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.

Synthesis and biological evaluation of cholic acid-conjugated oxaliplatin as a new prodrug for liver cancer.

A cholic acid-conjugated oxaliplatin, LLC-202, is developed as a novel prodrug for liver cancer. The conjugate is obtained by using 3-NH2-cyclobutane-1,1-dicarboxylate as a linker between the oxaliplatin analogue and cholic acid moiety and cholic acid is strongly bonded to the linker via an amide bond. Pharmacokinetic experiment shows that LLC-202 is mainly distributed and accumulated in the liver after intravenous administration to Sprague-Dawley rats, revealing the liver-targeting profile. Compared to oxaliplatin, LLC-202 is more easily taken up by human liver cancer cells than normal human liver cells. LLC-202 exhibits higher in vitro anticancer activity and higher efficacy comparable to oxaliplatin in treating primary hepatocellular carcinoma in C57BL/6 mice. It can significantly prolong the survival time of tumor-bearing mice by inducing apoptosis and inhibiting proliferation of cancer cells. In addition, LLC-202 shows less cytotoxicity toward normal human liver cells than oxaliplatin. Its acute toxicity in healthy Kunming (KM) mice after i.v. administration is comparable to oxaliplatin. Histopathological examination reveals that the main toxicity of LLC-202 in mice is the depression of bone marrow hematopoietic cells. The results suggest that LLC-202 has great potential for further development as a new prodrug specific for liver cancer.

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.

3D Genome of macaque fetal brain reveals evolutionary innovations during primate corticogenesis.

Elucidating the regulatory mechanisms of human brain evolution is essential to understanding human cognition and mental disorders. We generated multi-omics profiles and constructed a high-resolution map of 3D genome architecture of rhesus macaque during corticogenesis. By comparing the 3D genomes of human, macaque, and mouse brains, we identified many human-specific chromatin structure changes, including 499 topologically associating domains (TADs) and 1,266 chromatin loops. The human-specific loops are significantly enriched in enhancer-enhancer interactions, and the regulated genes show human-specific expression changes in the subplate, a transient zone of the developing brain critical for neural circuit formation and plasticity. Notably, many human-specific sequence changes are located in the human-specific TAD boundaries and loop anchors, which may generate new transcription factor binding sites and chromatin structures in human. Collectively, the presented data highlight the value of comparative 3D genome analyses in dissecting the regulatory mechanisms of brain development and evolution.

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.

Convergent genomic signatures of high-altitude adaptation among domestic mammals.

Abundant and diverse domestic mammals living on the Tibetan Plateau provide useful materials for investigating adaptive evolution and genetic convergence. Here, we used 327 genomes from horses, sheep, goats, cattle, pigs and dogs living at both high and low altitudes, including 73 genomes generated for this study, to disentangle the genetic mechanisms underlying local adaptation of domestic mammals. Although molecular convergence is comparatively rare at the DNA sequence level, we found convergent signature of positive selection at the gene level, particularly the EPAS1 gene in these Tibetan domestic mammals. We also reported a potential function in response to hypoxia for the gene C10orf67, which underwent positive selection in three of the domestic mammals. Our data provide an insight into adaptive evolution of high-altitude domestic mammals, and should facilitate the search for additional novel genes involved in the hypoxia response pathway.

YTHDF1 links hypoxia adaptation and non-small cell lung cancer progression.

Hypoxia occurs naturally at high-altitudes and pathologically in hypoxic solid tumors. Here, we report that genes involved in various human cancers evolved rapidly in Tibetans and six Tibetan domestic mammals compared to reciprocal lowlanders. Furthermore, m6A modified mRNA binding protein YTHDF1, one of evolutionary positively selected genes for high-altitude adaptation is amplified in various cancers, including non-small cell lung cancer (NSCLC). We show that YTHDF1 deficiency inhibits NSCLC cell proliferation and xenograft tumor formation through regulating the translational efficiency of CDK2, CDK4, and cyclin D1, and that YTHDF1 depletion restrains de novo lung adenocarcinomas (ADC) progression. However, we observe that YTHDF1 high expression correlates with better clinical outcome, with its depletion rendering cancerous cells resistant to cisplatin (DDP) treatment. Mechanistic studies identified the Keap1-Nrf2-AKR1C1 axis as the downstream mediator of YTHDF1. Together, these findings highlight the critical role of YTHDF1 in both hypoxia adaptation and pathogenesis of NSCLC.

Author Correction: Comprehensive integrative analyses identify GLT8D1 and CSNK2B as schizophrenia risk genes.

In the original version of this Article, the affiliation details for Qiushuo Shen incorrectly omitted 'Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, 650204, China'. This has now been corrected in both the PDF and HTML versions of the Article.

Comprehensive integrative analyses identify GLT8D1 and CSNK2B as schizophrenia risk genes.

Recent genome-wide association studies (GWAS) have identified multiple risk loci that show strong associations with schizophrenia. However, pinpointing the potential causal genes at the reported loci remains a major challenge. Here we identify candidate causal genes for schizophrenia using an integrative genomic approach. Sherlock integrative analysis shows that ALMS1, GLT8D1, and CSNK2B are schizophrenia risk genes, which are validated using independent brain expression quantitative trait loci (eQTL) data and integrative analysis method (SMR). Consistently, gene expression analysis in schizophrenia cases and controls further supports the potential role of these three genes in the pathogenesis of schizophrenia. Finally, we show that GLT8D1 and CSNK2B knockdown promote the proliferation and inhibit the differentiation abilities of neural stem cells, and alter morphology and synaptic transmission of neurons. These convergent lines of evidence suggest that the ALMS1, CSNK2B, and GLT8D1 genes may be involved in pathophysiology of schizophrenia.

GRK5 functions as an oncogenic factor in non-small-cell lung cancer.

Lung cancer is the leading cause of cancer-related deaths worldwide, and non-small-cell lung cancer (NSCLC) accounts for about 80% of all cases, which is the major subgroup of lung cancer. G protein-coupled receptor kinase 5 (GRK5) has been demonstrated to play pivotal roles in both development and progression of several pathological conditions including cancer. Here, we found that GRK5 expression was significantly increased in 539 NSCLC cancerous tissues than that in 99 normal non-cancerous tissues by immunohistochemistry analysis; we also showed intensive higher positive staining percentage in female and adenocarcinoma (ADC) NSCLC patients than that in male and squamous cell carcinoma (SCC) patients, respectively. In addition, GRK5 high expression NSCLC patients had a worse overall survival rate than the low expression patients. We provided evidence showing that both the mRNA and protein expression levels of GRK5 were increased in NSCLC cancerous cell lines (GLC-82, SPC-A-1, H520, H838, H358, A549, and H1299) comparing with that in normal human bronchial epithelium cell line (BEAS-2B), and identified many GRK5 mutations in NSCLC cancerous tissues. In addition, we found that depletion of GRK5 inhibited NSCLC cancerous cell proliferation, migration in vitro, and xenograft tumor formation in vivo. Furthermore, GRK5 knockdown promoted cell cycle arrest at G2/M phase and induced cellular apoptosis. In summary, our data reveal an oncogenic role of GRK5 in NSCLC progression, indicating that GRK5 could be used as a new therapeutic target in future.

Establishment of basal cell carcinoma animal model in Chinese tree shrew (Tupaia belangeri chinensis).

Basal cell carcinoma (BCC) is the most common skin cancer worldwide, with incidence rates continuing to increase. Ultraviolet radiation is the major environmental risk factor and dysregulation of the Hedgehog (Hh) signaling pathway has been identified in most BCCs. The treatment of locally advanced and metastatic BBCs is still a challenge and requires a better animal model than the widely used rodents for drug development and testing. Chinese tree shrews (Tupaia belangeri chinensis) are closely related to primates, bearing many physiological and biochemical advantages over rodents for characterizing human diseases. Here, we successfully established a Chinese tree shrew BCC model by infecting tail skins with lentiviral SmoA1, an active form of Smoothened (Smo) used to constitutively activate the Hh signaling pathway. The pathological characteristics were verified by immunohistochemical analysis. Interestingly, BCC progress was greatly enhanced by the combined usage of lentiviral SmoA1 and shRNA targeting Chinese tree shrew p53. This work provides a useful animal model for further BCC studies and future drug discoveries.

A Normalization-Free and Nonparametric Method Sharpens Large-Scale Transcriptome Analysis and Reveals Common Gene Alteration Patterns in Cancers.

Heterogeneity in transcriptional data hampers the identification of differentially expressed genes (DEGs) and understanding of cancer, essentially because current methods rely on cross-sample normalization and/or distribution assumption-both sensitive to heterogeneous values. Here, we developed a new method, Cross-Value Association Analysis (CVAA), which overcomes the limitation and is more robust to heterogeneous data than the other methods. Applying CVAA to a more complex pan-cancer dataset containing 5,540 transcriptomes discovered numerous new DEGs and many previously rarely explored pathways/processes; some of them were validated, both in vitro and in vivo, to be crucial in tumorigenesis, e.g., alcohol metabolism (ADH1B), chromosome remodeling (NCAPH) and complement system (Adipsin). Together, we present a sharper tool to navigate large-scale expression data and gain new mechanistic insights into tumorigenesis.

NCAPH plays important roles in human colon cancer.

Colon cancer (CC) is one of the major malignancies worldwide, whose pathogenesis is complex and requires the accumulated alteration of multiple genes and signaling pathways. Condensins are multi-protein complexes that play pivotal roles in chromosome assembly and segregation during mitosis, meiosis and even tumorigenesis. Using tissue microarrays by immunohistochemistry and hematoxylin-eosin staining, we found that non-SMC condensin I complex subunit H (NCAPH) in colon cancerous tissues was higher than that in all corresponding adjacent non-cancerous tissues. We then characterized the exact function of the NCAPH in CC. We provided evidences showing that NCAPH is highly expressed in colorectal cancer cell lines comparing with normal human colonic epithelial cells, and identified many NCAPH mutations in CC patients. We found that depletion of NCAPH inhibits CC cell proliferation, migration in vitro and xenograft tumor formation in vivo. Furthermore, NCAPH knockdown promotes cell apoptosis and cell cycle arrest at G2/M phase. Interestingly, the NCAPH high expression in tumor tissues of colon patients had a significantly better prognosis and survival rate than low-expression patients, suggesting that NCAPH high expression promotes colonic cancerous cell proliferation; on the other hand, it may also sensitize these cells responding to chemo- or radio-therapies. Collectively, these findings reveal an important role of NCAPH in CC, indicating that NCAPH could be used as a new therapeutic target in future.

Indole alkaloids with new skeleton activating neural stem cells.

Alstoscholarisines A-E (1-5), five unprecedented monoterpenoid indole alkaloids with 6/5/6/6/6 fused-bridge rings, were isolated from Alstonia scholaris. They promoted adult neuronal stem cells (NSCs) proliferation significantly, in which the most active one (1) functioned from a concentration of 0.1 µg/mL in a dosage-dependent manner. Furthermore, 1 enhanced NSC sphere formation and neurogenic fate commitment through activation of a Wnt signaling pathway and promoted NSC differentiation but did not affect proliferation of neuroblastoma cells.