Lai-Chu Wu

Sp1/NFκB/HDAC/miR-29b Regulatory Network in KIT-Driven Myeloid Leukemia

The biologic and clinical significance of KIT overexpression that associates with KIT gain-of-function mutations occurring in subsets of acute myeloid leukemia (AML) (i.e., core binding factor AML) is unknown. Here, we show that KIT mutations lead to MYC-dependent miR-29b repression and increased levels of the miR-29b target Sp1 in KIT-driven leukemia. Sp1 enhances its own expression by participating in a NFkappaB/HDAC complex that further represses miR-29b transcription. Upregulated Sp1 then binds NFkappaB and transactivates KIT. Therefore, activated KIT ultimately induces its own transcription. Our results provide evidence that the mechanisms of Sp1/NFkappaB/HDAC/miR-29b-dependent KIT overexpression contribute to leukemia growth and can be successfully targeted by pharmacological disruption of the Sp1/NFkappaB/HDAC complex or synthetic miR-29b treatment in KIT-driven AML.

Aberrant Overexpression of IL-15 Initiates Large Granular Lymphocyte Leukemia through Chromosomal Instability and DNA Hypermethylation

How inflammation causes cancer is unclear. Interleukin-15 (IL-15) is a pro-inflammatory cytokine elevated in human large granular lymphocyte (LGL) leukemia. Mice overexpressing IL-15 develop LGL leukemia. Here, we show that prolonged in vitro exposure of wild-type (WT) LGL to IL-15 results in Myc-mediated upregulation of aurora kinases, centrosome aberrancies, and aneuploidy. Simultaneously, IL-15 represses miR-29b via induction of Myc/NF-κBp65/Hdac-1, resulting in Dnmt3b overexpression and DNA hypermethylation. All this is validated in human LGL leukemia. Adoptive transfer of WT LGL cultured with IL-15 led to malignant transformation in vivo. Drug targeting that reverses miR-29b repression cures otherwise fatal LGL leukemia. We show how excessive IL-15 initiates cancer and demonstrate effective drug targeting for potential therapy of human LGL leukemia.

Modulation of DNA Methylation by a Sesquiterpene Lactone Parthenolide

Hypermethylation of 5′-cytosine-guanosine islands of tumor suppressor genes resulting in their silencing has been proposed to be a hallmark of various tumors. Modulation of DNA methylation with DNA methylation inhibitors has been shown to result in cancer cell differentiation or apoptosis and represents a novel strategy for chemotherapy. Currently, effective DNA methylation inhibitors are mainly limited to decitabine and 5-azacytidine, which still show unfavorable toxicity profiles in the clinical setting. Thus, discovery and development of novel hypomethylating agents, with a more favorable toxicity profile, is essential to broaden the spectrum of epigenetic therapy. Parthenolide, the principal bioactive sesquiterpene lactone of feverfew, has been shown to alkylate Cys38 of p65 to inhibit nuclear factor-κB activation and exhibit anti-tumor activity in human malignancies. In this article, we report that parthenolide 1) inhibits DNA methyltransferase 1 (DNMT1) with an IC50 of 3.5 μM, possibly through alkylation of the proximal thiolate of Cys1226 of the catalytic domain by its γ-methylene lactone, and 2) down-regulates DNMT1 expression possibly associated with its SubG1 cell-cycle arrest or the interruption of transcriptional factor Sp1 binding to the promoter of DNMT1. These dual functions of parthenolide result in the observed in vitro and in vivo global DNA hypomethylation. Furthermore, parthenolide has been shown to reactivate tumor suppressor HIN-1 gene in vitro possibly associated with its promoter hypomethylation. Hence, our study established parthenolide as an effective DNA methylation inhibitor, representing a novel prototype for DNMT1 inhibitor discovery and development from natural structural-diversified sesquiterpene lactones.

Curcumin Down-Regulates DNA Methyltransferase 1 and Plays an Anti-Leukemic Role in Acute Myeloid Leukemia

Bioactive components from dietary supplements such as curcumin may represent attractive agents for cancer prevention or treatment. DNA methylation plays a critical role in acute myeloid leukemia (AML) development, and presents an excellent target for treatment of this disease. However, it remains largely unknown how curcumin, a component of the popular Indian spice turmeric, plays a role in DNA hypomethylation to reactivate silenced tumor suppressor genes and to present a potential treatment option for AML. Here we show that curcumin down-regulates DNMT1 expression in AML cell lines, both in vitro and in vivo, and in primary AML cells ex vivo. Mechanistically, curcumin reduced the expression of positive regulators of DNMT1, p65 and Sp1, which correlated with a reduction in binding of these transcription factors to the DNMT1 promoter in AML cell lines. This curcumin-mediated down-regulation of DNMT1 expression was concomitant with p15INK4B tumor suppressor gene reactivation, hypomethylation of the p15INK4B promoter, G1 cell cycle arrest, and induction of tumor cell apoptosis in vitro. In mice implanted with the human AML MV4–11 cell line, administration of curcumin resulted in remarkable suppression of AML tumor growth. Collectively, our data indicate that curcumin shows promise as a potential treatment for AML, and our findings provide a basis for future studies to test the clinical efficacy of curcumin – whether used as a single agent or as an adjuvant – for AML treatment.

Lenalidomide-mediated enhanced translation of C/EBPα-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia

Recently, we showed that increased miR-181a expression was associated with improved outcomes in cytogenetically normal acute myeloid leukemia (CN-AML). Interestingly, miR-181a expression was increased in CN-AML patients harboring CEBPA mutations, which are usually biallelic and associate with better prognosis. CEBPA encodes the C/EBPα transcription factor. We demonstrate here that the presence of N-terminal CEBPA mutations and miR-181a expression are linked. Indeed, the truncated C/EBPα-p30 isoform, which is produced from the N-terminal mutant CEBPA gene or from the differential translation of wild-type CEBPA mRNA and is commonly believed to have no transactivation activity, binds to the miR-181a-1 promoter and up-regulates the microRNA expression. Furthermore, we show that lenalidomide, a drug approved for myelodysplastic syndromes and multiple myeloma, enhances translation of the C/EBPα-p30 isoform, resulting in higher miR-181a levels. In xenograft mouse models, ectopic miR-181a expression inhibits tumor growth. Similarly, lenalidomide exhibits antitumorigenic activity paralleled by increased miR-181a expression. This regulatory pathway may explain an increased sensitivity to apoptosis-inducing chemotherapy in subsets of AML patients. Altogether, our data provide a potential explanation for the improved clinical outcomes observed in CEBPA-mutated CN-AML patients, and suggest that lenalidomide treatment enhancing the C/EBPα-p30 protein levels and in turn miR-181a may sensitize AML blasts to chemotherapy.

MicroRNAs activate natural killer cells through Toll-like receptor signaling.

MicroRNAs (miRNAs) bind to complementary sequences of target mRNAs, resulting in translational repression or target degradation and thus gene silencing. miRNAs are abundant in circulating blood, yet it is not known whether, as a class of regulatory molecules, they interact with human natural killer (NK) cells. Here we found that the treatment of human NK cells with several mature miRNAs in the presence of a low concentration of interleukin-12 induced CD69 expression, interferon-γ production, and degranulation marker CD107a expression. In vivo, infusion of several miRNAs alone in murine peripheral blood also resulted in comparable NK-cell activation, but not T-cell activation. Furthermore, miRNA administration significantly protected mice from tumor development in an NK cell-dependent manner. Mechanistically, we found that miRNA stimulation led to downstream activation of nuclear factor κB (NF-κB), an effect that was blunted by a block in Toll-like receptor 1(TLR1) signaling and attenuated in lymphoma patients. Knockdown of TLR1 resulted in less activation by miRNAs. Collectively, we show that miRNAs have a capacity to selectively activate innate immune effector cells that is, at least in part, via the TLR1-NF-κB signaling pathway. This may be important in the normal host defense against infection and/or malignant transformation.

IL-27 enhances the survival of tumor antigen-specific CD8+ T cells and programs them into IL-10-producing, memory precursor-like effector cells.

IL‐27 is a member of the IL‐12 family of cytokines that is comprised of an IL‐12 p40‐related protein subunit, EBV‐induced gene 3, and a p35‐related subunit, p28. IL‐27 functions through IL‐27R and has been shown to have potent antitumor activity via activation of a variety of cellular components, including antitumor CD8+ T‐cell responses. However, the exact mechanisms of how IL‐27 enhances antitumor CD8+ T‐cell responses remain unclear. Here we show that IL‐27 significantly enhances the survival of activated tumor antigen‐specific CD8+ T cells in vitro and in vivo, and programs tumor antigen‐specific CD8+ T cells into memory precursor‐like effector cells, characterized by upregulation of Bcl‐6, SOCS3, Sca‐1, and IL‐10. While STAT3 activation and the CTL survival‐enhancing effects can be independent of CTL IL‐10 production, we show here that IL‐27‐induced CTL IL‐10 production contributes to memory precursor cell phenotype induction, CTL memory, and tumor rejection. Thus, IL‐27 enhances antitumor CTL responses via programming tumor antigen‐specific CD8+ T cells into a unique memory precursor type of effector cells characterized by a greater survival advantage. Our results have important implications for designing immunotherapy against human cancer.

Transcriptome-wide gene regulation by gentle treadmill walking during the progression of monoiodoacetate-induced arthritis.

OBJECTIVE Physiotherapies are the most widely recommended conservative treatment for arthritic diseases. The present study was undertaken to examine the molecular mechanisms underlying the effects of gentle treadmill walking (GTW) on various stages of monoiodoacetate-induced arthritis (MIA) to elucidate the basis for the success or failure of such therapies in joint damage. METHODS Knees were obtained from untreated control rats, rats with MIA that did not undergo GTW, rats with MIA in which GTW regimens were started 1 day post-MIA induction, and rats with MIA in which GTW regimens were started after cartilage damage had progressed to grade 1 or grade 2. The cartilage was examined macroscopically, microscopically, and by microfocal computed tomography imaging. Transcriptome-wide gene expression analysis was performed, and microarray data were assessed by Ingenuity Pathways Analysis to identify molecular functional networks regulated by GTW. RESULTS GTW intervention started on day 1 post-MIA induction significantly prevented the progression of MIA, but its efficacy was reduced when implemented on knees exhibiting close to grade 1 cartilage damage. GTW accelerated cartilage damage in knees with close to grade 2 damage. Transcriptome-wide gene expression analysis revealed that GTW intervention started 1 day post-MIA inception significantly suppressed inflammation-associated genes and up-regulated matrix-associated gene networks. However, delayed GTW intervention after grade 1 damage had occurred was less effective in suppressing proinflammatory genes or up-regulating matrix synthesis. CONCLUSION The present findings suggest that GTW suppresses proinflammatory gene networks and up-regulates matrix synthesis to prevent progression of cartilage damage in MIA-affected knees. However, the extent of cartilage damage at the initiation of GTW may be an important determinant of the success or failure of such therapies.

Reactivation of RASSF1A in Breast Cancer Cells by Curcumin

Reactivation of tumor suppressor genes (TSGs) involved in carcinogenesis by nontoxic bioactive food component represents a promising strategy for cancer chemoprevention. Recently, curcumin has been demonstrated to inhibit a bacterial DNA methyltransferase (M. Sss I) activity, induce global DNA hypomethylation in leukemia cells, and reactivate several hypermethylation silenced genes in lung and prostate cancer cells. Herein, we demonstrated that curcumin can enhance the mRNA and protein levels of ras-association domain family protein 1A (RASSF1A), 1 hypermethylation-silenced TSG, and decrease its promoter methylation in breast cancer cells. Mechanistic study demonstrated that curcumin can decrease DNA methylation activity of nuclear extract and downregulate the mRNA and protein levels of DNMT1 in MCF-7 cells, which may be associated with curcumin-induced disruption of NF-κB/Sp1 complex bound to the promoter region of DNMT1. Altogether, this study reveals a novel molecular mechanism of curcumin as a chemo-preventive agent for breast cancer through hypomethylation reactivation of RASSF1A.

Transcriptional control of human T-BET expression : The role of Sp1

Murine T‐bet (T‐box expressed in T cells) is a master regulator of IFN‐γ gene expression in NK and T cells. T‐bet also plays a critical role in autoimmunity, asthma and other diseases. However, cis elements or trans factors responsible for regulating T‐bet expression remain largely unknown. Here, we report on our discovery of six Sp1‐binding sites within the proximal human T‐BET promoter that are highly conserved among mammalian species. Electrophoretic mobility shift assays demonstrate a physical association between Sp1 and the proximal T‐BET promoter with a direct dose response between Sp1 expression and T‐BET promoter activity. Ectopic overexpression of Sp1 also enhanced T‐BET expression and cytokine‐induced IFN‐γ secretion in NK cells and T cells. Mithramycin A, which blocks the binding of Sp1 to the T‐BET promoter, diminished both T‐BET expression and IFN‐γ protein production in monokine‐stimulated primary human NK cells. Collectively, our results suggest that Sp1 is a positive transcriptional regulator of T‐BET. As T‐BET and IFN‐γ are critically important in inflammation, infection, and cancer, targeting Sp1, possibly with mithramycin A, may be useful for preventing and/or treating diseases associated with aberrant T‐BET or IFN‐γ expression.