Reprogramming of pyrimidine nucleotide metabolism supports vigorous cell proliferation of normal and malignant T cells.

ABSTRACT: Adult T-cell leukemia/lymphoma (ATL) is triggered by infection with human T-cell lymphotropic virus-1 (HTLV-1). Here, we describe the reprogramming of pyrimidine biosynthesis in both normal T cells and ATL cells through regulation of uridine-cytidine kinase 2 (UCK2), which supports vigorous proliferation. UCK2 catalyzes the monophosphorylation of cytidine/uridine and their analogues during pyrimidine biosynthesis and drug metabolism. We found that UCK2 was overexpressed aberrantly in HTLV-1-infected T cells but not in normal T cells. T-cell activation via T-cell receptor (TCR) signaling induced expression of UCK2 in normal T cells. Somatic alterations and epigenetic modifications in ATL cells activate TCR signaling. Therefore, we believe that expression of UCK2 in HTLV-1-infected cells is induced by dysregulated TCR signaling. Recently, we established azacitidine-resistant (AZA-R) cells showing absent expression of UCK2. AZA-R cells proliferated normally in vitro, whereas UCK2 knockdown inhibited ATL cell growth. Although uridine and cytidine accumulated in AZA-R cells, possibly because of dysfunction of pyrimidine salvage biosynthesis induced by loss of UCK2 expression, the amount of UTP and CTP was almost the same as in parental cells. Furthermore, AZA-R cells were more susceptible to an inhibitor of dihydroorotic acid dehydrogenase, which performs the rate-limiting enzyme of de novo pyrimidine nucleotide biosynthesis, and more resistant to dipyridamole, an inhibitor of pyrimidine salvage biosynthesis, suggesting that AZA-R cells adapt to UCK2 loss by increasing de novo pyrimidine nucleotide biosynthesis. Taken together, the data suggest that fine-tuning pyrimidine biosynthesis supports vigorous cell proliferation of both normal T cells and ATL cells.

A Combination of Alectinib and DNA-Demethylating Agents Synergistically Inhibits Anaplastic-Lymphoma-Kinase-Positive Anaplastic Large-Cell Lymphoma Cell Proliferation.

The recent evolution of molecular targeted therapy has improved clinical outcomes in several human malignancies. The translocation of anaplastic lymphoma kinase (ALK) was originally identified in anaplastic large-cell lymphoma (ALCL) and subsequently in non-small cell lung carcinoma (NSCLC). Since ALK fusion gene products act as a driver of carcinogenesis in both ALCL and NSCLC, several ALK tyrosine kinase inhibitors (TKIs) have been developed. Crizotinib and alectinib are first- and second-generation ALK TKIs, respectively, approved for the treatment of ALK-positive ALCL (ALK+ ALCL) and ALK+ NSCLC. Although most ALK+ NSCLC patients respond to crizotinib and alectinib, they generally relapse after several years of treatment. We previously found that DNA-demethylating agents enhanced the efficacy of ABL TKIs in chronic myeloid leukemia cells. Moreover, aberrant DNA methylation has also been observed in ALCL cells. Thus, to improve the clinical outcomes of ALK+ ALCL therapy, we investigated the synergistic efficacy of the combination of alectinib and the DNA-demethylating agent azacytidine, decitabine, or OR-2100 (an orally bioavailable decitabine derivative). As expected, the combination of alectinib and DNA-demethylating agents synergistically suppressed ALK+ ALCL cell proliferation, concomitant with DNA hypomethylation and a reduction in STAT3 (a downstream target of ALK fusion proteins) phosphorylation. The combination of alectinib and OR-2100 markedly altered gene expression in ALCL cells, including that of genes implicated in apoptotic signaling, which possibly contributed to the synergistic anti-ALCL effects of this drug combination. Therefore, alectinib and OR-2100 combination therapy has the potential to improve the outcomes of patients with ALK+ ALCL.

Cancer progression by the okadaic acid class of tumor promoters and endogenous protein inhibitors of PP2A, SET and CIP2A.

PURPOSE: Okadaic acid class of tumor promoters are transformed into endogenous protein inhibitors of PP2A, SET, and CIP2A in human cancers. This indicates that inhibition of PP2A activity is a common mechanism of cancer progression in humans. It is important to study the roles of SET and CIP2A vis-à-vis their clinical significance on the basis of new information gathered from a search of PubMed. RESULTS AND DISCUSSION: The first part of this review introduces the carcinogenic roles of TNF-α and IL-1, which are induced by the okadaic acid class of compounds. The second part describes unique features of SET and CIP2A in cancer progression for several types of human cancer: (1) SET-expressing circulating tumor cells (SET-CTCs) in breast cancer, (2) knockdown of CIP2A and increased PP2A activity in chronic myeloid leukemia, (3) CIP2A and epidermal growth factor receptor (EGFR) activity in erlotinib sensitive- and resistant-non-small cell lung cancer, (4) SET antagonist EMQA plus radiation therapy against hepatocellular carcinoma, (5) PP2A inactivation as a common event in colorectal cancer, (6) prostate cancer susceptibility variants, homeobox transcription factor (HOXB13 T) and CIP2A T, and (7) SET inhibitor OP449 for pre-clinical investigation of pancreatic cancer. In the Discussion, the binding complex of SET is briefly introduced, and overexpression of SET and CIP2A proteins is discussed in relation to age-associated chronic inflammation (inflammaging). CONCLUSION: This review establishes the concept that inhibition of PP2A activity is a common mechanism of human cancer progression and activation of PP2A activity leads to effective anticancer therapy.

Combination of a New Oral Demethylating Agent, OR2100, and Venetoclax for Treatment of Acute Myeloid Leukemia.

The standard treatment for elderly patients with acute myeloid leukemia (AML) is venetoclax (Ven), a BCL-2-selective inhibitor, combined with hypomethylating agents (HMA) such as azacitidine or decitabine. This regimen results in low toxicity, high response rates, and potentially durable remission; however, because of low oral bioavailability, these conventional HMAs must be administered intravenously or subcutaneously. A combination of oral HMAs and Ven would provide a therapeutic advantage over parenteral administration of drugs and improve quality of life by reducing the number of hospital visits. Previously, we showed the promising oral bioavailability and antileukemia effects of a new HMA, OR2100 (OR21). Here, we investigated the efficacy and underlying mechanism of OR21 when used in combination with Ven to treat AML. OR21/Ven showed synergistic antileukemia effects in vitro, and significantly prolonged survival without increasing toxicity in a human leukemia xenograft mice model. RNA sequencing following combination therapy revealed downregulation of VAMP7, which is involved in autophagic maintenance of mitochondrial homeostasis. Combination therapy led to accumulation of reactive oxygen species, leading to increased apoptosis. The data suggest that the combination of OR21 plus Ven is a promising candidate oral therapy for AML. Significance: The standard treatment for elderly patients with AML is Ven combined with HMAs. OR21, a new oral HMA plus Ven showed synergistic antileukemia effects in vitro and vivo, suggesting that the combination of OR2100 plus Ven is a promising candidate oral therapy for AML.

Dual targeting of aberrant DNA and histone methylation synergistically suppresses tumor cell growth in ATL.

Adult T-cell leukemia/lymphoma (ATL) is a malignancy of mature CD4+ T cells caused by human T-cell lymphotropic virus type 1 (HTLV-1)-induced T-cell transformation. After infection with HTLV-1, it takes several decades for HTLV-1 carriers to develop ATL. The prognosis of ATL remains poor despite several new agents being approved in the last few years. Recently, it has been noted that epigenetic abnormalities, both DNA methylation and trimethylation at histone H3Lys27 (H3K27me3), contribute to ATL leukemogenesis. Here, we investigated the effect of combination treatment with DNA demethylating agents (azacitidine [AZA], decitabine (DAC), and OR-2100 (OR21), which is a silylated derivative of DAC) and inhibitors of enhancer of zeste homolog 2 (EZH2) (EPZ-6438 and DS-3201b), which catalyze trimethylation of H3K27, in ATL. The combination of DAC and OR21 but not AZA with EZH inhibitors exhibited synergistic anti-ATL effects in vitro and in vivo, concomitant with DNA demethylation and reduction of H3K27me3. The combination induced gene expression reprogramming. Dual-specificity phosphatase 5 (DUSP5), an extracellular signal-regulated kinase (ERK)-specific phosphatase, was identified as a key molecule that mediated the inhibitory effect of combination treatment by inactivating the ERK signaling pathway. DUSP5 was downregulated by DNA methylation and H3K27me3 accumulation in the promoter region in HTLV-1-infected cells from patients with ATL during ATL leukemogenesis. The present results demonstrate that dual targeting of aberrant DNA and histone methylation synergistically suppresses tumor cell growth by restoring DUSP5, and that dual targeting of aberrant DNA and histone methylation is a feasible therapeutic approach for ATL.

Diffuse Large B-cell Lymphoma Involving an Abundant Infiltration of T Follicular Helper Cells.

A 76-year-old man presented with skin plaque and splenic nodules, and diffuse large B-cell lymphoma (DLBCL) with infiltration of T-cells was suspected based on the skin lesions. The disease showed indolent clinical behavior for three months, when systemic lymphadenopathy rapidly evolved. An inguinal lymph node biopsy revealed DLBCL with abundant infiltration of T follicular helper (TFH) cells. A polymerase chain reaction-based analysis of immunoglobulin variable heavy chain showed that the skin, splenic nodules, and inguinal lymph node shared the same clone. This case indicates that the dysregulated infiltration of TFH cells in the tumor microenvironment accelerates the lymphomagenesis and progression of DLBCL.

Targeting DNMT1 by demethylating agent OR-2100 increases tyrosine kinase inhibitors-sensitivity and depletes leukemic stem cells in chronic myeloid leukemia.

ABL1 tyrosine kinase inhibitors (TKIs) dramatically improve the prognosis of chronic myeloid leukemia (CML), but 10-20% of patients achieve suboptimal responses with low TKIs sensitivity. Furthermore, residual leukemic stem cells (LSCs) are involved in the molecular relapse after TKIs discontinuation. Aberrant DNA hypermethylation contributes to low TKIs sensitivity and the persistence of LSCs in CML. DNMT1 is a key regulator of hematopoietic stem cells, suggesting that aberrant DNA hypermethylation targeting DNMT1 represents a potential therapeutic target for CML. We investigated the efficacy of OR-2100 (OR21), the first orally available single-compound prodrug of decitabine. OR21 exhibited anti-tumor effects as a monotherapy, and in combination therapy it increased TKI-induced apoptosis and induction of tumor suppressor genes including PTPN6 encoding SHP-1 in CML cells. OR21 in combination with imatinib significantly suppressed tumor growth in a xenotransplant model. OR21 and combination therapy decreased the abundance of LSCs and inhibited engraftment in a BCR-ABL1-transduced mouse model. These results demonstrate that targeting DNMT1 using OR21 exerts anti-tumor effects and impairs LSCs in CML. Therefore, combination treatment of TKIs and OR21 represents a promising treatment strategy in CML.

Adult T-cell leukemia-lymphoma acquires resistance to DNA demethylating agents through dysregulation of enzymes involved in pyrimidine metabolism.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive neoplasm derived from T-cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Recently, we reported that regional DNA hypermethylation in HTLV-1-infected T-cells reflects the disease status of ATL and the anti-ATL effects of DNA demethylating agents, including azacitidine (AZA), decitabine (DAC) and a new DAC prodrug, OR-2100 (OR21), which we developed. Here, to better understand the mechanisms underlying drug resistance, we generated AZA-, DAC- and OR21-resistant (AZA-R, DAC-R and OR21-R, respectively) cells from the ATL cell line TL-Om1 and the HTLV-1-infected cell line MT-2 via long-term drug exposure. The efficacy of OR21 was almost the same as that of DAC, indicating that the pharmacodynamics of OR21 were due to release of DAC from OR21. Resistant cells did not show cellular responses observed in parental cells induced by treatment with drugs, including growth suppression, depletion of DNA methyltransferase DNMT1 and DNA hypomethylation. We also found that reduced expression of deoxycytidine kinase (DCK) correlated with lower susceptibility to DAC/OR21 and that reduced expression of uridine cytidine kinase2 (UCK2) correlated with reduced susceptibility to AZA. DCK and UCK2 catalyze phosphorylation of DAC and AZA, respectively; reconstitution of expression reversed the resistant phenotypes. A large homozygous deletion in DCK and a homozygous splice donor site mutation in UCK2 were identified in DAC-R TL-Om1 and AZA-R TL-Om1, respectively. Both genomic mutations might lead to loss of protein expression. Thus, inactivation of UCK2 and DCK might be a putative cause of phenotypes that are resistant to AZA and DAC/OR21, respectively.

Silylation of Deoxynucleotide Analog Yields an Orally Available Drug with Antileukemia Effects.

DNA methyltransferase inhibitors have improved the prognosis of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). However, because these agents are easily degraded by cytidine deaminase (CDA), they must be administered intravenously or subcutaneously. Recently, two orally bioavailable DNA methyltransferase inhibitors, CC-486 and ASTX727, were approved. In previous work, we developed 5-O-trialkylsilylated decitabines that resist degradation by CDA. However, the effects of silylation of a deoxynucleotide analog and enzymatic cleavage of silylation have not been fully elucidated. Enteric administration of OR21 in a cynomolgus monkey model led to high plasma concentrations and hypomethylation, and in a mouse model, oral administration of enteric-coated OR21 led to high plasma concentrations. The drug became biologically active after release of decitabine (DAC) from OR21 following removal of the 5'-O-trisilylate substituent. Toxicities were tolerable and lower than those of DAC. Transcriptome and methylome analysis of MDS and AML cell lines revealed that OR21 increased expression of genes associated with tumor suppression, cell differentiation, and immune system processes by altering regional promoter methylation, indicating that these pathways play pivotal roles in the action of hypomethylating agents. OR21 induced cell differentiation via upregulation of the late cell differentiation drivers CEBPE and GATA-1 Thus, silylation of a deoxynucleotide analog can confer oral bioavailability without new toxicities. Both in vivo and in vitro, OR21 exerted antileukemia effects, and had a better safety profile than DAC. Together, our findings indicate that OR21 is a promising candidate drug for phase I study as an alternative to azacitidine or decitabine.

Role of TNF-α-Inducing Protein Secreted by Helicobacter pylori as a Tumor Promoter in Gastric Cancer and Emerging Preventive Strategies.

The tumor necrosis factor-α (TNF-α)-inducing protein (tipα) gene family, comprising Helicobacter pylori membrane protein 1 (hp-mp1) and tipα, has been identified as a tumor promoter, contributing to H. pylori carcinogenicity. Tipα is a unique H. pylori protein with no similarity to other pathogenicity factors, CagA, VacA, and urease. American H. pylori strains cause human gastric cancer, whereas African strains cause gastritis. The presence of Tipα in American and Euro-Asian strains suggests its involvement in human gastric cancer development. Tipα secreted from H. pylori stimulates gastric cancer development by inducing TNF-α, an endogenous tumor promoter, through its interaction with nucleolin, a Tipα receptor. This review covers the following topics: tumor-promoting activity of the Tipα family members HP-MP1 and Tipα, the mechanism underlying this activity of Tipα via binding to the cell-surface receptor, nucleolin, the crystal structure of rdel-Tipα and N-terminal truncated rTipα, inhibition of Tipα-associated gastric carcinogenesis by tumor suppressor B-cell translocation gene 2 (BTG2/TIS21), and new strategies to prevent and treat gastric cancer. Thus, Tipα contributes to the carcinogenicity of H. pylori by a mechanism that differs from those of CagA and VacA.

Stiffening of Cancer Cell Membranes Is a Key Biophysical Mechanism of Primary and Tertiary Cancer Prevention with Green Tea Polyphenols.

Over the past 30 years, research of green tea polyphenols, especially (-)-epigallocatechin gallate (EGCG), has revealed that consumption of green tea is a practical and effective primary cancer prevention method for the general population. More recently, we believe that green tea polyphenols are beneficial for tertiary cancer prevention using green tea alone or combined with anticancer drugs because EGCG has the potential to inhibit metastatic progression and stemness, and enhance antitumor immunity. In an effort to identify a common underlying mechanism responsible for EGCG's multifunctional effects on various molecular targets, we studied the biophysical effects of EGCG on cell stiffness using atomic force microscopy. We found that EGCG acts to stiffen the membranes of cancer cells, leading to inhibition of signaling pathways of various receptors. Stiffening of membranes with EGCG inhibited AXL receptor tyrosine kinase, a stimulator of cell softening, motility and stemness, and expression of programmed cell death-ligand 1. This review covers the following: i) primary cancer prevention using EGCG or green tea, ii) tertiary cancer prevention by combining EGCG and anticancer drugs, iii) inhibition of metastasis with EGCG by stiffening the cell membrane, iv) inhibition of AXL receptor tyrosine kinase, a stimulator of cell softening and motility, with EGCG, v) inhibition of stemness properties with EGCG, and vi) EGCG as an alternative chemical immune checkpoint inhibitor. Development of new drugs that enhance stiffening of cancer cell membranes may be an effective strategy for tertiary cancer prevention and treatment.

Targeting aberrant DNA hypermethylation as a driver of ATL leukemogenesis by using the new oral demethylating agent OR-2100.

Adult T-cell leukemia-lymphoma (ATL) is an aggressive hematological malignancy of CD4+ T cells transformed by human T-cell lymphotropic virus-1 (HTLV-1). Most HTLV-1-infected individuals are asymptomatic, and only 3% to 5% of carriers develop ATL. Here, we describe the contribution of aberrant DNA methylation to ATL leukemogenesis. HTLV-1-infected T-cells and their uninfected counterparts were separately isolated based on CADM1 and CD7 expression status, and differentially methylated positions (DMPs) specific to HTLV-infected T cells were identified through genome-wide DNA methylation profiling. Accumulation of DNA methylation at hypermethylated DMPs correlated strongly with ATL development and progression. In addition, we identified 22 genes downregulated because of promoter hypermethylation in HTLV-1-infected T cells, including THEMIS, LAIR1, and RNF130, which negatively regulate T-cell receptor (TCR) signaling. Phosphorylation of ZAP-70, a transducer of TCR signaling, was dysregulated in HTLV-1-infected cell lines but was normalized by reexpression of THEMIS. Therefore, we hypothesized that DNA hypermethylation contributes to growth advantages in HTLV-1-infected cells during ATL leukemogenesis. To test this idea, we investigated the anti-ATL activities of OR-1200 and OR-2100 (OR21), novel decitabine (DAC) prodrugs with enhanced oral bioavailability. Both DAC and OR21 inhibited cell growth, accompanied by global DNA hypomethylation, in xenograft tumors established by implantation of HTLV-1-infected cells. OR21 was less hematotoxic than DAC, whereas tumor growth inhibition was almost identical between the 2 compounds, making it suitable for long-term treatment of ATL patient-derived xenograft mice. Our results demonstrate that regional DNA hypermethylation is functionally important for ATL leukemogenesis and an effective therapeutic target.

Naked antisense double-stranded DNA oligonucleotide efficiently suppresses BCR-ABL positive leukemic cells.

Oligonucleotide-based gene silencing, using molecules such as antisense oligonucleotides (ASOs), small interfering RNA, and aptamers, is widely studied. Another approach uses DNA/RNA heteroduplex oligonucleotides (HDOs). Here, we developed an antisense double-stranded DNA oligonucleotide (ADO) by modification of the complementary RNA in an HDO to generate DNA for increasing resistance to nucleases. Naked BCR-ABL-targeting ADO was significantly more potent than siRNA at reducing BCR-ABL chimeric mRNA expression in chronic myeloid leukemia (CML) cell lines. Further, naked BCR-ABL-targeting ADO suppressed BCR-ABL protein levels in a dose-dependent manner, inhibited CML cell proliferation, and augmented the inhibitory effects of imatinib mesylate. In conclusion, ADO technology is an attractive method for therapeutic application.

Meeting report of the 14th Japan-Korea joint symposium on cancer and aging research: current status of translational research and approaches to precision medicine.

PURPOSE: The 14th Japan-Korea joint symposium on cancer and aging research was held at an auditorium of Saga University, Japan, May 31-Jun 2, 2018. Participants presented 31 oral and 21 poster presentations, two lectures at a luncheon seminar, plus special lectures from two Korean Emeritus Professors and founders of our joint symposia. The essential parts of the lectures are reviewed here. RESULTS: This Symposium was called Japan-Korea, because the host country comes first. Our symposia are organized every 18 months and the program includes keynote and plenary lectures, and oral and poster presentations. (1) Subjects related to cancer development at this symposium were: prostate cancer progression, molecules activating GSK3ß, suppressing the activation of cancer stem cells, profiling human B cell receptor repertoires, and hereditary gastrointestinal cancer syndrome. (2) Subjects related to treatment were: G-quadruplex ligands for glioma stem cells, tankyrase inhibitor for colorectal cancer, and eradication of ATL. (3) Cancer prevention subjects were: physical adsorption of EGCG to cell membrane, inhibition of immune evasion of cancer cells with EGCG, and prevention with antidiabetic agents. (4) Aging subjects were life span extension with Toll-like receptor 5 vaccine and reversal of senescence with inhibitors of ATM and ROCK. (5) The results of epidemiology focused on aldehyde dehyrogenase-2 and alcohol consumption. CONCLUSION: The 14th symposium demonstrated the cutting-edge of presentations with discussion of numerous ideas by the participants.

The concept of the okadaic acid class of tumor promoters is revived in endogenous protein inhibitors of protein phosphatase 2A, SET and CIP2A, in human cancers.

PURPOSE: The okadaic acid class of tumor promoters, which are inhibitors of protein phosphatases 1 and 2A (PP1 and PP2A), induced tumor promotion in mouse skin, rat glandular stomach, and rat liver. Endogenous protein inhibitors of PP2A, SET and CIP2A, were up-regulated in various human cancers, so it is vital to review the essential mechanisms of tumor promotion by the okadaic acid class compounds, together with cancer progression by SET and CIP2A in humans. RESULTS AND DISCUSSION: The first part of this review introduces the okadaic acid class compounds and the mechanism of tumor promotion: (1) inhibition of PP1 and PP2A activities of the okadaic acid class compounds; (2) some topics of tumor promotion; (3) TNF-α gene expression as a central mediator in tumor promotion; (4) exposure to the okadaic acid class of tumor promoters in relation to human cancer. The second part emphasizes the overexpression of SET and CIP2A in cancer progression, and the anticancer activity of SET antagonists as follows: (5) isolation and characterization of SET; (6) isolation and characterization of CIP2A; (7) progression of leukemia with SET; (8) progression of breast cancer with SET and CIP2A; (9) progression of lung cancer with SET; (10) anti-carcinogenic effects of SET antagonists OP449 and FTY720; and also (11) TNF-α-inducing protein of Helicobacter pylori, which is a clinical example of the okadaic acid pathway. CONCLUSIONS: The overexpression of endogenous protein inhibitors of PP2A, SET and CIP2A, is tightly linked to the progression of various human cancers, as well as Alzheimer's disease.

Deep sequencing of the T cell receptor visualizes reconstitution of T cell immunity in mogamulizumab-treated adult T cell leukemia.

Although the anti-CCR4 antibody mogamulizumab (moga) shows striking antitumor activity against adult T cell leukemia (ATL), it can also cause fatal immunological pathology such as severe skin rash and graft-versus-host disease, which might be attributed to depletion of CCR4+ regulatory T cells. We previously showed that next generation sequencing enables precise analysis of the T cell receptor (TCR) repertoire, and we here used the technique to reveal the immunological dynamics in moga-treated ATL patients. Treatment with moga resulted in remarkable reduction or elimination of clonal cells, and enhanced reconstitution of non-tumor polyclonal CD4+ T cells and oligoclonal CD8+ T cells. Interestingly, cutaneous T cells infiltrating moga-related skin rashes did not share the same major clones in peripheral blood, which minimizes the possibility of cross-reaction. Thus, deep sequencing of the TCR can reveal the immune reconstitution of moga-treated ATL and provides powerful insights into its mode of action.

Inhibition of TNFα-interacting protein α (Tipα)-associated gastric carcinogenesis by BTG2/TIS21 via downregulating cytoplasmic nucleolin expression.

To understand the regulation of Helicobacter pylori (H. pylori)-associated gastric carcinogenesis, we examined the effect of B-cell translocation gene 2 (BTG2) expression on the biological activity of Tipα, an oncoprotein secreted from H. pylori. BTG2, the human ortholog of mouse TIS21 (BTG2/TIS21), has been reported to be a primary response gene that is transiently expressed in response to various stimulations. Here, we report that BTG2 is constitutively expressed in the mucous epithelium and parietal cells of the gastric gland in the stomach. Expression was increased in the mucous epithelium following H. pylori infection in contrast to its loss in human gastric adenocarcinoma. Indeed, adenoviral transduction of BTG2/TIS21 significantly inhibited Tipα activity in MKN-1 and MGT-40, human and mouse gastric cancer cells, respectively, thereby downregulating tumor necrosis factor-α (TNFα) expression and Erk1/2 phosphorylation by reducing expression of nucleolin, a Tipα receptor. Chromatin immunoprecipitation proved that BTG2/TIS21 inhibited Sp1 expression and its binding to the promoter of the nucleolin gene. In addition, BTG2/TIS21 expression significantly reduced membrane-localized nucleolin expression in cancer cells, and the loss of BTG2/TIS21 expression induced cytoplasmic nucleolin availability in gastric cancer tissues, as evidenced by immunoblotting and immunohistochemistry. Higher expression of BTG2 and lower expression of nucleolin were accompanied with better overall survival of poorly differentiated gastric cancer patients. This is the first report showing that BTG2/TIS21 inhibits nucleolin expression via Sp1 binding, which might be associated with the inhibition of H. pylori-induced carcinogenesis. We suggest that BTG2/TIS21 is a potential inhibitor of nucleolin in the cytoplasm, leading to inhibition of carcinogenesis after H. pylori infection.

Cancer Prevention with Green Tea and Its Principal Constituent, EGCG: from Early Investigations to Current Focus on Human Cancer Stem Cells.

Cancer preventive activities of green tea and its main constituent, (-)-epigallocatechin gallate (EGCG) have been extensively studied by scientists all over the world. Since 1983, we have studied the cancer chemopreventive effects of EGCG as well as green tea extract and underlying molecular mechanisms. The first part of this review summarizes ground-breaking topics with EGCG and green tea extract: 1) Delayed cancer onset as revealed by a 10-year prospective cohort study, 2) Prevention of colorectal adenoma recurrence by a double-blind randomized clinical phase II trial, 3) Inhibition of metastasis of B16 melanoma cells to the lungs of mice, 4) Increase in the average value of Young's moduli, i.e., cell stiffness, for human lung cancer cell lines and inhibition of cell motility and 5) Synergistic enhancement of anticancer activity against human cancer cell lines with the combination of EGCG and anticancer compounds. In the second part, we became interested in cancer stem cells (CSCs). 1) Cancer stem cells in mouse skin carcinogenesis by way of introduction, after which we discuss two subjects from our review on human CSCs reported by other investigators gathered from a search of PubMed, 2) Expression of stemness markers of human CSCs compared with their parental cells, and 3) EGCG decreases or increases the expression of mRNA and protein in human CSCs. On this point, EGCG inhibited self-renewal and expression of pluripotency-maintaining transcription factors in human CSCs. Human CSCs are thus a target for cancer prevention and treatment with EGCG and green tea catechins.

Functional analysis of Discoidin domain receptor 2 mutation and expression in squamous cell lung cancer.

OBJECTIVES: Discoidin domain receptor (DDR) 2 mutations have recently been reported to be candidate targets of molecular therapy in lung squamous cell carcinoma (SQCC). However, the status of DDR2 expression and mutations, as well as their precise roles in lung SQCC, have not been clarified. We here report DDR2 mutation and expression status in clinical samples and its role of lung SQCC. MATERIALS AND METHODS: We investigated DDR2 expression and mutation status in 44 human clinical samples and 7 cell lines. Biological functions of DDR2 were assessed by in vitro cell invasion assay and animal model experiments. RESULTS: Endogenous DDR2 protein expression levels were high in one cell line, PC-1, and immunohistochemistry of lung cancer tissue array showed high levels of DDR2 protein in 29% of lung SQCC patients. A mutation (T681I) identified in lung SQCC and the cell line EBC-1 was detected among 44 primary lung SQCC samples and 7 lung SQCC cell lines. Although Forced expression of DDR2 and its mutant (T681I) led to induce SQCC cell invasion in vitro, only wild type DDR2 enhanced lung metastasis in an animal model. We also found that ectopic expression of DDR2 induced MMP-1 mRNA expression accompanied by phosphorylation of c-Jun after treatment with its ligand, collagen type I, but DDR2 with the T681I mutation did not, suggesting that T681I mutation is an inactivating mutation. CONCLUSION: Overexpression of DDR2 might contribute to tumor progression in lung SQCC. The overexpression of DDR2 could be potential molecular target of lung SQCC.

Down-regulation of histone deacetylase 4, -5 and -6 as a mechanism of synergistic enhancement of apoptosis in human lung cancer cells treated with the combination of a synthetic retinoid, Am80 and green tea catechin.

(-)-Epigallocatechin gallate (EGCG), a green tea catechin, acts as a synergist with various anticancer drugs, including retinoids. Am80 is a synthetic retinoid with a different structure from all-trans-retinoic acid: Am80 is now clinically utilized as a new drug for relapsed and intractable acute promyelocytic leukemia patients. Our experiments showed that the combination of EGCG and Am80 synergistically induced both apoptosis in human lung cancer cell line PC-9 and up-regulated expressions of growth arrest and DNA damage-inducible gene 153 (GADD153), death receptor 5, and p21waf1 genes in the cells. To understand the mechanisms of synergistic anticancer activity of the combination, we gave special attention to the lysine acetylation of proteins. Proteomic analysis using nanoLC-ESI-MS/MS revealed that PC-9 cells treated with the combination contained 331 acetylated proteins, while nontreated cells contained 553 acetylated proteins, and 59 acetylated proteins were found in both groups. Among them, the combination increased acetylated-p53 and acetylated-α-tubulin through reduction of histone deacetylase (HDAC) activity in cytosol fraction, although the levels of acetylation in histones H3 or H4 did not change, and the combination reduced protein levels of HDAC4, -5 and -6 by 20% to 80%. Moreover, we found that a specific inhibitor of HDAC4 and -5 strongly induced p21waf1 gene expression, and that of HDAC6 induced both GADD153 and p21waf1 gene expression, which resulted in apoptosis. All results demonstrate that EGCG in combination with Am80 changes levels of acetylation in nonhistone proteins via down-regulation of HDAC4, -5 and -6 and stimulates apoptotic induction.