Lung Adenocarcinoma Metastasis Research Articles

HMOX1 promotes lung adenocarcinoma metastasis by affecting macrophages and mitochondrion complexes.

BackgroundMetastasis is the leading cause of lung adenocarcinoma (LUAD) patient death. However, the mechanism of metastasis is unclear. We performed bioinformatic analyses for HMOX1 (Heme oxygenase-1), aiming to explore its role in LUAD metastasis.MethodsPan-cancer analysis was first used to identify the metastasis-associated role of HMOX1 in LUAD. HMOX1-related genomic alterations were then investigated. Based on functional enrichment, we systematically correlated HMOX1 with immunological characteristics and mitochondrial activities. Furthermore, weighted gene co-expression network analysis (WGCNA) was applied to construct the HMOX1-mediated metastasis regulatory network, which was then validated at the proteomic level. Finally, we conducted the survival analysis and predicted the potential drugs to target the HMOX1 network.ResultsHMOX1 expression was significantly associated with epithelial-mesenchymal transition (EMT) and lymph and distant metastasis in LUAD. High HMOX1 levels exhibited higher macrophage infiltration and lower mitochondrial complex expression. WGCNA showed a group of module genes co-regulating the traits mentioned above. Subsequently, we constructed an HMOX1-mediated macrophage-mitochondrion-EMT metastasis regulatory network in LUAD. The network had a high inner correlation at the proteomic level and efficiently predicted prognosis. Finally, we predicted 9 potential drugs targeting HMOX1-mediated metastasis in LUAD, like chloroxine and isoliquiritigenin.ConclusionsOur analysis elaborates on the role of HMOX1 in LUAD metastasis and identified a highly prognostic HMOX1-mediated metastasis regulatory network. Novel potential drugs targeting the HMOX1 network were also proposed, which should be tested for their activity against LUAD metastasis in future studies.

Glutathione peroxidase 8 expression on cancer cells and cancer-associated fibroblasts facilitates lung cancer metastasis.

Lung cancer is the leading cause of cancer death worldwide, of which lung adenocarcinoma (LUAD) is the most common subtype. Metastasis is the major cause of poor prognosis and mortality for lung cancer patients, which urgently needs great efforts to be further explored. Herein, glutathione peroxidase 8 (GPX8) was identified as a novel potential pro‐metastatic gene in LUAD metastatic mice models from GEO database. GPX8 was highly expressed in tumor tissues, predicting poor prognosis in LUAD patients. Knockdown of GPX8 inhibited LUAD metastasis in vitro and in vivo, while it did not obviously affect tumor growth. Knockdown of GPX8 decreased the levels of p‐FAK and p‐Paxillin and disturbed the distribution of focal adhesion. Furthermore, GPX8 was overexpressed in cancer‐associated fibroblast (CAF) and associated with CAF infiltration in tumor microenvironment of lung cancer. GPX8 silence on fibroblasts suppressed lung cancer cell migration in the coculture system. BRD2 and RRD4 were the potential transcriptionally regulators for GPX8. Bromodomain extra‐terminal inhibitor JQ1 downregulated GPX8 expression and suppressed lung cancer cell migration. Our findings indicate that highly expressed GPX8 in lung cancer cells and fibroblasts functions as a pro‐metastatic factor in lung cancer. JQ1 is identified as a potential inhibitor against GPX8‐mediated lung cancer metastasis.

METTL7B serves as a prognostic biomarker and promotes metastasis of lung adenocarcinoma cells.

BackgroundTo research the correlation between the prognosis of patients suffering from lung adenocarcinoma (LUAD) and methyltransferase like 7B (METTL7B) expression.MethodsThe Cancer Genome Atlas (TCGA) database was utilized to verify METTL7B expression, and The Human Protein Atlas database was utilized to verify METTL7B expression at the tissue level. The relationship between METTL7B and LUAD prognostic data was then analyzed using the KM-plotter database. The correlation between METTL7B expression and immune cells was demonstrated through the TIMER database. For exploring the possible mechanism of action, gene set enrichment analysis (GSEA) was performed. Finally, the role of METTL7B in the adverse biological events of LUAD was further explored by in vitro experiments such as proliferation and invasion assays.ResultsAs per the TCGA database, METTL7B expression was increased in cancerous tissues compared with paracancerous tissues, and it was mostly located in the cytoplasm. Patients suffering from LUAD who had low METTL7B expression had a relatively better overall survival (OS) and disease-specific survival (DSS) according to the Kaplan-Meier-plotter (KM-plotter) database. METTL7B expression was significantly associated with immune cell infiltration in LUAD patients, as shown by correlation analysis. GSEA revealed that METTL7B may affect the physiological events of LUAD by playing a part in cell cycle regulation. In vitro cytological experiments demonstrated that METTL7B can markedly affect the metastasis of LUAD cells.ConclusionsThe reduction of METTL7B expression can prolong OS and DSS in LUAD patients. It may be utilized as a novel predictive biomarker of LUAD, and may be associated with immune infiltration of LUAD. Interfering with METTL7B expression can significantly cause inhibition of LUAD by modulating the ability of cells to proliferate and migrate. These results point to a possible target for developing anti-cancer therapies against LUAD.

LINC02535/miR-30a-5p/GALNT3 axis contributes to lung adenocarcinoma progression via the NF- κ B signaling pathway

ABSTRACT Long non-coding RNAs (LncRNA) play important roles in multiple types of cancers. We addressed the role of LINC02535 by regulating the miR-30a-5p /GalNAc Transferase 3 (GALNT3) axis to promote the proliferation, migration, and invasion in lung adenocarcinoma (LUAD) cells. The Cancer Genome Atlas (TCGA) database screened differentially expressed lncRNAs. Quantitative real-time PCR analysis (qRT-PCR) confirmed that LINC02535 is highly expressed in LUAD tissues and cells. In vitro experiments showed that LINC02535 promotes the proliferation, migration, and invasion of LUAD cells. A xenograft mouse model was used to show that LINC02535 promotes tumor growth in vivo. RNA immunoprecipitation (RIP) and Dual-luciferase reporter assay results confirmed that LINC02535 targets miR-30a-5p. The Vicia villosa lectin (VVA) pull-down assay indicated that MUC1 is the glycosylation target of GALNT3, and western blot verified that NF-κB is the downstream signaling pathway of MUC1. We found that LINC02535 was increased in LUAD tissues and cells, and LINC02535 was correlated with the poor prognosis of LUAD patients. miR-30a-5p acts as a tumor suppressor in LUAD by targeting GALNT3. We also demonstrated that LINC02535 might function as the sponge of miR-30a-5p to up-regulate GALNT3, and consequently promote the proliferation and metastasis of LUAD. LINC02535 acts as a competing endogenous RNA (ceRNA) to interact with miR-30a-5p, thereby upregulating the expression of GALNT3, enhancing the function of MUC1, and activating the NF-κB signaling pathway, promoting the malignant progression of LUAD cells. Abbreviations: LncRNA:long non-coding RNA; LUAD: lung adenocarcinoma; TCGA: The Cancer Genome Atlas; GALNT3: GalNAc Transferase 3; qRT-PCR: quantitative real-time PCR analysis; RIP: RNA immunoprecipitation; SPF: specific pathogen-free; VVA: Vicia villosa lectin; ceRNA: competing endogenous RNA; MiRNAs: microRNAs; FBS: fetal bovine serum; PBS: Phosphate buffered saline; CCK-8: Cell Counting Kit-8; NSCLC: non-small cell lung cancer; OC: ovarian cancer; HCC: hepatocellular carcinoma.

Genomic Features of Organ-Specific Metastases in Lung Adenocarcinoma.

BackgroundThe genomic features of cancer cells may confer the metastatic ability of lung adenocarcinoma (LUAD) to metastasize to specific organs. We aimed to identify the differences in genomic alterations between patients with primary LUAD with and without metastases and to elucidate the metastatic biology that may help developing biomarker-directed therapies for advanced or metastatic disease.MethodsA retrospective cohort of 497 patients with LUAD including 388 primary tumors (PR), 53 bone metastases (MT-bone), 30 liver metastases (MT-liver), and 26 brain metastases (MT-brain) was tested for genomic alterations by a next-generation sequencing assay.ResultsThe EGFR, TP53, TERT, LRP1B, CDKN2A, ERBB2, ALK, and KMT2C genes had a high frequency of mutations, and the mutations were shared by PR and metastases groups. TP53 and EGFR were the most common mutated genes. In comparison with PR, KRAS, STK11, ATM, NPM1, and ROS1 were significantly mutated in MT-brain, and TP53, MYC, RSPO2, CDKN2a, and CDKN2B were significantly mutated in MT-liver. The frequencies of TP53, CDKN2A, MTAP, PRKCI, and APC mutations were higher in MT-bone than that in PR. The ERBB, phosphoinositide-3-kinase/protein kinase B (PI3K-AKT), cell cycle, Fibroblast growth factor (FGF), and homologous recombination deficiency signaling pathways were affected in both PR and metastases, and there is higher frequency of mutations in metastases. Moreover, the co-mutations in patients with PR and metastasis were respectively analyzed. In addition, the programmed death ligand 1 (PD-L1) level was obviously related to tumor stage and tumor metastases, and the tumor mutational burden was correlated to clinicopathological features including age, gender, pathological stages, and tumor metastases. FGFR1, KAT6A, MYC, RAD21, TP53, and DAXX were also dramatically correlated to the tumor mutational burden.ConclusionMetastases are the most devastating stage of tumors and the main cause of cancer-related deaths. Our results provided a clinically relevant view of the tumor-intrinsic mutational landscape of patients with metastatic LUAD.

Identification of metastasis-related long non-coding RNAs in lung cancer through a novel tumor mesenchymal score

Long non-coding RNAs (lncRNAs) have been proven to play critical roles in epithelial-mesenchymal transition (EMT) and metastasis of lung cancer. However, the biological functions and related mechanisms of lncRNAs are unclear. In addition, the EMT-based prognosis prediction in lung cancer still lacks investigation. Here, we established the methodology of identifying critical metastasis-related lncRNAs using comprehensive datasets of cancer transcriptome, genome and epigenome, and also provided tools for prognosis prediction in lung cancer. Initially, important mesenchymal marker genes were identified to compose the tumor mesenchymal score, which predicted patient prognosis in lung cancer, especially lung adenocarcinoma (LUAD). The score was also correlated with several crucial biological and physiological processes, such as tumor immune and hypoxia. Based on the score, lung cancer patients was classified into epithelial and mesenchymal subtypes, and lncRNAs which exhibited expressional dysregulation, promotor methylation alteration and copy number variation between the two subtypes in LUAD were identified and underwent further prognostic analyses. Finally, we identified 14 lncRNAs as EMT-related and significant biomarkers in prognosis prediction of LUAD. As validation, lncRNA RBPMS-AS1 was proven to be co-expressed with epithelial biomarkers, suppressive for A549 cell migration, invasion and EMT, and also significantly associated with better outcomes of LUAD patients, suggesting the potential of RBPMS-AS1 to serve as a lncRNA epithelial biomarker in metastasis of LUAD. Based on the identified lncRNAs, an EMT-linked lncRNA prognostic signature was further established. Taken together, our study provides robust predictive tools, potential lncRNA targets and feasible screening strategies for future study of lung cancer metastasis.

GPR87 promotes tumor cell invasion and mediates the immunogenomic landscape of lung adenocarcinoma

The purpose of this study is to examine the association between G protein-coupled receptor 87 (GPR87) and lung adenocarcinoma (LUAD) metastasis and immune infiltration. The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) datasets extract clinical data. According to the TCGA database, increased GPR87 expression predicts poor overall survival, progression-free interval, and disease-specific survival in LUAD patients. The meta-analysis also reveals a significant association between high GPR87 expression and poor overall survival. Moreover, functional experiments demonstrate that GPR87 silencing reduces LUAD cell invasion and migration. Immunoblotting shows that GPR87 knockdown decreased Vimentin and N-cadherin expression and increased E-cadherin expression in LUAD cells. GPR87 expression in LUAD is positively correlated with immune infiltration. In addition, GPR87 expression is associated with immune and chemotherapy resistance in LUAD patients. Our findings indicate that GPR87 promotes tumor progression and is correlated with immune infiltration, suggesting GPR87 as a possible biomarker for prognosis prediction in LUAD.

ERO1L promotes the proliferation and metastasis of lung adenocarcinoma via the Wnt2/β‐catenin signaling pathway

This study aimed to explore the role and underlying mechanism of action of Endoplasmic reticulum oxidoreductin-1 L (ERO1L) in lung adenocarcinoma (LUAD). The Gene expression profiling interactive analysis database was used to analyze the expression of ERO1L in LUAD cases. The expression of ERO1L and Wnt2 in LUAD tissue was evaluated using immunohistochemistry. We also used western blotting to assess the expression of ERO1L or Wnt2 and the phosphorylation of β-catenin in LUAD cell lines. Plasmid transfection and small interfering RNA were used for overexpression and knockdown of ERO1L in LUAD cells, respectively. The proliferation, invasion and migration of LUAD cells were analyzed using cell viability, Transwell invasion and wound healing assays. The growth of LUAD tumors in animal models was assessed using tumor xenograft experiments. This study revealed that elevated ERO1L expression was associated with a poor prognosis in LUAD patients. In addition, ERO1L expression was significantly associated with lymph-node metastasis, TNM stage and tumor size. The expression of Wnt2 was positively associated with ERO1L expression in LUAD tissue samples and cell lines. ERO1L overexpression upregulated the expression of Wnt2 and β-catenin phosphorylation in vitro. Additionally, ERO1L was essential for the ubiquitination of Wnt2. Last, ERO1L promoted the proliferation and metastasis of LUAD via the Wnt2 signaling pathway in vitro and in vivo. These findings suggest that ERO1L was highly expressed in LUAD tissue, and it promoted the proliferation and metastasis of LUAD by activating the Wnt2/β-catenin signaling pathway.

ANKRD49 promotes the invasion and metastasis of lung adenocarcinoma via a P38/ATF-2 signalling pathway.

Lung adenocarcinoma (LUAD) is the most challenging neoplasm to treat in clinical practice. Ankyrin repeat domain 49 protein (ANKRD49) is highly expressed in several carcinomas; however, its pattern of expression and role in LUAD are not known. Tissue microarrays, immunohistochemistry, χ 2 test, Spearman correlation analysis, Kaplan–Meier, log‐rank test, and Cox's proportional hazard model were used to analyse the clinical cases. The effect of ANKRD49 on the LUAD was investigated using CCK‐8, clonal formation, would healing, transwell assays, and nude mice experiment. Expressions of ANKRD49 and its associated downstream protein molecules were verified by real‐time PCR, Western blot, immunohistochemistry, and/or immunofluorescence analyses. ANKRD49 expression was highly elevated in LUAD. The survival rate and Cox's modelling analysis indicated that there may be an independent prognostic indicator for LUAD patients. We also found that ANKRD49 promoted the invasion and migration in both in in vitro and in vivo assays, through upregulating matrix metalloproteinase (MMP)‐2 and MMP‐9 activities via the P38/ATF‐2 signalling pathway Our findings suggest that ANKRD49 is a latent biomarker for evaluating LUAD prognosis and promotes the metastasis of A549 cells via upregulation of MMP‐2 and MMP‐9 in a P38/ATF‐2 pathway‐dependent manner.

Increased expression of FAT4 suppress metastasis of lung adenocarcinoma through regulating MAPK pathway and associated with immune cells infiltration.

FAT4 is an extremely large atypical cadherin with crucial roles in the control of planar cell polarity (PCP) and regulation of the Hippo signaling pathway. Our study aims to clarify the FAT4 expression patterns, as well as the significance of FAT4 in predicting the prognosis and cancer immunity to non-small cell lung cancer (NSCLC). FAT4 mRNA and protein expressions were both underregulated in NSCLC and associated with poor prognosis in both lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). In addition, overexpress FAT4 with jujuboside A (JUA) or knockdown FAT4 with siRNA regulated the metastasis of LUAD through MAPK pathways. Moreover, the FAT4 expression included multiple immunological components to promote an immunosuppressive tumor microenvironment (TME). Furthermore, a study of the TCGA-LUAD cohort's DNA methylation results showed that most FAT4 DNA CpG sites were typically hypermethylated in NSCLC relative to the normal lung tissue. The DNA CpG sites cg25879360 and cg26389756 of FAT4 were found to be strongly associated with FAT4 expression in LUAD through the correlation study. In conclusion, this is the first to report the potential function of FAT4 in NSCLC. Hence, FAT4 could be used as a promising prognostic and immunological biomarker for NSCLC.

CD73 (NT5E) Promotes the Proliferation and Metastasis of Lung Adenocarcinoma through the EGFR/AKT/mTOR Pathway.

Introduction Lung cancer is the most common malignant tumor and the main cause of tumor-related death globally. As the 5-year survival rate of lung adenocarcinoma (LUAD) remains low, it is necessary to investigate novel molecular markers and therapeutic targets for LUAD. Materials and Methods The protein expression of CD73 (NT5E) in LUAD specimens was analyzed using immunohistochemistry. Reverse transcription-quantitative PCR and western blot analysis were used to analyze the mRNA and protein expression levels of several genes in LUAD cells. The proliferation of LUAD cells was evaluated using proliferation and colony formation assays and apoptosis analysis. Wound healing and Transwell invasion assays were used to analyze the migration and invasion of the A549 cells, respectively. In addition, overexpression plasmids and small interfering RNAs were used to overexpress or knockdown the expression levels of CD73 in the A549 cell line, respectively. Finally, the interaction between CD73 and EGFR in the A549 cell line was analyzed using immunoprecipitation. Results Our research emphasized the importance of CD73 in the prognosis of LUAD and highlighted it as a potential therapeutic target. We also found that the mRNA and protein expression levels of CD73 are increased in LUAD specimens and cell lines and were associated with a poor prognosis in patients with LUAD. Furthermore, it was revealed that CD73 may promote the proliferation, migration, and invasion of the A549 cell line. Finally, we demonstrated that CD73 could bind epidermal growth factor receptor (EGFR) to further regulate the activation of the AKT/mTOR signaling pathway. Conclusions CD73 promotes LUAD proliferation and metastasis through EGFR/AKT/mTOR axis.

Extra Spindle Pole Bodies-Like 1 Serves as a Prognostic Biomarker and Promotes Lung Adenocarcinoma Metastasis

Extra spindle pole bodies-like 1 (ESPL1), a cysteine endopeptidase, plays a vital role in chromosome inheritance. However, the association of ESPL1 with prognosis and immune infiltration in lung adenocarcinoma (LUAD) has not yet been explored. Here, we analyzed the expression level, prognostic values, diagnostic value, and immune infiltration level in LUAD using various databases. Immunohistochemistry (IHC) and quantitative real-time PCR (qRT-PCR) assays were used to detect the expression of ESPL1 in LUAD tissues and cell lines. In this study, we found that ESPL1 was upregulated in LUAD and a higher expression of ESPL1 was correlated with unfavorable prognosis in LUAD. Meanwhile, Cox hazard regression analysis results suggested that ESPL1 may be an independent prognostic factor for LUAD. Moreover, we demonstrated that ESPL1 expression was significantly correlated with immune infiltration of Th2 and dendritic cells in LUAD. We also confirmed that DNA copy number amplification and DNA hypo-methylation were positively correlated with ESPL1 expression in LUAD. Additionally, DNA copy number amplification was significantly associated with adverse clinical outcomes in LUAD. Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene set enrichment analysis (GSEA) confirmed that ESPL1 was mainly involved in the DNA replication and glycolysis signaling pathway. Finally, we revealed that ESPL1 was highly expressed in LUAD tissues and cell lines. Knockdown of ESPL1 significantly inhibited cell migration and the invasion abilities of LUAD. Our study comprehensively confirmed that ESPL1 expression may serve as a novel prognostic biomarker for both the clinical outcome and immune cell infiltration in LUAD.

Gene amplification-driven RNA methyltransferase KIAA1429 promotes tumorigenesis by regulating BTG2 via m6A-YTHDF2-dependent in lung adenocarcinoma.

BackgroundEpigenetic alterations have been shown to contribute immensely to human carcinogenesis. Dynamic and reversible N6‐methyladenosine (m6A) RNA modification regulates gene expression and cell fate. However, the reasons for activation of KIAA1429 (also known as VIRMA, an RNA methyltransferase) and its underlying mechanism in lung adenocarcinoma (LUAD) remain largely unexplored. In this study, we aimed to clarify the oncogenic role of KIAA1429 in the tumorigenesis of LUAD.MethodsWhole‐genome sequencing and transcriptome sequencing of LUAD data were used to analyze the gene amplification of RNA methyltransferase. The in vitro and in vivo functions of KIAA1429 were investigated. Transcriptome sequencing, methylated RNA immunoprecipitation sequencing (MeRIP‐seq), m6A dot blot assays and RNA immunoprecipitation (RIP) were performed to confirm the modified gene mediated by KIAA1429. RNA stability assays were used to detect the half‐life of the target gene.ResultsCopy number amplification drove higher expression of KIAA1429 in LUAD, which was correlated with poor overall survival. Manipulating the expression of KIAA1429 could regulate the proliferation and metastasis of LUAD. Mechanistically, the target genes of KIAA1429‐mediated m6A modification were confirmed by transcriptome sequencing and MeRIP‐seq assays. We also revealed that KIAA1429 could regulate BTG2 expression in an m6A‐dependent manner. Knockdown of KIAA1429 significantly decreased the m6A levels of BTG2 mRNA, leading to enhanced YTH m6A RNA binding protein 2 (YTHDF2, the m6A “reader”)‐dependent BTG2 mRNA stability and promoted the expression of BTG2; thus, participating in the tumorigenesis of LUAD.ConclusionsOur data revealed the activation mechanism and important role of KIAA1429 in LUAD tumorigenesis, which may provide a novel view on the targeted molecular therapy of LUAD.

Abstract 676: ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis

Abstract Lung cancer is the leading cause of cancer-related death worldwide due to the ability of cancer cells to metastasize. Therefore, it is essential to expand our current knowledge of the biological processes that contribute to metastasis to guide the discovery of novel therapeutic modalities. The Epithelial-to-mesenchymal transition (EMT) is a mechanism for metastasis, which changes polarized epithelial cells into invasive mesenchymal cells. High expression of the Zinc finger E-box binding homeobox 1 (ZEB1) transcription factor is correlated to poor outcomes in cancer, including therapeutic resistance and EMT-mediated metastasis. ZEB1 has a predicted molecular weight of 125kDa; however, multiple groups have reported discrepancies in the observed molecular weight (approximately 190-250kDa). This has been attributed to dimerization mediated by post-translational modifications (PTMs). Therefore, we performed mass spectrometry and identified a novel PTM - K811 acetylation - that may regulate ZEB1 dimerization and function. To define the role of ZEB1 acetylation, we generated ZEB1 acetyl mimetic (K811Q) and deficient (K811R) mutants in a panel of lung adenocarcinoma cell lines. We hypothesize that ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis. We determined that the acetyl-deficient mutant (125kDa) exhibits a decreased half-life compared to WT and acetylated ZEB1, suggesting that disruption of acetylation hinders dimerization and protein stability. However, the acetyl-mimetic mutant (250kDa) protects ZEB1 from proteasomal degradation brought by the action of the E3 ubiquitin ligase SIAH1. siRNA-mediated silencing of SIAH1 increased the mRNA and protein levels of ZEB1 WT and both acetylation mutants. Importantly, silencing of SIAH1 markedly increased the half-life of 250kDa ZEB1 WT from 18.0hrs to 25.4hrs and acetyl-mimetic from 12.3hrs to 24.0hrs, as well as 125kDa ZEB1 acetyl-deficient from 9.7hrs to 18.3hrs. Although we determined that 250kDa ZEB1 is significantly more stable than 125kDa ZEB1, the increase in the stability by silencing SIAH1 contributes to ZEB1 acetyl-deficient binding at the promoter of its target genes mir200c-141 and SEMA3F. 250kDa ZEB1 WT and acetyl-mimetic recruits the histone deacetylases (HDACs) containing the nucleosome remodeling and deacetylase (NuRD) complex, to promote lung adenocarcinoma invasion and metastasis compared to ZEB1 acetyl-deficient. We further established that the stability of ZEB1 is integral to promoting these mesenchymal features. Our results suggest that ZEB1_K811ac regulates its dimerization and stability to promote lung adenocarcinoma via EMT. Future studies are focused on understanding the role of ZEB1 protein stability to recruit NuRD co-repressor and subsequently mediate tumorigenesis and metastasis. Citation Format: Mabel G. Perez-Oquendo, Roxsan Manshouri, Don Gibbons. ZEB1 acetylation regulates dimerization and protein stability to promote lung adenocarcinoma progression and metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 676.

Abstract 2675: Discovery of potent CNS-penetrant covalent KRAS G12C inhibitors

Abstract The RAS family of GTPases, which include KRAS, NRAS, and HRAS, are key regulators of cellular signaling, acting as molecular switches downstream of receptor tyrosine kinases (RTKs) and as key regulators of the RAS/MAPK pathway. RAS cycles between inactive (GDP) and active (GTP) states to regulate the activity of the RAS/MAPK pathway. In many cancers, activating KRAS mutations enable KRAS to persist in the active GTP-bound state, resulting in hyperactive RAS/MAPK pathway signaling that drives cell growth and survival. The KRAS G12C mutation occurs in 14% of lung adenocarcinoma, a cancer type that frequently metastasizes to the brain (40%). To address the high prevalence of KRAS G12C mutant CNS metastases in lung adenocarcinoma, we are developing CNS-penetrant covalent KRAS G12C inhibitors (“ERAS G12Ci’s”) to treat both CNS and systemic disease. ERAS G12Ci’s rapidly form covalent adducts with the KRAS G12C protein in its inactive GDP state and exhibit low nanomolar IC50’s in a nucleotide exchange assay. ERAS G12Ci’s demonstrate high selectivity in a cell-based proteome selectivity assay and inhibit the growth of RAS Initiative KRAS G12C mutant cells, and not RAS Initiative KRAS WT cells. ERAS G12Ci’s potently inhibit cell proliferation in 3-dimensional Cell-Titer Glo (3D-CTG) assays in KRAS G12C mutant lung and pancreatic cell lines (NCI-H1373, NCI-H2122, and MIA PaCa-2). Mechanistically, ERAS G12Ci’s blocked RAS-RAF complex formation and inhibited ERK1/2 phosphorylation. This in vitro activity translates in vivo where ERAS G12Ci’s induce pharmacodynamic modulation in the pancreatic cancer MIA PaCa-2 model. ERAS G12Ci’s significantly inhibit tumor growth in NCI-H1373 and NCI-H2122 CDX lung adenocarcinoma and MIA PaCa-2 PDAC models. No meaningful body weight loss or clinical adverse events were observed with any of these compounds. Human efflux transporter substrate assessments indicate that ERAS G12Ci’s are either not substrates or weak/modest substrates of P-gp, a crucial efflux transporter that can limit CNS penetration. In rat CNS studies, these inhibitors exhibit good CNS penetration performance as measured by brain-to-plasma partition coefficients, which are comparable to those of approved CNS-active small molecule inhibitors. Daily oral administration demonstrates dose-dependent tumor regression in intracranial and intra-carotid injection (ICA) KRAS G12C CDX models. We are optimizing multiple covalent CNS-penetrant KRAS G12C inhibitors that exhibit both CNS and systemic activity in vivo. Citation Format: Jae Hyun Bae, Erin D. Lew, Jun Feng, Marcos Gonzalez-Lopez, Joanne Oh, Patrick Fagan, Matt Salie, Nick Isley, Richard Lam, Adriana Irimia, Robin Nevarez, Bingzhen Lin, Taylor Congdon, Jingchuan Zhang, Dawei Xuan, Ping Chen, Jean-Michel Vernier, Robert Shoemaker. Discovery of potent CNS-penetrant covalent KRAS G12C inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2675.

Abstract 2181: Genomic correlates of Metastasis in KRAS mutant lung adenocarcinoma

Abstract Background: Lung adenocarcinoma (LUAD) is a disease classified by molecular markers. In KRAS-mutant LUAD, STK11 and KEAP1 mutations are associated with decreased overall survival (OS), but predictors of metastasis have been poorly defined. In this study, we identify clinical and genomic predictors of metastatic KRAS-mutant LUAD. Methods: Patients with KRAS-mutant LUAD profiled by targeted next generation sequencing (OncoPanel) were included. Stage, histology, recurrence-free and overall survival were assessed. Clinical and genomic features between metastatic vs non-metastatic samples were compared. KRAS-mutant LUAD samples profiled using MSK-IMPACT in the AACR GENIE database were used to validate our findings. Continuous variables were compared using the Mann-Whitney U test and categorical variables were compared using the Fisher’s Exact test. Survival analysis was performed using the Cox Proportional Hazards model. WExT was used to identify co-occurring and mutually exclusive genetic alterations. Benjamini-Hochberg was used to correct for multiple comparisons. P-values < 0.05 and q-values < 0.10 were considered significant. Results: In the OncoPanel cohort (metastatic n=290; non-metastatic n=324), tumor mutational burden (TMB) (p = .001) and KEAP1 mutations (q = 0.05) were enriched in metastatic samples, while NFKBIA amplifications (q = 0.07) were enriched in non-metastatic samples. KEAP1/STK11 mutations significantly co-occurred (q < 1e-8). Compared to double wild-type samples: KEAP1/STK11 co-mutations were significantly enriched in metastatic samples (n = 72, p = 0.0002, OR 3.4); KEAP1-mutant samples trended towards enrichment in metastatic samples, (n = 21,p = 0.07, OR 2.47); STK11 mutations did not associate with stage (n = 53, p = 0.88, OR = 0.94). In multivariable survival analysis, metastasis (p < 0.005), KEAP1 mutation (p=0.01), and STK11 mutation (p=0.02) were associated with worse OS. In the MSK-IMPACT validation cohort (metastatic site n=417, primary site n = 781), KEAP1 was the only gene enriched in metastatic samples (q < 0.001) at q < 0.05. Compared to double wild type samples: KEAP/STK11 co-mutations (n=138, p < 0.0001, OR 2.1) and KEAP1 mutations (n=59, p = 0.04, OR 1.77) were enriched in metastatic samples; STK11-mutations did not associate with metastasis (n = 190, p = 0.34, OR 0.83). Other predictors of metastasis included Fraction Genome Altered (FGA) (p < 1e-5), TMB (p < 1e-5), and CDKN2A/B deletions (q < 0.003). Conclusion: While both KEAP1 and STK11 mutations are associated with decreased OS in KRAS-mutant LUAD, we find in two independent cohorts that only KEAP1 mutations and KEAP1/STK11 co-mutations, but not STK11 mutations, are associated with metastasis. We also found that FGA, TMB, CDKN2A/B deletions are strongly associated with metastasis. Further research is necessary to understand the influence of KEAP1 mutations, independent of and in-conjunction with STK11 mutations, on metastasis. Citation Format: Daniel Boiarsky, Christine A. Lydon, Emily Chambers, Pasi A. Janne, Mark M. Awad, Eliezer M. Van Allen, David Barbie, Natalie I. Vokes. Genomic correlates of Metastasis in KRAS mutant lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2181.

Abstract 695: Patient derived organoids model lung adenocarcinoma metastases

Abstract Introduction: While lung cancer patient-derived xenografts (PDX) models predict response to therapy, PDX tumors rarely metastasize thus offering little personalized assessment of the biology of metastases. We hypothesize that the development of an in vivo patient-derived organoid (PDO) model will improve our understanding of the underlying genomic and transcriptomic alterations that help drive the biology of metastases for specific lung adenocarcinoma (LUAD) histologic subtypes. Methods: Fresh LUAD specimens were processed and cultured using optimized organoid culture conditions. Established PDOs were characterized by histologic analyses, RNA-sequencing and whole genomic sequencing (WGS). Response to therapy of PDOs was evaluated by treatment with combined chemotherapies, targeted therapies, or co-culturing with patient-matched peripheral blood mononuclear cells (PMBC) in vitro and/or in vivo. The metastatic capabilities of PDOs were assessed in vivo using intracardiac injection and scoring for tumor outgrowth in distant organs using in vivo bioluminescence imaging system. Metastatic tumors from specific organs were isolated, PDOs reestablished, and those tumors underwent additional genomic and transcriptomic characterization. Phylogenetic analyses of tumor metastatic progression and putative metastasis markers were investigated by WGS and RNA-sequencing. Results: We successfully generated 14 PDOs from early-stage (I-II) primary LUAD specimens and 12 from human LUAD metastatic sites (brain, bone and pleural effusion). All PDOs recapitulate genetic alterations and transcriptomic signatures of their original tumors. Moreover, we found that individual PDOs exhibit distinct responses to different chemotherapy regimens and targeted therapies. Pre-treatment of PDOs with low doses of platinum-based doublet chemotherapy regimens activated patient-matched tumor specific T cells and enhanced PD-1 checkpoint inhibitor Nivolumab induced CD8+ T cell-mediated tumor killing. Importantly, utilizing our in vivo PDO metastasis model we successfully developed a platform to evaluate: 1) metastatic potential, 2) response to targeted therapies, 3) tumor evolution with respect of genomic instability, tumor phylogeny, and colonization, and 4) molecular signatures promoting tumor metastases. Conclusion: Our PDO system reliably models human LUAD in vitro and in vivo, by recapitulating histologic architectures, genomic alterations and transcriptomic signatures of the parent LUAD. Using LUAD PDOs we have successfully created multiple platforms to assess response to chemo- and targeted- therapies, develop an in vivo PDO metastasis model to assess tumor evolution, and use of patient matched PMBCs and PDOs to assess immune-priming strategies for individual patient tumors. Citation Format: Yuan Liu, David Jones. Patient derived organoids model lung adenocarcinoma metastases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 695.

Re-evaluating the need for mediastinal lymph node dissection and exploring lncRNAs as biomarkers of N2 metastasis in T1 lung adenocarcinoma.

BackgroundAlthough a well-acknowledged component of curative surgery for lung cancer, investigators have recently questioned the need for mediastinal lymph node dissection (MLND) in early-stage lung cancer cases. As such, the accurate prediction of N2 stage prior to surgery has become increasingly critical. But diagnostic biomarkers predicting N2 metastases are deficient, which are urgently needed.MethodsWe extracted the data of non-small cell lung cancer (NSCLC) patients whose clinical information and follow-up data are complete and without preoperative induction therapy from the Surveillance, Epidemiology, and End Results (SEER) database. The SEER program registries routinely collect demographic and clinic data on patients. And the prognostic differences were analyzed according to the presence or absence of MLND in their lung resection using the R package. Subsequently, the correlations between pN2 metastasis and clinical characteristics were analyzed. In parallel, the long non-coding RNAs (lncRNAs) associated with pN2 status were screened in The Cancer Genome Atlas (TCGA) database by expression difference analysis between pN0-N1 and pN2 patients using limma. Their diagnostic efficiency for detecting N2 metastases was evaluated using receiver operating characteristic (ROC) curves, and a combined diagnostic model was constructed using logistic regression and ROC curve analyses in lung adenocarcinoma (LUAD).ResultsThere were 16,772 patients in MLND group, and 2,699 cases in no-MLND group. The clinical data from SEER showed that the incidence of N2 metastasis was low in pT1 NSCLC (1,023/16,772, 6.10%), but the prognosis of no-MLND patients was poorer than those who underwent MLND (P<0.001, HR =1.605). Pathological N2 metastasis was correlated with age, histologic type, and tumor size. On the other hand, five lncRNAs (LINC00892, AC099522.2, LINC01481, SCAMP1-AS1, and AC004812.2) were screened and confirmed as potential diagnostic biomarkers for detecting N2 metastasis in pT1 LUAD. The AUC of the combined indicators was 0.857.ConclusionsMLND may be oncologically necessary for selected T1 NSCLC patients based on the metastasis incidence and prognosis. A diagnostic model combining LINC00892, AC099522.2, LINC01481, SCAMP1-AS1, and AC004812.2 expression levels may have the potential to be a diagnostic biomarker for detecting N2 metastasis in pT1 LUAD. This study suggests that MLND might be omitted in patients with lower expression level of this diagnostic model.

Pan-cancer analysis of altered glycosyltransferases confers poor clinical outcomes.

Pan-cancer analysis of altered glycosyltransferases confers poor clinical outcomes.

MiR-17-5p/HOXA7 Is a Potential Driver for Brain Metastasis of Lung Adenocarcinoma Related to Ferroptosis Revealed by Bioinformatic Analysis.

ObjectivesPresent study aims to identify the essential mRNAs responsible for the development of brain neurovascular-related metastases (BNM) among lung adenocarcinoma (LUAD) patients. Further, we attempted to predict brain metastases more accurately and prevent their development in LUAD patients.MethodsTranscriptome data analysis was used to identify differentially expressed mRNAs (DEMs) associated with brain metastasis, and thereby the ferroptosis index (FPI) is calculated using a computational model. Meanwhile, the DEmRNAs linked with FPI, and brain metastasis were derived by the intersection of these two groups of DEMs. We also constructed a ceRNA network containing these DEmRNAs, identifying the HCP5 /hsa-miR-17-5p/HOXA7 axis for analysis. Further, a clinical cohort was employed to validate the regulatory roles of molecules involved in the ceRNA regulatory axis.ResultsHere we report the development of a ceRNA network based on BNM-associated DEMs and FPI-associated DEmRNAs which includes three core miRNAs (hsa-miR-338-3p, hsa-miR-429, and hsa-miR-17-5p), three mRNAs (HOXA7, TBX5, and TCF21), and five lncRNAs (HCP5, LINC00460, TP53TG1). Using gene set enrichment analysis (GSEA) and survival analysis, the potential axis of HCP5 /hsa-miR-17-5p/HOXA7 was further investigated. It is found that HOXA7 and ferroptosis index are positively correlated while inhibiting tumor brain metastasis. It may be that HCP5 binds competitively with miR-17-5p and upregulates HOXA7 to increase iron death limiting brain cancer metastasesConclusionsThe expression of both HOXA7 and HCP5 is positively correlated with FPI, indicating a possible link between ferroptosis and BNM. According to the results of our study, the ferroptosis-related ceRNA HCP5 /hsa-miR-17-5p/HOXA7 axis may contribute to the development of BNM in LUAD patients.