LET-767 determines lipid droplet protein targeting and lipid homeostasis.

Lipid droplets (LDs) are composed of a core of neutral lipids wrapped by a phospholipid (PL) monolayer containing several hundred proteins that vary between different cells or organisms. How LD proteins target to LDs is still largely unknown. Here, we show that RNAi knockdown or gene mutation of let-767, encoding a member of hydroxysteroid dehydrogenase (HSD), displaced the LD localization of three well-known LD proteins: DHS-3 (dehydrogenase/reductase), PLIN-1 (perilipin), and DGAT-2 (diacylglycerol O-acyltransferase 2), and also prevented LD growth in Caenorhabditis elegans. LET-767 interacts with ARF-1 (ADP-ribosylation factor 1) to prevent ARF-1 LD translocation for appropriate LD protein targeting and lipid homeostasis. Deficiency of LET-767 leads to the release of ARF-1, which further recruits and promotes translocation of ATGL-1 (adipose triglyceride lipase) to LDs for lipolysis. The displacement of LD proteins caused by LET-767 deficiency could be reversed by inhibition of either ARF-1 or ATGL-1. Our work uncovers a unique LET-767 for determining LD protein targeting and maintaining lipid homeostasis.

ANKRD29, as a new prognostic and immunological biomarker of non-small cell lung cancer, inhibits cell growth and migration by regulating MAPK signaling pathway.

BACKGROUND: The predominant cancer-related deaths worldwide are caused by lung cancer, particularly non-small cell lung cancer (NSCLC), despite the fact that numerous therapeutic initiatives have been devised to improve the outcomes. Ankyrin repeat domain (ANKRD) is one of the widespread protein structural motifs in eukaryotes but the functions of ANKRD proteins in NSCLC progression remains unclear. METHODS: We performed integrative bioinformatical analysis to determine the dysregulated expression of ANKRDs in multiple tumors and the association between ANKRD29 expression and the NSCLC tumor environment. Quantitative real-time PCR (qRT-PCR), western blot, immunohistochemistry (IHC), and tissue microarray (TMA) assays were used to investigate the expression of ANKRD29 in NSCLC cell lines. The role of ANKRD29 in NSCLC cell proliferation and migration in vitro was deteceted by 5-bromodeoxyuridine (BrdU) incorporation, colony formation, flow cytometry, would-healing, trans-well, and western blot experiment. RNA-seq technology was applied to deciper the molecular mechanism regulated by ANKRD29 in NSCLC. RESULTS: We constructed a valuable risk-score system for predicting the overall survival outcomes of NSCLC patients based on the expression of five hub ANKRD genes. And we found that the hub gene ANKRD29 was remarkedly decreased in NSCLC tissues and cell lines due to the promoter hypermethylation, and revealed that high ANKRD29 expression obviously correlated with patients' better clinical outcome. Overexpression of ANKRD29 significantly inhibited cell proliferation and migration, promoted the cancerous cells' sensitivity to carboplatin and enhanced the killing ability of T cells in NSCLC cells. Interestingly, ANKRD29 can be served as a biomarker to predict the response to immunotherapy in NSCLC. Mechanically, RNA-seq results showed that ANKRD29 could regulate MAPK signaling pathway. Moreover, we screened two potential agonists for ANKRD29. CONCLUSIONS: ANKRD29 functions as a new tumor suppressor in NSCLC tumorigenesis and could be developed as a biomarker for prognostic prediction, immunotherapy response, and drug susceptibility evaluation of NSCLC in the future.

IKZF4 acts as a novel tumor suppressor in non-small cell lung cancer by suppressing Notch signaling pathway.

Non-small cell lung cancer (NSCLC) is the predominant cause of cancer-related mortality globally, although many clinical efforts have been developed to improve the outcomes. The Ikaros zing-finger family transcription factors (IKZFs) have been proved to play pivotal roles in lymphopoiesis and myeloma progression, but their roles in solid tumors development remain unclear. We performed integrative bioinformatical analysis to determine the dysregulation expression of IKZFs in multiple tumors and the correlation between IKZF4 and NSCLC tumor environment. We showed that IKZFs were dysregulated in multiple tumors and IKZF4 was significantly decreased in NSCLC tissues and cell lines due to promoter hypermethylation. We found that low IKZF4 expression obviously correlated with patients' poor clinical outcome. We revealed that IKZF4 overexpression inhibited NSCLC cell growth, migration and xenograft tumor growth, supporting the inhibitory role of IKZF4 in NSCLC tumorigenesis. Additionally, integrative bioinformatical analysis showed that IKZF4 was involved in NSCLC tumor microenvironment. Mechanically, RNA-seq results showed that IKZF4 forced-expression remarkably suppressed Notch signaling pathway in NSCLC, which was validated by qRT-PCR and immunoblot assays. Moreover, we screened several potential agonists for IKZF4.

MrgprF acts as a tumor suppressor in cutaneous melanoma by restraining PI3K/Akt signaling.

The incidence of cutaneous melanoma (CM) has been increasing annually worldwide. In this study, we identify that MrgprF, a MAS related GPR family member, is decreased in cutaneous melanoma tissues and cell lines due to hypermethylation of its promoter region, and show that patients with CM expressing high levels of MrgprF exhibit an improved clinical outcome. We demonstrate that MrgprF forced expression inhibits tumor cell proliferation, migration, xenograft tumor growth, and metastasis. On the contrary, MrgprF knockdown promotes tumor cell proliferation and transformation of immortalized human keratinocyte-HaCaT cells, supporting the inhibitory role of MrgprF during tumor progression. Mechanistic studies reveal that MrgprF reduces the phosphoinositol­3­kinase (PI3K) complex formation between p101 and p110γ subunits, the critical step for phosphatidylinositol-(3, 4)-P2 (PIP2) conversion to phosphatidylinositol-(3, 4, 5)-P3 (PIP3), and then reduces the activation of PI3K/Akt signaling. This effect can be reversed by Akt specific agonist SC79. In addition, AMG 706, a previously documented inhibitor for endothelial cell proliferation, is identified as a potential agonist for MrgprF, and can impede tumor growth both in vitro and in vivo. Taken together, our findings suggest that MrgprF, a novel tumor suppressor in cutaneous melanoma, may be useful as a therapeutic target in the future.

Hypoxia-induced GLT8D1 promotes glioma stem cell maintenance by inhibiting CD133 degradation through N-linked glycosylation.

Gliomas are the most aggressive primary brain tumors. However, no significant improvement in survival has been achieved with the addition of temozolomide (TMZ) or radiation as initial therapy, although many clinical efforts have been carried out to target various signaling pathways or putative driver mutations. Here, we report that glycosyltransferase 8 domain containing 1 (GLT8D1), induced by HIF-1α under a hypoxic niche, significantly correlates with a higher grade of glioma, and a worse clinical outcome. Depletion of GLT8D1 inhibits self-renewal of glioma stem cell (GSC) in vitro and represses tumor growth in glioma mouse models. GLT8D1 knockdown promotes cell cycle arrest at G2/M phase and cellular apoptosis with or without TMZ treatment. We reveal that GLT8D1 impedes CD133 degradation through the endosomal-lysosomal pathway by N-linked glycosylation and protein-protein interaction. Directly blocking the GLT8D1/CD133 complex formation by CD133N1~108 (referred to as FECD133), or inhibiting GLT8D1 expression by lercanidipine, suppresses Wnt/ß-catenin signaling dependent tumorigenesis both in vitro and in patient-derived xenografts mouse model. Collectively, these findings offer mechanistic insights into how hypoxia promotes GLT8D1/CD133/Wnt/ß-catenin signaling during glioma progression, and identify GLT8D1 as a potential therapeutic target in the future.

LncRNA PKMYT1AR promotes cancer stem cell maintenance in non-small cell lung cancer via activating Wnt signaling pathway.

BACKGROUND: Non-small cell lung cancer (NSCLC) is the most common type of human lung cancers, which has diverse pathological features. Although many signaling pathways and therapeutic targets have been defined to play important roles in NSCLC, limiting efficacies have been achieved. METHODS: Bioinformatics methods were used to identify differential long non-coding RNA expression in NSCLC. Real-time RT-PCR experiments were used to examine the expression pattern of lncRNA PKMYT1AR, miR-485-5p. Both in vitro and in vivo functional assays were performed to investigate the functional role of PKMYT1AR/miR-485-5p/PKMYT1 axis on regulating cell proliferation, migration and tumor growth. Dual luciferase reporter assay, fluorescent in situ hybridization (FISH), immunoblot, co-immunoprecipitation experiments were used to verify the molecular mechanism. RESULT: Here, we identify a human-specific long non-coding RNA (lncRNA, ENST00000595422), termed PKMYT1AR (PKMYT1 associated lncRNA), that is induced in NSCLC by Yin Yang 1 (YY1) factor, especially in cancerous cell lines (H358, H1975, H1299, H1650, A549 and SPC-A1) compared to that in normal human bronchial epithelium cell line (BEAS-2B). We show that PKMYT1AR high expression correlates with worse clinical outcome, and knockdown of PKMYT1AR inhibits tumor cell proliferation, migration and xenograft tumor formation abilities. Bioinformatic analysis and a luciferase assay demonstrate that PKMYT1AR directly interacts with miR-485-5p to attenuate the inhibitory role on its downstream oncogenic factor PKMYT1 (the protein kinase, membrane-associated tyrosine/threonine 1) in NSCLC. Furthermore, we uncover that miR-485-5p is downregulated in both cancerous cell lines and peripheral blood serum isolated from NSCLC patients compared to reciprocal control groups. Consistently, forced expression of miR-485-5p inhibits the proliferation and migration abilities of tumor cells. Moreover, we provide evidence showing that PKMYT1AR targeting antisense oligonucleotide (ASO) dramatically inhibit tumor growth in vivo. Mechanistic study shows that PKMYT1AR/ miR-485-5p /PKMYT1 axis promotes cancer stem cells (CSCs) maintenance in NSCLC via inhibiting ß-TrCP1 mediated ubiquitin degradation of ß-catenin proteins, which in turn causes enhanced tumorigenesis. CONCLUSIONS: Our findings reveal the critical role of PKMYT1AR/miR-485-5p /PKMYT1 axis during NSCLC progression, which could be used as novel therapeutic targets in the future.

RETSAT Mutation Selected for Hypoxia Adaptation Inhibits Tumor Growth.

Hypoxia occurs not only in natural environments including high altitude, underground burrows and deep sea, but also in human pathological conditions, such as hypoxic solid tumors. It has been well documented that hypoxia related signaling pathway is associated with a poor clinical outcome. Our group has recently identified multiple novel genes critical for solid tumor growth comparing the genome-wide convergent/parallel sequence evolution of highland mammals. Among them, a single mutation on the retinol saturase gene (RETSAT) containing amino acid switch from glutamine (Q) to arginine (R) at the position 247 was identified. Here, we demonstrate that RETSAT is mostly downregulated in multiple types of human cancers, whose lower expression correlates with worse clinical outcome. We show that higher expression of RETSAT is positively associated with immune infiltration in different human cancers. Furthermore, we identify that the promoter region of RETSAT is highly methylated, which leads to its decreased expressions in tumor tissues comparing to normal tissues. Furthermore, we show that RETSAT knockdown promotes, while its overexpression inhibits, the cell proliferation ability of mouse embryonic fibroblasts (MEFs) and B16 in vitro. In addition, the mice carrying homozygous Q247R mutation (RETSATR/R) is more resistant to xenograft tumor formation, as well as DMBA/TPA induced cutaneous keratinocyte carcinoma formation, compared to littermate wild-type (RETSATQ/Q) mice. Mechanistic study uncovers that the oncogenic factor, the prolyl isomerase (PPIase) Pin1 and its related downstream signaling pathway, were both markedly repressed in the mutant mice compared to the wild-type mice. In summary, these results suggest that interdisciplinary study between evolution and tumor biology can facilitate identification of novel molecular events essential for hypoxic solid tumor growth in the future.

PAQR4 promotes chemoresistance in non-small cell lung cancer through inhibiting Nrf2 protein degradation.

Purpose: Lung cancer is the leading cause of cancer related deaths worldwide. We have previously identified many differentially expressed genes (DEGs) from large scale pan-cancer dataset using the Cross-Value Association Analysis (CVAA) method. Here we focus on Progestin and AdipoQ Receptor 4 (PAQR4), a member of the progestin and adipoQ receptor (PAQR) family localized in the Golgi apparatus, to determine their clinical role and mechanism in the development of non-small cell lung cancer (NSCLC). Methods: The protein expression profile of PAQR4 was examined by IHC using tissue microarrays, and the effects of PAQR4 on cell proliferation, colony formation and xenograft tumor formation were tested in NSCLC cells. Real-time RT-PCR, co-immunoprecipitation (co-IP) and GST-pulldown assays were used to explore the mechanism of action of PAQR4. Results: We provided evidence showing that PAQR4 is increased in NSCLC cancer cell lines (A549, H1299, H1650, H1975, H358, GLC-82 and SPC-A1), and identified many mutations in PAQR4 in non-small cell lung cancer (NSCLC) tissues. We demonstrated that PAQR4 high expression correlates with a worse clinical outcome, and that its knockdown suppresses cell proliferation by inducing apoptosis. Importantly, overexpressed PAQR4 physically interacts with Nrf2 in NSCLC cells, blocking the interaction between Nrf2 and Keap1. Conclusion: Our results suggest that PAQR4 depletion enhances the sensitivity of cancerous cell to chemotherapy both in vitro and xenograft tumor formation in vivo, by promoting Nrf2 protein degradation through a Keap1-mediated ubiquitination process.