マイクロRNA449aの欠損は大腸での腫瘍形成を促進する

MicroRNAs have broad roles in tumorigenesis and cell differentiation through regulation of target genes. Notch signaling also controls cell differentiation and tumorigenesis. However, the mechanisms through which Notch mediates microRNA expression are still unclear. In this study, we aimed to identify microRNAs regulated by Notch signaling. Our analysis found that microRNA-449a (miR-449a) was indirectly regulated by Notch signaling. Although miR-449a-deficient mice did not show any Notch-dependent defects in immune cell development, treatment of miR-449a-deficient mice with azoxymethane (AOM) or dextran sodium sulfate (DSS) increased the numbers and sizes of colon tumors. These effects were associated with an increase in intestinal epithelial cell proliferation following AOM/DSS treatment. In patients with colon cancer, miR-449a expression was inversely correlated with disease-free survival and histological scores and was positively correlated with the expression of MLH1 for which loss-of function mutations have been shown to be involved in colon cancer. Colon tissues of miR-449a-deficient mice showed reduced Mlh1 expression compared with those of wild-type mice. Thus, these data suggested that miR-449a acted as a key regulator of colon tumorigenesis by controlling the proliferation of intestinal epithelial cells. Additionally, activation of miR-449a may represent an effective therapeutic strategy and prognostic marker in colon cancer

Control of embryonic stem cells self-renewal and differentiation via coordinated splicing and translation of YY2

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Influence of EGFR-activating mutations on sensitivity to tyrosine kinase inhibitors in a KRAS mutant non-small cell lung cancer cell line.

In non-small cell lung cancer (NSCLC), oncogenic driver mutations including those in KRAS and EGFR are typically mutually exclusive. However, recent reports indicate that multiple driver mutations are found in a certain percentage of cancers, and that the therapeutic responses of such cases with co-mutations of driver genes are largely unclear. Here, using CRISPR-Cas9-mediated genome editing, we generated isogenic cell lines harboring one or two copies of an EGFR-activating mutation from the human NSCLC cell line A549, which is known to harbor a homozygous KRAS gene mutation. In comparison with parent cells with KRAS mutation alone, cells with concomitant EGFR mutation exhibited higher sensitivity to EGFR-tyrosine kinase inhibitors (TKIs) but not to conventional anti-cancer drugs. In particular, cells with two copies of EGFR mutation were markedly more sensitive to EGFR-TKIs compared with parent cells. Thus, the presence of concomitant EGFR mutation can affect the TKI response of KRAS-mutated cells, implying that EGFR-TKI may represent an effective treatment option against NSCLC with EGFR/KRAS co-mutation

TBL2 Is a Novel PERK-Binding Protein that Modulates Stress-Signaling and Cell Survival during Endoplasmic Reticulum Stress

<div><p>Under ER stress, PKR-like ER-resident kinase (PERK) phosphorylates translation initiation factor eIF2α, resulting in repression of global protein synthesis and concomitant upregulation of the translation of specific mRNAs such as activating transcription factor 4 (ATF4). This PERK function is important for cell survival under ER stress and poor nutrient conditions. However, mechanisms of the PERK signaling pathway are not thoroughly understood. Here we identify transducin (beta)-like 2 (TBL2) as a novel PERK-binding protein. We found that TBL2 is an ER-localized type-I transmembrane protein and preferentially binds to the phosphorylated form of PERK, but not another eIF2α kinase GCN2 or ER-resident kinase IRE1, under ER stress. Immunoprecipitation analysis using various deletion mutants revealed that TBL2 interacts with PERK via the N-terminus proximal region and also associates with eIF2α via the WD40 domain. In addition, TBL2 knockdown can lead to impaired ATF4 induction under ER stress or poor nutrient conditions such as glucose and oxygen deprivation. Consistently, TBL2 knockdown rendered cells vulnerable to stresses similarly to PERK knockdown. Thus, TBL2 serves as a potential regulator of the PERK pathway.</p></div

TBL2 interacts with PERK in response to ER stress.

<p>(A) 293T cells were transiently co-transfected with pTBL2 (V5-tag) and pFLAG-PERK, and then were treated with 300 nM thapsigargin for 1, or 3 h. The cell lysates were immunoprecipitated with anti-V5 antibody and immunoblotted with anti-FLAG or anti-V5 antibody. (B) 293T cells were transiently transfected with pFLAG-PERK or together with pTBL2 (non-tag). After immunoprecipitation with anti-FLAG conjugated beads, PERK-bound TBL2 protein was detected with anti-TBL2 antibody. (C) 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with 300 nM thapsigargin for 1 hour, 1 mM DTT for 30 min or with 5 mM histidinol (HisOH) for 4 hour. After immunoprecipitation with anti-FLAG antibody-conjugated beads, each protein was immunoblotted with the indicated antibody.</p

TBL2 knockdown impairs ATF4 induction under glucose- and oxygen-deprived conditions.

<p>(A) 293T cells were transiently transfected with pFLAG-TBL2. Then, the cells were incubated for 4 h under glc(−) and/or hypoxic conditions (Hy). After immunoprecipitation with anti-FLAG-conjugated beads, each sample was subjected to immunoblot analysis. (B) Control-, TBL2- or PERK-shRNA-expressing 786-O cells were incubated for 2 h under glc(−) and/or hypoxic conditions (Hy). Each sample was subjected to immunoblot analysis. (C) Control or TBL2 shRNA-expressing cells were exposed to glc(−) and/or hypoxia (Hy) for 2 h in the presence or absence of 10 µM MG132. (D) TBL2- or PERK-shRNA-expressing cells were incubated for the indicated times under glc(−) and hypoxic conditions (Hy). <i>ATF4</i> mRNA induction under glc(−)/hypoxic conditions was measured by qRT-PCR. <i>β-actin</i> mRNA levels were used for normalization.</p

Preferential binding of TBL2 to phospho-PERK.

<p>(A) 293T cells were transiently co-transfected with pTBL2 (V5-tag) and either pFLAG-PERK, pFLAG-PERK(K621A) or pFLAG-IRE1 and then were treated with 300 nM thapsigargin (Tg) for 2 h. The cell lysates were immunoprecipitated with anti-V5 antibody and immunoblotted with anti-FLAG or anti-V5 antibody. (B) 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with 300 nM thapsigargin (Tg), 4 µg/ml tunicamycin (Tu) or 10 mM 2-deoxyglucose (2DG) for 2 h. Endogenous PERK protein was detected with anti-PERK or anti–phospho-PERK antibody. (C) 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with the indicated doses of hydrogen peroxide (H₂O₂) for 4 hour. After immunoprecipitation with anti-FLAG antibody-conjugated beads, each protein was immunoblotted with the indicated antibody. (D) 786-O, 293 and 293T cells were transiently transfected with pFLAG-TBL2 and then were treated with 300 nM thapsigargin (Tg) for 1 hour. After immunoprecipitation with anti-FLAG antibody-conjugated beads, each protein was immunoblotted with the indicated antibody.</p