and pancreatic carcinoma (Panc‐1) cells. TGF‐β1 caused morphological changes characteristic of EMT, and immunostaining showed loss of E‐cadherin from cell‐to‐cell junction complexes in addition to the upregulation of the mesenchymal marker vimentin. During scanning electron microscopy (SEM) with an ionic liquid, we observed EMT‐specific morphological changes, including the formation of various cell protrusions. Interestingly, filopodia in mitotic cells were clearly observed by SEM, and the number of these filopodia in TFG‐β1‐treated mitotic cells was reduced significantly. We conclude that this reduction in such mitotic protrusions is a novel effect of TGF‐β1 and may contribute to EMT. Microsc. Res. Tech., 2011.

Glycogen synthase kinase-3β is a pivotal mediator of cancer invasion and resistance to therapy.

Tumor cell invasion and resistance to therapy are the most intractable biological characteristics of cancer and, therefore, the most challenging for current cancer research and treatment paradigms. Refractory cancers, including pancreatic cancer and glioblastoma, show an inextricable association between the highly invasive behavior of tumor cells and their resistance to chemotherapy, radiotherapy and targeted therapies. These aggressive properties of cancer share distinct cellular pathways that are connected to each other by several molecular hubs. There is increasing evidence to show that glycogen synthase kinase (GSK)‐3β is aberrantly activated in various cancer types and this has emerged as a potential therapeutic target. In many but not all cancer types, aberrant GSK3β sustains the survival, immortalization, proliferation and invasion of tumor cells, while also rendering them insensitive or resistant to chemotherapeutic agents and radiation. Here we review studies that describe associations between therapeutic stimuli/resistance and the induction of pro‐invasive phenotypes in various cancer types. Such cancers are largely responsive to treatment that targets GSK3β. This review focuses on the role of GSK3β as a molecular hub that connects pathways responsible for tumor invasion and resistance to therapy, thus highlighting its potential as a major cancer therapeutic target. We also discuss the putative involvement of GSK3β in determining tumor cell stemness that underpins both tumor invasion and therapy resistance, leading to intractable and refractory cancer with dismal patient outcomes.

Glycogen synthase kinase 3β inhibition sensitizes pancreatic cancer cells to gemcitabine

BackgroundPancreatic cancer is obstinate and resistant to gemcitabine, a standard chemotherapeutic agent for the disease. We previously showed a therapeutic effect of glycogen synthase kinase-3β (GSK3β) inhibition against gastrointestinal cancer and glioblastoma. Here, we investigated the effect of GSK3β inhibition on pancreatic cancer cell sensitivity to gemcitabine and the underlying molecular mechanism.MethodsExpression, phosphorylation, and activity of GSK3β in pancreatic cancer cells (PANC-1) were examined by Western immunoblotting and in vitro kinase assay. The combined effect of gemcitabine and a GSK3β inhibitor (AR-A014418) against PANC-1 cells was examined by isobologram and PANC-1 xenografts in mice. Changes in gene expression in PANC-1 cells following GSK3β inhibition were studied by cDNA microarray and reverse transcription (RT)-PCR.ResultsPANC-1 cells showed increased GSK3β expression, phosphorylation at tyrosine 216 (active form), and activity compared with non-neoplastic HEK293 cells. Administration of AR-A014418 at pharmacological doses attenuated proliferation of PANC-1 cells and xenografts, and significantly sensitized them to gemcitabine. Isobologram analysis determined that the combined effect was synergistic. DNA microarray analysis detected GSK3β inhibition-associated changes in gene expression in gemcitabine-treated PANC-1 cells. Among these changes, RT-PCR and Western blotting showed that expression of tumor protein 53-induced nuclear protein 1, a gene regulating cell death and DNA repair, was increased by gemcitabine treatment and substantially decreased by GSK3β inhibition.ConclusionsThe results indicate that GSK3β inhibition sensitizes pancreatic cancer cells to gemcitabine with altered expression of genes involved in DNA repair. This study provides insight into the molecular mechanism of gemcitabine resistance and thus a new strategy for pancreatic cancer chemotherapy.

Depletion of RNA-binding protein RBM8A (Y14) causes cell cycle deficiency and apoptosis in human cells

RBM8A (Y14) contains an RNA-binding motif and forms a tight heterodimer with Magoh. The heterodimer is known to be a member of the exon junction complex that forms on mRNA before export and it is required for mRNA metabolism processes such as splicing, mRNA export and nonsense-mediated mRNA decay. Recently, deficient cellular proliferation has been observed in RBM8A- or Magoh-depleted cells. These results prompted us to study the role of RBM8A in cell cycle progression of human tumour cells. The depletion of RBM8A in A549 cells resulted in poor cell survival and the accumulation of mitotic cells. After release from G1/S arrest induced by a double thymidine block, the RBM8A-silenced cells could not proceed to the next G1 phase beyond G2/M phase. Finally, the sub-G1 population increased and the apoptosis markers caspases 3/7 were activated. Silenced cells exhibited an increased frequency of multipolar or monopolar centrosomes, which may have caused the observed deficiency in cell cycle progression. Finally, silencing of either RBM8A or Magoh resulted in mutual downregulation of the other protein. These results illustrate that the RBM8A-Magoh mRNA binding complex is required for M phase progression and both proteins may be novel targets for anticancer therapy.

Functional promoter polymorphisms of NFKB1 influence susceptibility to the diffuse type of gastric cancer

In the present study, we report an association between gastric cancer and polymorphisms in NFKB1 (rs28362941 and rs78696119). We employed the PCR-SSCP method to detect gene polymorphisms in 479 gastric cancer cases and 880 controls. The rs28362941 del/del homozygote was significantly associated with gastric cancer development; in particular it was closely associated with diffuse type gastric cancer. The rs78696119 GG homozygote was also associated with the diffuse type of gastric cancer. In young subjects, both polymorphisms were significantly associated with the development of gastric cancer. In addition, both polymorphisms were related to tumor progression such as tumor invasion and lymph node metastasis. The inflammatory cell infiltration into non-cancerous gastric mucosa was greater in the subjects with the rs28362491 del/del or rs78696119 GG genotype when compared to those with the other genotypes. In conclusion, functional polymorphisms of NFKB1 are associated with an increased risk of gastric cancer; in particular they are closely associated with the development of diffuse type of gastric cancer via severe gastric inflammation. These polymorphisms also appear to be associated with gastric cancer progression.

Human SMG-1 is involved in gemcitabine-induced primary microRNA-155/BIC up-regulation in human pancreatic cancer PANC-1 cells.

Objectives: Human primary microRNA-155/B-cell integration cluster (BIC) transcript is the precursor of microRNA-155. The overexpression of them has been widely observed in the progression of various types of tumors. Our objective was to investigate the effect of anticancer agents on the expression of BIC and possible signal pathways that involved in. Methods: Quantitative real-time reverse transcriptase polymerase chain reaction was used to measure the expression of BIC. Chemical inhibitors against c-Jun N-terminal kinase 1, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2, protein kinase C, checkpoint kinase 1, and phosphatidylinositol 3 kinase (PI3K) were used for the evaluation of involved signal pathways. RNA interference was used to knock down the expression of ataxia-telangiectasia mutated, ataxia-telangiectasia and Rad3 related, and suppressor of morphogenesis in genitalia-1 (SMG-1), and Western blot was carried out to evaluate the knockdown effect. Results: B-cell integration cluster expression was induced by a representative anti-pancreatic cancer drug, gemcitabine, in human pancreatic cancer PANC-1 cells. The mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 and c-Jun N-terminal kinase inhibitors, but not the checkpoint kinase 1 and protein kinase C inhibitors, suppressed the up-regulation of BIC. B-cell integration cluster up-regulation was also significantly inhibited by the PI3K inhibitor wortmannin. RNA interference studies showed that wortmannin-sensitive SMG-1 but not ataxia-telangiectasia mutated or ataxia-telangiectasia and Rad3 related was involved in the up-regulation. Conclusions: Our results show that multiple pathways can be involved in the up-regulation of BIC. Furthermore, we demonstrate for the first time that PI3K SMG-1 is required for gemcitabine-induced up-regulation of BIC transcript.Abbreviations: ACTB - &bgr;-actin, ATM - ataxia-telangiectasia mutated, ATR - ataxia-telangiectasia and Rad3 related, BIC - B-cell integration cluster, Chk1 - checkpoint kinase 1, EB - Epstein-Barr, JNK - c-Jun N-terminal kinase, MEK1/2 - mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2, miRNA - microRNA, miR-155 - miRNA-155, PI3K - phosphatidylinositol 3 kinase, PKC - protein kinase C, RT-PCR - reverse transcriptase polymerase chain reaction, siRNA - small interfering RNA, SMG-1 - suppressor of morphogenesis in genitalia-1

Characterization of proteins secreted by pancreatic cancer cells with anticancer drug treatment in vitro

Pancreatic cancer is one of the most lethal cancers, with an incidence equaling mortality. It is a heterogeneous group of neoplasms in which pancreatic ductal adenocarcinoma is most common. Pancreatic cancer cannot be cured even if detected early. When treatment is initiated, a suitable method of administration of anticancer drugs must be chosen. Anticancer drugs kill tumor cells. However, side effects including initiation are problematic in anticancer drug therapy. Improved methods for the diagnosis of side effects of pancreatic cancer by using sensitive and specific tumor markers are highly desirable. Therefore, efficient strategies for biomarker discovery are urgently needed. Here, we present an approach based on direct experimental access to proteins released by PANC-1 human pancreatic cancer cells in vitro. A two-dimensional (2-D) map and catalog of this subproteome, herein termed the secretome, were established comprising more than 1,000 proteins observed by ‘2-D difference in-gel electrophoresis analysis using cyanine dye’. We investigated 22 spots that were 1.20-fold upregulated and 31 spots that were 0.66-fold downregulated by gemcitabine chloride treatment. Proteins in these spots were identified by nano-high-performance liquid chromatography electrospray ionization time of flight mass spectrometry/mass spectrometry. Most secretome constituents were nominally cellular proteins. By mass spectrometry screening, 14-3-3 protein sigma (14-3-3 σ), protein S100-A8, protein S100-A9, galectin-7, lactotransferrin (lactoferrin, LF) precursor, serotransferrin (transferrin) precursor, and vitamin D binding protein precursor were identified. Western blotting confirmed the presence of 14-3-3 σ and LF. We found that upregulation of 14-3-3 σ was associated with apoptosis, and downregulation of LF was found to suppress tumorigenesis.