PERK-Mediated Cholesterol Excretion from IDH Mutant Glioma Determines Anti-Tumoral Polarization of Microglia

Isocitrate dehydrogenase (IDH) mutation, a known pathologic classifier, initiates metabolic reprogramming in glioma cells and has been linked to the reaction status of glioma-associated microglia/macrophages (GAMs). However, it remains unclear how IDH genotypes contribute to GAM phenotypes. Here, it is demonstrated that gliomas expressing mutant IDH determine M1-like polarization of GAMs, while archetypal IDH induces M2-like polarization. Intriguingly, IDH-mutant gliomas secrete excess cholesterol, resulting in cholesterol-rich, pro-inflammatory GAMs without altering their cholesterol biosynthesis, and simultaneously exhibiting low levels of tumoral cholesterol due to expression remodeling of cholesterol transport molecules, particularly upregulation of ABCA1 and downregulation of LDLR. Mechanistically, a miR-19a/LDLR axis-mediated novel post-transcriptional regulation of cholesterol uptake is identified, modulated by IDH mutation, and influencing tumor cell proliferation and invasion. IDH mutation-induced PERK activation enhances cholesterol export from glioma cells via the miR-19a/LDLR axis and ABCA1/APOE upregulation. Further, a synthetic PERK activator, CCT020312 is introduced, which markedly stimulates cholesterol efflux from IDH wild-type glioma cells, induces M1-like polarization of GAMs, and consequently suppresses glioma cell invasion. The findings reveal an essential role of the PERK/miR-19a/LDLR signaling pathway in orchestrating gliomal cholesterol transport and the subsequent phenotypes of GAMs, thereby highlighting a novel potential target pathway for glioma therapy

Gremlin-1 Promotes Colorectal Cancer Cell Metastasis by Activating ATF6 and Inhibiting ATF4 Pathways

Cancer cell survival, function and fate strongly depend on endoplasmic reticulum (ER) proteostasis. Although previous studies have implicated the ER stress signaling network in all stages of cancer development, its role in cancer metastasis remains to be elucidated. In this study, we investigated the role of Gremlin-1 (GREM1), a secreted protein, in the invasion and metastasis of colorectal cancer (CRC) cells in vitro and in vivo. Firstly, public datasets showed a positive correlation between high expression of GREM1 and a poor prognosis for CRC. Secondly, GREM1 enhanced motility and invasion of CRC cells by epithelial–mesenchymal transition (EMT). Thirdly, GREM1 upregulated expression of activating transcription factor 6 (ATF6) and downregulated that of ATF4, and modulation of the two key players of the unfolded protein response (UPR) was possibly through activation of PI3K/AKT/mTOR and antagonization of BMP2 signaling pathways, respectively. Taken together, our results demonstrate that GREM1 is an invasion-promoting factor via regulation of ATF6 and ATF4 expression in CRC cells, suggesting GREM1 may be a potential pharmacological target for colorectal cancer treatment

Cyclophilin J Is a Novel Peptidyl-Prolyl Isomerase and Target for Repressing the Growth of Hepatocellular Carcinoma

<div><p>Cyclophilin J (CYPJ) is a new member of the peptidyl-prolyl <i>cis/trans</i>-isomerase (PPIase) identified with upregulated expression in human glioma. However, the biological function of CYPJ remained unclear. We aimed to study the role of CYPJ in hepatocellular carcinoma (HCC) carcinogenesis and its therapeutic potential. We determined the expression of CYPJ in HCC/adjacent normal tissues using Western blot, Northern blot and semi-quantitative RT-PCR, analyzed the biochemical characteristics of CYPJ, and resolved the 3D-structure of CYPJ/Cyclosporin A (CsA) complex. We also studied the roles of CYPJ in cell cycle, cyclin D1 regulation, <i>in vitro</i> and <i>in vivo</i> tumor growth. We found that CYPJ expression was upregulated in over 60% HCC tissues. The PPIase activity of CYPJ could be inhibited by the widely used immunosuppressive drug CsA. CYPJ was found expressed in the whole cell of HCC with preferential location at the cell nucleus. CYPJ promoted the transition of cells from G1 phase to S phase in a PPIase-dependent manner by activating cyclin D1 promoter. CYPJ overexpression accelerated liver cell growth <i>in vitro</i> (cell growth assay, colony formation) and <i>in vivo</i> (xenograft tumor formation). Inhibition of CYPJ by its inhibitor CsA or CYPJ-specific RNAi diminished the growth of liver cancer cells <i>in vitro</i> and <i>in vivo</i>. In conclusion, CYPJ could facilitate HCC growth by promoting cell cycle transition from G1 to S phase through the upregulation of cyclin D1. Suppression of CYPJ could repress the growth of HCC, which makes CYPJ a potential target for the development of new strategies to treat this malignancy.</p></div

CYPJ regulates the transcription of cyclin D1.

<p>(A) Effects of CYPJ overexpression on the expression of several cell cycle controllers. The mRNA levels of each gene were evaluated by quantitative real-time RT-PCR, and subsequently normalized by internal <i>GAPDH</i>. ** <i>P</i><0.01. (B) Knockdown of CYPJ expression decreased the expression level of cyclin D1 mRNA. ** <i>P</i><0.01. (C) Knockdown of CYPJ expression decreased the expression level of cyclin D1 protein. (D) Regulation of cyclin D1 promoter by CYPJ, CYPA and their mutants. HEK-293T cells were cotransfected with expression vectors encoding CYPJ, CYPJ mutant (R44A&F49A), CYPA, CYPA mutant (R55A&F60A) or vector control alone (pCMV-HA) and cyclin D1 promoter reporter -962CD1 or the basic luciferase control vector (pGL3-basic) as indicated. The pRL-SV40 plasmids were used as internal control to normalize transfection efficiency. The exogenous CYPJ, CYPA and their mutants were detected by western blot using anti-HA monoclonal antibodies. ** <i>P</i><0.01. (E) Graphics indicate the -962 cyclin D1 promoter (-962CD1) and its deletion mutants. (F) HEK-293T cells were cotransfected with vectors encoding CYPJ, CYPA or pCMV-HA vector control and cyclin D1 promoter reporter -962CD1, or various mutants. Mean and SD of relative fluorescence activities were obtained from four independent experiments. The relative fluorescence activities were normalized to control vector transfected cells. ** <i>P</i><0.01.</p

Overexpression of CYPJ promoted the growth of human cell line SK-Hep1 and L02.

<p>(A) Cell clones with CYPJ protein by stable cell transfection were identified by western blot using anti-myc monoclonal antibodies. L3, L4 and S1 are negative control cells stably transfected with empty vector pcDNA3.1-myc. (B) Growth curves of the recombinant cells with or without exogenous CYPJ were obtained from MTS assays. Each sample was tested in triplicate and the error bars are included. (C) and (D), promotion of colony formation by CYPJ in normal liver cell line L02. (C) Expression of CYPJ promoted the colony formation in L02 cells. L02 cells were transfected with either pcDNA3.1-myc vector (left) or with CYPJ-expression vector (right). (D) Percentage of G418 resistant colonies. Data were results of 3 independent experiments. ** <i>P</i><0.01. (E), (F) and (G), CYPJ promoted <i>in vivo</i> tumorigenicity of L02 and SK-Hep1 cells. (E) Recombinant cells with or without exogenous CYPJ were injected into each side of nude mice, respectively, and tumor weight was measured. (F) Compared with control tumors, tumors originated from cells with overexpressed CYPJ were significantly heavier. ** <i>P</i><0.01. (G) Immunohistochemical staining using anti-myc monoclonal antibody indicated the expression of exogenous CYPJ. Scale bar indicated 50 μm.</p

Targeting CYPJ diminishes the growth of liver cancer cells.

<p>(A) and (B), CsA inhibited the growth of several liver cancer cell lines. (A) Cell viability of liver cancer cell lines in CsA treatment. (B) Microscopic view of SK-Hep1 cells treated with different concentrations of CsA. (C), (D) and (E) Lentivirus-mediated CYPJ knockdown resulted in slower tumor growth <i>in vivo</i>. (C) Tumor growth curves of SK-Hep1 liver cancer cell line originated from tumors injected with LV-CYPJ-RNAi or LV-non-silencing control. ** <i>P</i><0.01. (D) Comparison of the hepatic tumor weight and volume 32 days after injection of Lentivirus between CYPJ knockdown mice and control. (E) Immunofluorescence and pathological analysis of mouse liver cancer cells with/without CYPJ knockdown. For immunofluorescence, transducted cells were labeled with GFP marker and cell nucleus were labeled with DAPI. For pathological analysis, tissue sections were stained with H&E. Scale bar below indicated 100 μm.</p

Effects of CYPJ on cell cycle distribution.

<p>(A) Effects of CYPJ and CYPJ(R44A&F49A) overexpression on cell cycle distribution of SK-Hep1 cells. ** <i>P</i><0.01, N = 3. (B) Knockdown of exogenously enforced CYPJ expression by three different short interference RNAs. The most effective siRNA complex si-J-1 was selected for further analyses. (C) Knockdown of endogenous <i>CYPJ</i> expression by short interference RNA. The expression of <i>CYPA</i> was not affected by the si-J-1 transfection. β-actin was used as internal control. (D) Effects of CYPJ knockdown on cell cycle distribution of SK-Hep1 cells. ** <i>P</i><0.01, N = 3. (E) Effects of CsA inhibition on cell cycle distribution of SK-Hep1 cells. Cells were treated with 4 μM CsA for 48 h before the experiment. ** <i>P</i><0.01, N = 3.</p

Biochemical characteristics of CYPJ protein and the 3D-structure of CYPJ/CsA complex.

<p>(A) Expression and purification of CYPJ. Line 1: un-induced <i>E</i>.<i>coli</i> lysate; lines 2–5: transformed <i>E</i>.<i>coli</i> was induced by 0.2 mM IPTG for 2, 3, 4 and 5 h, respectively; line 6: purified tagged CYPJ; line 7: the chitin tag was removed by DTT treatment. (B) Michaelis-Menten kinetics of CYPJ. (C) Effects of several site mutations (R44A, R44A&F49A, and K120A) to the peptidyl-prolyl <i>cis/trans</i>-isomerase activity of CYPJ. ** <i>P</i><0.01. (D) Catalytic curves of inhibition. a, PPIase assay with 13.1 μM recombinant CYPJ protein; b-e, PPIase assays with 13.1 μM CYPJ and 1, 4, 8 or 16μM CsA, respectively; f, reference assay with no CYPJ or CsA. (D) and (E), 3D-structure of CYPJ-CsA complex. (D) CYPJ-CsA complex structure. CYPJ is shown as ribbon, and CsA is shown in green. The structure of CYPJ-CsA complex refined at 2.4 Å resolution contains two molecules of the complex in an asymmetric unit. In the final structure model the two CYPJ chains consist of 159 and 160 amino acid residues, respectively (the last two C-terminal residues in molecule A and the last one in molecule B could not be located). (E) A part of hydrogen bonding interactions between CYPJ and CsA. The side chains of Arg44, Gln52, Asn92, His110 and Tyr115 are shown in red and CsA in green. Hydrogen bonds are shown as dotted lines.</p

Subcellular localization of CYPJ.

<p>(A) Hela cells were transfected with pEGFP-CYPJ. After 48 h, cells were fixed and stained with DAPI to indicate the nuclei. (B) Hela cells were transfected with pCMV-CYPJ-HA, and 48 h later, the cells were fixed and stained with anti-HA monoclonal antibody. (C) and (D) Immunohistochemical stain of human HCC tissue section using anti-PPIL3 antibody. (C) ×100 magnification. (D) ×400 magnification.</p