Jun Wan

Interleukin-17 receptor A signaling in transformed enterocytes promotes early colorectal tumorigenesis

Interleukin-17A (IL-17A) is a pro-inflammatory cytokine linked to rapid malignant progression of colorectal cancer (CRC) and therapy resistance. IL-17A exerts its pro-tumorigenic activity through its type A receptor (IL-17RA). However, IL-17RA is expressed in many cell types, including hematopoietic, fibroblastoid, and epithelial cells, in the tumor microenvironment, and how IL-17RA engagement promotes colonic tumorigenesis is unknown. Here we show that IL-17RA signals directly within transformed colonic epithelial cells (enterocytes) to promote early tumor development. IL-17RA engagement activates ERK, p38 MAPK, and NF-κB signaling and promotes the proliferation of tumorigenic enterocytes that just lost expression of the APC tumor suppressor. Although IL-17RA signaling also controls the production of IL-6, this mechanism makes only a partial contribution to colonic tumorigenesis. Combined treatment with chemotherapy, which induces IL-17A expression, and an IL-17A neutralizing antibody enhanced the therapeutic responsiveness of established colon tumors. These findings establish IL-17A and IL-17RA as therapeutic targets in colorectal cancer.

MiR-124 Suppresses Growth of Human Colorectal Cancer by Inhibiting STAT3

Emerging evidence indicate that microRNAs (miRNAs) may play important roles in cancer. Aberrant expression of miRNAs has been frequently identified in different human malignancies, including colorectal cancer (CRC). However, the mechanism by which deregulated miRNAs impact the development of CRC remains largely elusive. In this study, we show that miR-124 is significantly down-regulated in CRC compared to adjacent non-tumor colorectal tissues. MiR-124 suppresses the expression of STAT3 by directly binding to its 3′-untranslated region (3′-UTR). Overexpression of miR-124 led to increased apoptosis of CRC cells and reduced tumor growth in vitro and in vivo. Knocking down STAT3 expression by specific siRNA suppressed the growth of CRC cells in vitro and in vivo, resembling that of miR-124 overexpression. Moreover, overexpression of STAT3 in miR-124-transfected CRC cells effectively rescued the inhibition of cell proliferation caused by miR-124. These data suggest that miR-124 serves as a tumor suppressor by targeting STAT3, and call for the use of miR-124 as a potential therapeutic tool for CRC, where STAT3 is often hyper-activated.

miR-203, a tumor suppressor frequently down-regulated by promoter hypermethylation in Rhabdomyosarcoma

Background: Rhabdomyosarcoma (RMS) is a pediatric tumor that expresses several muscle-specific proteins with poor terminal differentiation. Results: miR-203 was frequently down-regulated in RMS, and its re-expression in RMS cells inhibited their growth and migration and promoted terminal differentiation. Conclusion: miR-203 is a tumor suppressor down-regulated in RMS. Significance: miR-203 can serve as a potential target for therapeutic treatment of RMS. Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma found in children and young adults. It is characterized by the expression of a number of skeletal muscle-specific proteins, including MyoD and muscle α-actin. However, unlike normal myoblasts, RMS cells differentiate poorly both in vivo and in culture. As microRNAs are known to regulate tumorigenesis, intensive efforts have been made to identify microRNAs that are involved in RMS development. In this work, we found that miR-203 was frequently down-regulated by promoter hypermethylation in both RMS cell lines and RMS biopsies and could be reactivated by DNA-demethylating agents. Re-expression of miR-203 in RMS cells inhibited their migration and proliferation and promoted terminal myogenic differentiation. Mechanistically, miR-203 exerts its tumor-suppressive effect by directly targeting p63 and leukemia inhibitory factor receptor in RMS cells, which promotes myogenic differentiation by inhibiting the Notch and the JAK1/STAT1/STAT3 pathways, respectively. Our work reveals that miR-203 functions as a tumor suppressor in RMS development.

Endophilin B1 as a Novel Regulator of Nerve Growth Factor/ TrkA Trafficking and Neurite Outgrowth

Neurotrophins and their cognate receptors Trks are important regulators of neuronal survival and differentiation. Recent studies reveal that internalization and trafficking of Trks play a critical role in neurotrophin-mediated signaling. At present, little is known of the molecular events that mediate this process. In the current study, we show that endophilin B1 is a novel regulator of nerve growth factor (NGF) trafficking. We found that endophilin B1 interacts with both TrkA and early endosome marker EEA1. Interestingly, knockdown of endophilin B1 results in enlarged EEA1-positive vesicles in NGF-treated PC12 cells. This is accompanied by increased lysosomal targeting of NGF/TrkA and TrkA degradation, and reduced total TrkA levels. In addition, knockdown of endophilin B1 attenuates Erk1/2 activation in the endosomal fraction after NGF treatment. This is accompanied by a marked inhibition of NGF-induced gene transcription and neurite outgrowth in endophilin B1-knocked down cells. Our observations implicate endophilin B1 as a novel regulator of NGF trafficking, thereby affecting TrkA levels and downstream signaling on endosomes to mediate biological functions of NGF.

BS69, a Specific Adaptor in the Latent Membrane Protein 1-Mediated c-Jun N-Terminal Kinase Pathway

ABSTRACT We previously demonstrated that the Epstein-Barr virus-encoded latent membrane protein 1 (LMP1) potently activates the cellular c-Jun N-terminal kinase (JNK) pathway by sequentially engaging an unknown adaptor, TRAF6, TAB1/TAK1, and JNKKs. We now show that BS69, a MYND domain-containing cellular protein, is the missing adaptor that bridges LMP1 and TRAF6, as the MYND domain and a separate region of BS69 bind to the carboxyl termini of LMP1 and TRAF6, respectively. While LMP1 promotes the interaction between BS69 and TRAF6, the complex formation between LMP1 and TRAF6 is BS69 dependent. A fraction of LMP1 and BS69 is constitutively colocalized in the membrane lipid rafts. Importantly, knockdown of BS69 by small interfering RNAs specifically inhibits JNK activation by LMP1 but not tumor necrosis factor alpha. Although overexpression of either BS69 or a mutant LMP1 without the cytoplasmic carboxyl tail is not sufficient to activate JNK, interestingly, when BS69 is covalently linked to the mutant LMP1, the chimeric protein restores the ability to activate JNK. This indicates that the recruitment and aggregation of BS69 is a prerequisite for JNK activation by LMP1.

Functional elucidation and methylation‐mediated downregulation of ITGA5 gene in breast cancer cell line MDA‐MB‐468

Expression level of integrin α5 in tumor cells has been indicated to be involved in cell proliferation and organ‐specific metastasis. We previously demonstrated that ITGA5 expression was downregulated in the high invasive MDA‐MB‐468 cells compared with other breast cancer cell lines. In this study, we found that the methylation status in the region around transcriptional start site of ITGA5 gene was increased in MDA‐MB‐468 cells. Overexpression of integrin α5 on MDA‐MB‐468 cells resulted in cell growth inhibition, which could be reversed by adhesion to fibronectin. Cell adhesion and spreading to fibronectin was enhanced after ITGA5 was overexpressed in MDA‐MB‐468 cells, while cell migration was attenuated. Knockdown of ITGA5 in MCF‐7 cells led to cell growth inhibition but had little influence on cell migration. These findings indicated the diverse roles of ITGA5 expression in breast cancer cells. J. Cell. Biochem. 110: 1130–1141, 2010. Published 2010 Wiley‐Liss, Inc.

Plasma microRNA-586 is a new biomarker for acute graft-versus-host disease

Acute graft-versus-host disease (aGVHD) is one of the major causes of morbidity and mortality in patients receiving allogeneic hematopoietic cell transplantation (allo-HSCT). MicroRNAs (miRs) were found to have the potential to be the new biomarkers of aGVHD. In this study, we collected samples from 98 patients who underwent allo-HSCT; 63 patients developed aGVHD, and 35 patients did not. Plasma samples were collected at three time points (before aGVHD, at the onset of aGVHD, and after aGVHD) from 52 patients, and the miR-586 expression level was detected by quantitative real-time PCR. We found that the plasma miR-586 level was decreased at the onset of grade I–II aGVHD (Pu2009=u20090.074). In contrast, when infections were detected, plasma miR-586 level was increased. Moreover, we detected the miR-586 expression level in patients who had infections but did not have aGVHD, and we found that miR-586 was upregulated (Pu2009=u20090.005). We also compared the plasma miR-586 level at day 7 after transplantation between aGVHD patients and control patients. In the aGVHD group, there was a considerably higher miR-586 expression in comparison with the non-aGVHD group (Pu2009<u20090.05). A more significant difference between the two groups was found when the patients with infections were excluded (Pu2009=u20090.004). Furthermore, receive operating characteristic (ROC) analysis indicated that a higher expression level of miR-586 at day 7 could predict impending aGVHD. The optimal cutoff value of miR-586 to predict aGVHD was 2200 copies/μL with a sensitivity of 87.5xa0% and specificity of 55.0xa0%, and the area under the curve (AUC) was 0.739 (95xa0% CI 0.598–0.880, Pu2009=u20090.004). Our study suggests that miR-586 might participate in the occurrence of aGVHD and could be a putative target for novel aGVHD therapy. The plasma level of miR-586 at day 7 after allo-HSCT would be a potential biomarker for predicting the occurrence of aGVHD.

Rpph1 Upregulates CDC42 Expression And Promotes Hippocampal Neuron Dendritic Spine Formation By Competing With MiR-330-5p

Alzheimer’s disease (AD) is a heterogeneous neurodegenerative disease. Recent studies employing microRNA-seq and genome-wide sequencing have identified some non-coding RNAs that are influentially involved in AD pathogenesis. Non-coding RNAs can compete with other endogenous RNAs by microRNA response elements (MREs) and manipulate biological processes, such as tumorigenesis. However, only a few non-coding RNAs have been reported in the pathogenesis of AD. In this study, we constructed the first competing endogenous RNA (ceRNA) network leveraging whole transcriptome sequencing and a previously studied microRNA-seq of APPswe/PS1ΔE9 transgenic mice. The underlying mechanisms for the involvement of ceRNA in AD were validated using the Dual Luciferase Reporter Assay, detection of transcription levels by quantitative RT-PCR and translation levels by Western blotting, and morphological examination in primary cultured neurons. In the ceRNA network, four lncRNAs (C030034L19Rik, Rpph1, A830012C17Rik, and Gm15477) and five miRNAs (miR-182-5p, miR-330-5p, miR-326-3p, miR-132-3p, and miR-484) are enriched in nine pathways and an AD-related gene pool. Among them, Ribonuclease P RNA component H1 (Rpph1) is upregulated in the cortex of APPswe/PS1ΔE9 mice compared to wild type controls. Rpph1 binds to miR326-3p/miR-330-5p and causes the release of their downstream target Cdc42, which leads to CDC42 upregulation. This effect was disrupted upon mutation of the MRE on Rpph1. Moreover, overexpression of Rpph1 increased dendritic spine density in primary cultured hippocampal pyramidal neurons, whereas knocking down of Rpph1 had the reverse effect. In conclusion, Rpph1 modulates CDC42 expression level in a ceRNA-dependent manner, which may represent a compensatory mechanism in the early stage of the AD pathogenesis.

MicroRNAs 99b-5p/100-5p Regulated by Endoplasmic Reticulum Stress are Involved in Abeta-Induced Pathologies

Alzheimer’s disease (AD) is the most common cause of dementia. Amyloid β (Abeta, Aβ) deposition and intracellular tangles are the pathological hallmarks of AD. MicroRNAs (miRNAs) are small non-coding RNAs, which have been found to play very important roles, and have the potential to serve as diagnostic markers during neuronal pathogenesis. In this study, we aimed to determine the roles of miR-99b-5p and miR-100-5p in Aβ-induced neuronal pathologies. We detected the expression levels of miR-99b-5p and miR-100-5p in the brains of APPswe/PS1ΔE9 double-transgenic mice (APP/PS1 mice) at different age stages and found that both miRNAs were decreased at early stages while increased at late stages of APP/PS1 mice when compared with the age-matched wild type (WT) mice. Similar phenomenon was also observed in Aβ-treated cultured cells. We also confirmed that mammalian target of rapamycin (mTOR) is one of the targets of miR-99b-5p/100-5p, which is consistent with previous studies in cancer. MiR-99b-5p/100-5p has been found to promote cell apoptosis with the Aβ treatment. This effect may be induced via the mTOR pathway. In our study, we find both miR-99b-5p and miR-100-5p affect neuron survival by targeting mTOR. We also speculate that dynamic change of miR-99b-5p/100-5p levels during Aβ-associated pathologies might be attributed to Aβ-induced endoplasmic reticulum stress (ER stress), suggesting the potential role of the “ER stress–miRNAs–mTOR” axis in Aβ-related AD pathogenesis.

Myosin III-mediated cross-linking and stimulation of actin bundling activity of Espin

Class III myosins (Myo3) and actin-bundling protein Espin play critical roles in regulating the development and maintenance of stereocilia in vertebrate hair cells, and their defects cause hereditary hearing impairments. Myo3 interacts with Espin1 through its tail homology I motif (THDI), however it is not clear how Myo3 specifically acts through Espin1 to regulate the actin bundle assembly and stabilization. Here we discover that Myo3 THDI contains a pair of repeat sequences capable of independently and strongly binding to the ankyrin repeats of Espin1, revealing an unexpected Myo3-mediated cross-linking mechanism of Espin1. The structures of Myo3 in complex with Espin1 not only elucidate the mechanism of the binding, but also reveal a Myo3-induced release of Espin1 auto-inhibition mechanism. We also provide evidence that Myo3-mediated cross-linking can further promote actin fiber bundling activity of Espin1. DOI: http://dx.doi.org/10.7554/eLife.12856.001