Yuanyuan Zheng

Identification of miRNomes in Human Liver and Hepatocellular Carcinoma Reveals miR-199a/b-3p as Therapeutic Target for Hepatocellular Carcinoma

The full scale of human miRNome in specific cell or tissue, especially in cancers, remains to be determined. An in-depth analysis of miRNomes in human normal liver, hepatitis liver, and hepatocellular carcinoma (HCC) was carried out in this study. We found nine miRNAs accounted for ∼88.2% of the miRNome in human liver. The third most highly expressed miR-199a/b-3p is consistently decreased in HCC, and its decrement significantly correlates with poor survival of HCC patients. Moreover, miR-199a/b-3p can target tumor-promoting PAK4 to suppress HCC growth through inhibiting PAK4/Raf/MEK/ERK pathway both in vitro and in vivo. Our study provides miRNomes of human liver and HCC and contributes to better understanding of the important deregulated miRNAs in HCC and liver diseases.

The cytosolic nucleic acid sensor LRRFIP1 mediates the production of type I interferon via a beta-catenin-dependent pathway.

Intracellular nucleic acid sensors detect microbial RNA and DNA and trigger the production of type I interferon. However, the cytosolic nucleic acid–sensing system remains to be fully identified. Here we show that the cytosolic nucleic acid–binding protein LRRFIP1 contributed to the production of interferon-β (IFN-β) induced by vesicular stomatitis virus (VSV) and Listeria monocytogenes in macrophages. LRRFIP1 bound exogenous nucleic acids and increased the expression of IFN-β induced by both double-stranded RNA and double-stranded DNA. LRRFIP1 interacted with β-catenin and promoted the activation of β-catenin, which increased IFN-β expression by binding to the C-terminal domain of the transcription factor IRF3 and recruiting the acetyltransferase p300 to the IFN-β enhanceosome via IRF3. Therefore, LRRFIP1 and its downstream partner β-catenin constitute another coactivator pathway for IRF3-mediated production of type I interferon.

Tumor-Educated CD11bhighIalow Regulatory Dendritic Cells Suppress T Cell Response through Arginase I

Tumors can induce generation and accumulation of the immunosuppressive cells such as regulatory T cells in the tumor microenvironment, contributing to tumor escape from immunological attack. Although dendritic cell (DC)-based cancer vaccine can initiate antitumor immune response, regulatory DC subsets involved in the tolerance induction attracted much attention recently. Our previous studies demonstrate that the stromal microenvironment of the spleen, lung, and liver can program generation of CD11clowCD11bhighIalow DCs with regulatory function (CD11bhighIalow regulatory DCs). However, whether and how the tumor microenvironment can program generation of CD11bhighIalow regulatory DCs remain to be investigated. In this study, we used the freshly isolated tumor cells to mimic tumor microenvironment to coculture DCs and found that the freshly isolated tumor cells could drive DCs to differentiate into regulatory DCs with a CD11clowCD11bhighIalow phenotype and high expression of IL-10, NO, vascular endothelial growth factor, and arginase I. Tumor-educated CD11bhighIalow regulatory DCs inhibited CD4+ T cell proliferation both in vitro and in vivo. 3LL lung cancer-derived TGF-β and PGE2 were responsible for the generation of regulatory DCs. PGE2 was the main inducer of arginase I in regulatory DCs. Arginase I played a major role in the suppression of T cell response by regulatory DCs induced by 3LL lung cancer. A natural counterpart of CD11bhighIalow DCs was identified in tumor tissue, and CD11bhighIalow DCs sorted from 3LL lung cancer tissue expressed arginase I and inhibited T cell response. Therefore, tumors can educate DCs to differentiate into a regulatory DC subset, which contributes to constitution of the immunosuppressive tumor microenvironment and promotes tumor immune escape.

CaMKII promotes TLR-triggered proinflammatory cytokine and type I interferon production by directly binding and activating TAK1 and IRF3 in macrophages

Calcium and its major downstream effector, calcium/calmodulin-dependent protein kinase II (CaMKII), are found to be important for the functions of immune cells. Lipopolysaccharide (LPS) has been shown to induce intracellular calcium release in macrophages; however, whether and how CaMKII is required for Toll-like receptor (TLR) signaling remain unknown. Here we demonstrate that TLR 4, 9, and 3 ligands markedly induce intracellular calcium fluxes and activate CaMKII-alpha in macrophages. Selective inhibition or RNA interference of CaMKII significantly suppresses TLR4, 9, 3-triggered production of interleukin-6 (IL-6), tumor necrosis factor-alpha, and interferon-alpha/beta (IFN-alpha/beta) in macrophages. Coincidently, overexpression of constitutively active CaMKII-alpha significantly enhances production of the above cytokines. In addition to the activation of mitogen-activated protein kinase and nuclear factor kappaB pathways, CaMKII-alpha can directly bind and phosphorylate transforming growth factor beta-activated kinase 1 (TAK1) and IFN regulatory factor 3 (IRF3; serine on 386) via the N-terminal part of its regulatory domain. Therefore, CaMKII can be activated by TLR ligands, and in turn promotes both myeloid differentiating factor 88 and Toll/IL-1 receptor domain-containing adaptor protein-inducing IFN-beta-dependent inflammatory responses by directly activating TAK1 and IRF3. The cross-talk with the calcium/CaMKII pathway is needed for full activation of TLR signaling in macrophages.

Hepatic RIG-I Predicts Survival and Interferon-α Therapeutic Response in Hepatocellular Carcinoma

In hepatocellular carcinoma (HCC), biomarkers for prediction of prognosis and response to immunotherapy such as interferon-α (IFN-α) would be very useful in the clinic. We found that expression of retinoic acid-inducible gene-I (RIG-I), an IFN-stimulated gene, was significantly downregulated in human HCC tissues. Patients with low RIG-I expression had shorter survival and poorer response to IFN-α therapy, suggesting that RIG-I is a useful prognosis and IFN-α response predictor for HCC patients. Mechanistically, RIG-I enhances IFN-α response by amplifying IFN-α effector signaling via strengthening STAT1 activation. Furthermore, we found that RIG-I deficiency promotes HCC carcinogenesis and that hepatic RIG-I expression is lower in men than in women. RIG-I may therefore be a tumor suppressor in HCC and contribute to HCC gender disparity.

Adaptor Protein LAPF Recruits Phosphorylated p53 to Lysosomes and Triggers Lysosomal Destabilization in Apoptosis

Evidence suggests a functional association between the tumor suppressor p53 and apoptosis-involved organelle lysosome; however, the detailed mechanisms remain poorly understood. We recently reported that a lysosome-targeting protein, LAPF (lysosome-associated and apoptosis-inducing protein containing PH and FYVE domains), could initiate apoptosis of L929 cells through a lysosomal-mitochondrial pathway. In this study, we show that LAPF specifically interacted with phosphorylated p53 (Ser(15/18)) both in vitro and in vivo, which could be enhanced by apoptotic stimuli, such as tumor necrosis factor alpha (TNF-alpha) and ionizing irradiation. The PH domain of LAPF and the transactivation domain of p53 mediated the interaction between both molecules. Phosphorylated p53 (Ser(15/18)) could translocate to lysosomes before lysosomal membrane permeabilization (LMP) in LAPF-initiated and TNF-induced apoptosis. Silencing of LAPF expression abrogated lysosomal translocation of phosphorylated p53 (Ser(15/18)), whereas silencing of p53 expression had no effect on lysosomal translocation of LAPF. Similar to that of LAPF silencing, silencing of endogenous p53 expression in L929 cells could significantly impair TNF-alpha-induced LMP and apoptosis. However, reexpression of wild-type p53, p53S15D (substitution of Ser(15) to Asp that mimics a phosphorylated state), and p53R175H (a transcription-deficient mutant) in p53-knockdown L929 cells could rescue the decrease in TNF-induced apoptosis. The data suggest that phosphorylated p53 (Ser(15/18)) might translocate to lysosome via forming complexes with adaptor protein LAPF and subsequently result in LMP and apoptosis, which might be in a transcription-independent manner.

A Novel Endogenous Human CaMKII Inhibitory Protein Suppresses Tumor Growth by Inducing Cell Cycle Arrest via p27 Stabilization

Calcium/calmodulin-dependent protein kinase II (CaMKII) regulates numerous physiological functions. Inhibition of CaMKII activity, mostly by synthetic reagents, has been proved to suppress cell growth in many cases. So far there are no reports about the physiological functions and underlying mechanisms of endogenous CaMKII inhibitory proteins in cell cycle progression. Here we report the characterization of a novel human endogenous CaMKII inhibitor, human CaMKII inhibitory protein α (hCaMKIINα), which directly interacts with activated CaMKII and effectively inhibits CaMKII activity. hCaMKIINα expression is negatively correlated with the severity of human colon adenocarcinoma. Overexpression of hCaMKIINα inhibits colon adenocarcinoma growth in vitro and in vivo by arresting the cell cycle at the S phase through its conserved inhibitory region (27CIR), whereas silencing the hCaMKIINα expression accelerates tumor growth and cell cycle progression. We found that the effect of hCaMKIINα on cell cycle is correlated with up-regulation of p27 expression, which may be due to the inhibition of proteasome degradation, but not transcriptional regulation, of p27. Moreover, hCaMKIINα deactivated MEK/ERK, which is prerequisite to the inhibition of Thr-187 phosphorylation and subsequent proteasomal degradation of p27, causing the inhibition of S-phase progression of cell cycle. The findings underscore a link between hCaMKIINα-mediated inhibition of CaMKII activity and p27-dependent pathways in controlling tumor cell growth and cell cycle and imply a potential application of hCaMKIINα in the therapeutics of colon cancers.

Rapamycin suppresses TLR4-triggered IL-6 and PGE2 production of colon cancer cells by inhibiting TLR4 expression and NF-κB activation

Toll-like receptor 4 (TLR4) signaling in tumor cells can mediate tumor cell immune escape and tumor progression, being regarded as one of the mechanisms for chronic inflammation in tumorigenesis and progression. So, intervention of TLR4-mediated immune escape and metastasis has been proposed as one of the approaches to cancer prevention and treatment. Rapamycin, an immunosuppressant agent widely used for treatment of autoimmune diseases and transplantation rejection, is recently used for cancer therapy. However, the underlying mechanisms remain to be fully understood. In the present study, we demonstrate that rapamycin can significantly inhibit TLR4-triggered IL-6 and PGE(2) production and invasion of colon cancer cells. Suppression of TLR4-induced IL-6 and PGE(2) production is responsible for the rapamycin-mediated decrease of TLR4-evoked invasion of colon cancer cells. Furthermore, disruption of NF-kappaB pathway contributes to the inhibition of TLR4-induced IL-6, PGE(2) production and invasion by rapamycin in colon cancer cells. Rapamycin can also downregulate TLR4 expression. Therefore, we demonstrate that rapamycin may abrogate TLR4-triggered tumor cell immune escape and invasion by downregulating TLR4 expression and inhibiting TLR4-activated NF-kappaB pathway, thus providing new mechanistic explanation for the antitumor effect of rapamycin.

TLR4 inactivation protects from graft-versus-host disease after allogeneic hematopoietic stem cell transplantation

Graft-versus-host disease (GVHD) is the most common complication after hematopoietic stem cell transplantation. To clarify the role of Toll-like receptor 4 (TLR4), which is a major receptor for bacterial lipopolysaccharides (LPS), in the development of acute GVHD, we used a TLR4-knockout (TLR4−/−) mouse GVHD model and analyzed the underlying immunological mechanisms. When TLR4−/− mice were used as bone marrow and splenocyte cell graft donors or recipients, GVHD symptom occurrence and mortality were delayed compared to wild-type (TLR4+/+) mice. In addition, histopathological analyses revealed that in TLR4−/−→BALB/c chimeras, liver and small intestine tissue damage was reduced with minimal lymphocytic infiltration. In contrast to TLR4+/+, TLR4−/− mice dendritic cells did not express CD80, CD86, CD40, MHC-II or IL-12 during LPS induction and remained in an immature state. Furthermore, the ability of TLR4−/− mice spleen dendritic cells to promote allogeneic T-cell proliferation and, in particular, T-helper cell 1 (Th1) development was obviously attenuated compared with TLR4+/+ mice dendritic cells, and the levels of interferon-γ (IFN-γ) and IL-10, Th2-cell specific cytokines, were significantly higher in the serum of TLR4−/−→BALB/c than in TLR4+/+→BALB/c chimeric mice. Overall, our data revealed that TLR4 may play a role in the pathogenesis of GVHD and that targeted TLR4 gene therapy might provide a new treatment approach to reduce the risk of GVHD.

Rapamycin reverses TLR4 signaling-triggered tumor apoptosis resistance by disrupting Akt-mediated Bcl-xL upregulation.

Toll-like receptor 4 (TLR4) signaling in tumor cells can promote tumor escape and tumor progression, for which TLR4-triggered resistance of tumor cells to apoptosis has been proposed as one of the mechanisms. Rapamycin is an immunosuppressant agent widely used for treatment of transplantation rejection and autoimmune diseases, and recently used for cancer therapy. However, the underlying mechanisms responsible for antitumor effects of rapamycin remain to be fully elucidated. Here we report that rapamycin can reverse TLR4-triggered resistance of colon cancer cells to oxaloplatin- or doxorubicin-induced apoptosis by disrupting Akt and subsequent NF-kappaB activation, suppressing upregulation of anti-apoptotic protein Bcl-xL. Furthermore, Akt/NF-kappaB inhibitors also reverses the apoptosis resistance, accordingly, Akt constitutive activation rescues NF-kappaB activation and Bcl-xL expression in rapamycin-pretreated colon cancer cells, suggesting Akt disruption is critical to the process. Therefore, rapamycin may abrogate TLR4-triggered tumor apoptosis resistance by inhibiting Akt/NF-kappaB pathways and Bcl-xL expression, providing experimental evidence for the anti-tumor effect of rapamycin.