Long non-coding RNA TILR constitutively represses TP53 and apoptosis in lung cancer.

Non-coding RNAs have an integral regulatory role in numerous functions related to lung cancer development. Here, we report identification of a novel lncRNA, termed TP53-inhibiting lncRNA (TILR), which was found to function as a constitutive negative regulator of p53 expression, including activation of downstream genes such as p21 and MDM2, and induction of apoptosis. A proteomic search for TILR-associated proteins revealed an association with PCBP2, while the mid-portion of TILR was found to be required for both PCBP2 and p53 mRNA binding. In addition, depletion of PCBP2 resulted in phenocopied effects of TILR silencing. TILR was also shown to suppress p53 expression in a post-transcriptional manner, as well as via a positive feedback loop involving p53 and Fanconi anemia pathway genes. Taken together, the present findings clearly demonstrate that TILR constitutively inhibits p53 expression in cooperation with PCBP2, thus maintaining p53 transcriptional activity at a level sufficiently low for avoidance of spurious apoptosis induction.

SRGN-Triggered Aggressive and Immunosuppressive Phenotype in a Subset of TTF-1-Negative Lung Adenocarcinomas.

BACKGROUND: Approximately 20% of lung adenocarcinoma (LUAD) is negative for the lineage-specific oncogene Thyroid transcription factor 1 (TTF-1) and exhibits worse clinical outcome with a low frequency of actionable genomic alterations. To identify molecular features associated with TTF-1-negative LUAD, we compared the transcriptomic and proteomic profiles of LUAD cell lines. SRGN , a chondroitin sulfate proteoglycan Serglycin, was identified as a markedly overexpressed gene in TTF-1-negative LUAD. We therefore investigated the roles and regulation of SRGN in TTF-1-negative LUAD. METHODS: Proteomic and metabolomic analyses of 41 LUAD cell lines were done using mass spectrometry. The function of SRGN was investigated in 3 TTF-1-negative and 4 TTF-1-positive LUAD cell lines and in a syngeneic mouse model (n = 5 to 8 mice per group). Expression of SRGN was evaluated in 94 and 105 surgically resected LUAD tumor specimens using immunohistochemistry. All statistical tests were 2-sided. RESULTS: SRGN was markedly overexpressed at mRNA and protein levels in TTF-1-negative LUAD cell lines (P < .001 for both mRNA and protein levels). Expression of SRGN in LUAD tumor tissue was associated with poor outcome (hazard ratio = 4.22, 95% confidence interval = 1.12 to 15.86, likelihood ratio test, P = .03), and with higher expression of Programmed cell death 1 ligand 1 (PD-L1) in tumor cells and higher infiltration of Programmed cell death protein 1-positive lymphocytes. SRGN regulated expression of PD-L1 as well as proinflammatory cytokines, including Interleukin-6, Interleukin-8, and C-X-C motif chemokine 1 in LUAD cell lines; increased migratory and invasive properties of LUAD cells and fibroblasts; and enhanced angiogenesis. SRGN was induced by DNA demethylation resulting from Nicotinamide N-methyltransferase-mediated impairment of methionine metabolism. CONCLUSIONS: Our findings suggest that SRGN plays a pivotal role in tumor-stromal interaction and reprogramming into an aggressive and immunosuppressive tumor microenvironment in TTF-1-negative LUAD.

CEBPγ facilitates lamellipodia formation and cancer cell migration through CERS6 upregulation.

Ceramide synthase 6 (CERS6) promotes lung cancer metastasis by stimulating cancer cell migration. To examine the underlying mechanisms, we performed luciferase analysis of the CERS6 promoter region and identified the Y-box as a cis-acting element. As a parallel analysis of database records for 149 non-small-cell lung cancer (NSCLC) cancer patients, we screened for trans-acting factors with an expression level showing a correlation with CERS6 expression. Among the candidates noted, silencing of either CCAAT enhancer-binding protein γ (CEBPγ) or Y-box binding protein 1 (YBX1) reduced the CERS6 expression level. Following knockdown, CEBPγ and YBX1 were found to be independently associated with reductions in ceramide-dependent lamellipodia formation as well as migration activity, while only CEBPγ may have induced CERS6 expression through specific binding to the Y-box. The mRNA expression levels of CERS6, CEBPγ, and YBX1 were positively correlated with adenocarcinoma invasiveness. YBX1 expression was observed in all 20 examined clinical lung cancer specimens, while 6 of those showed a staining pattern similar to that of CERS6. The present findings suggest promotion of lung cancer migration by possible involvement of the transcription factors CEBPγ and YBX1.

Conditional Ror1 knockout reveals crucial involvement in lung adenocarcinoma development and identifies novel HIF-1α regulator.

We previously reported that ROR1 is a crucial downstream gene for the TTF-1/NKX2-1 lineage-survival oncogene in lung adenocarcinoma, while others have found altered expression of ROR1 in multiple cancer types. Accumulated evidence therefore indicates ROR1 as an attractive molecular target, though it has yet to be determined whether targeting Ror1 can inhibit tumor development and growth in vivo. To this end, genetically engineered mice carrying homozygously floxed Ror1 alleles and an SP-C promoter-driven human mutant EGFR transgene were generated. Ror1 ablation resulted in marked retardation of tumor development and progression in association with reduced malignant characteristics and significantly better survival. Interestingly, gene set enrichment analysis identified a hypoxia-induced gene set (HALLMARK_HYPOXIA) as most significantly downregulated by Ror1 ablation in vivo, which led to findings showing that ROR1 knockdown diminished HIF-1α expression under normoxia and clearly hampered HIF-1α induction in response to hypoxia in human lung adenocarcinoma cell lines. The present results directly demonstrate the importance of Ror1 for in vivo development and progression of lung adenocarcinoma, and also identify Ror1 as a novel regulator of Hif-1α. Thus, a future study aimed at the development of a novel therapeutic targeting ROR1 for treatment of solid tumors such as seen in lung cancer, which are frequently accompanied with a hypoxic tumor microenvironment, is warranted.

Inhibition of heat shock protein 90 destabilizes receptor tyrosine kinase ROR1 in lung adenocarcinoma.

We have previously identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a direct transcriptional target of TTF-1/NKX2-1, a lineage-survival oncogene in lung adenocarcinoma. ROR1 sustains prosurvival signaling from multiple receptor tyrosine kinases including epidermal growth factor receptor, MET, and insulin-like growth factor 1 receptor in part by maintaining the caveolae structure as a scaffold protein of cavin-1 and caveolin-1. In this study, a high throughput screening of the natural product library containing 2560 compounds was undertaken using a cell-based FluoPPI assay detecting ROR1-cavin-1 interaction. As a result, geldanamycin (GA), a known inhibitor of heat shock protein 90 (HSP90), was identified as a potential inhibitor of ROR1. Geldanamycin, as well as two GA derivatives tested in the clinic, 17-allylamino-17-demethoxygeldanamycin (17-AAG) and 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreased ROR1 protein expression. We found that ROR1 physically interacted with HSP90α, but not with other HSP90 paralogs, HSP90ß or GRP94. Geldanamycin in turn destabilized and degraded ROR1 protein in a dose- and time-dependent manner through the ubiquitin/proteasome pathway, resulting in a significant suppression of cell proliferation in lung adenocarcinoma cell lines, for which the kinase domain of ROR1, but not its kinase activity or N-glycosylation, was required. Our findings indicate that HSP90 is required to sustain expression of ROR1 crucial for lung adenosarcoma survival, suggesting that inhibition of HSP90 could be a promising therapeutic strategy in ROR1-positive lung adenocarcinoma.

Divergent lncRNA MYMLR regulates MYC by eliciting DNA looping and promoter-enhancer interaction.

Long non-coding RNAs (lncRNAs) function in a wide range of processes by diverse mechanisms, though their roles in regulation of oncogenes and/or tumor suppressors remain rather elusive. We performed a global search for lncRNAs affecting MYC activity using a systems biology-based approach with a K supercomputer and the GIMLET algorism based on local distance correlations. Consequently, MYMLR was identified and experimentally shown to maintain MYC transcriptional activity and cell cycle progression despite the low levels of expression. A proteomic search for MYMLR-binding proteins identified PCBP2, while it was also found that MYMLR places a 557-kb upstream enhancer region in the proximity of the MYC promoter in cooperation with PCBP2. These findings implicate a crucial role for MYMLR in regulation of the archetypical oncogene MYC and warrant future studies regarding the involvement of low copy number lncRNAs in regulation of other crucial oncogenes and tumor suppressor genes.

ROR1-CAVIN3 interaction required for caveolae-dependent endocytosis and pro-survival signaling in lung adenocarcinoma.

The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transcriptional target of the lineage-survival oncogene NKX2-1/TTF-1 in lung adenocarcinomas. In addition to its kinase-dependent role, ROR1 functions as a scaffold protein to facilitate interaction between caveolin-1 (CAV1) and CAVIN1, and consequently maintains caveolae formation, which in turn sustains pro-survival signaling toward AKT from multiple receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR), MET (proto-oncogene, receptor tyrosine kinase), and IGF-IR (insulin-like growth factor receptor 1). Therefore, ROR1 is an attractive target for overcoming EGFR-TKI resistance due to various mechanisms such as EGFR T790M double mutation and bypass signaling from other RTKs. Here, we report that ROR1 possesses a novel scaffold function indispensable for efficient caveolae-dependent endocytosis. CAVIN3 was found to bind with ROR1 at a site distinct from sites for CAV1 and CAVIN1, a novel function required for proper CAVIN3 subcellular localization and caveolae-dependent endocytosis, but not caveolae formation itself. Furthermore, evidence of a mechanistic link between ROR1-CAVIN3 interaction and consequential caveolae trafficking, which was found to utilize a binding site distinct from those for ROR1 interactions with CAV1 and CAVIN1, with RTK-mediated pro-survival signaling towards AKT in early endosomes in lung adenocarcinoma cells was also obtained. The present findings warrant future study to enable development of novel therapeutic strategies for inhibiting the multifaceted scaffold functions of ROR1 in order to reduce the intolerable death toll from this devastating cancer.

Thyroid transcription factor-1-regulated microRNA-532-5p targets KRAS and MKL2 oncogenes and induces apoptosis in lung adenocarcinoma cells.

Thyroid transcription factor-1 (TTF-1), also known as NKX2-1, plays a role as a lineage-survival oncogene in lung adenocarcinoma that possesses double-edged sword characteristics. Although evidence from previous studies has steadily accumulated regarding the roles of TTF-1 in transcriptional regulation of protein-coding genes, little is known about its regulatory relationship with microRNAs. Here, we utilized an integrative approach designed to extract maximal information from expression profiles of both patient tumors in vivo and TTF-1-inducible cell lines in vitro, which identified microRNA (miR)-532-5p as a novel transcriptional target of TTF-1. We found that miR-532-5p is directly regulated by TTF-1 through its binding to a genomic region located 8 kb upstream of miR-532-5p, which appears to impose transcriptional regulation independent of that of CLCN5, a protein-coding gene harboring miR-532-5p in its intron 3. Furthermore, our results identified KRAS and MKL2 as novel direct targets of miR-532-5p. Introduction of miR-532-5p mimics markedly induced apoptosis in KRAS-mutant as well as KRAS wild-type lung adenocarcinoma cell lines. Interestingly, miR-532-5p showed effects on MEK-ERK pathway signaling, specifically in cell lines sensitive to siKRAS treatment, whereas those miR-532-5p-mediated effects were clearly rendered as phenocopies by repressing expression or inhibiting the function of MKL2 regardless of KRAS mutation status. In summary, our findings show that miR-532-5p is a novel transcriptional target of TTF-1 that plays a tumor suppressive role by targeting KRAS and MKL2 in lung adenocarcinoma.

Receptor tyrosine kinase-like orphan receptor 1, a target of NKX2-1/TTF-1 lineage-survival oncogene, inhibits apoptosis signal-regulating kinase 1-mediated pro-apoptotic signaling in lung adenocarcinoma.

We previously identified receptor tyrosine kinase-like orphan receptor 1 (ROR1) as a transcriptional target of the NKX2-1/TTF-1 lineage-survival oncogene in lung adenocarcinoma. ROR1 consequently sustains a favorable balance between pro-survival phosphatidylinositol 3-kinase-protein kinase B and pro-apoptotic apoptosis signal-regulating kinase 1 (ASK1)-p38MAPK signaling. In contrast to recent advances in understanding how ROR1 sustains pro-survival signaling, the mechanism of ROR1 repression of pro-apoptotic signaling remains rather elusive. In the present study, we investigated the underlying mechanism of ROR1-mediated inhibition of the ASK1-p38MAPK signaling pathway. Growth inhibition mediated by siROR1 was partially but significantly alleviated by ASK1 co-knockdown in lung adenocarcinoma cell lines. Also, ASK1 phosphorylation at Thr845, which reflects its activated state, was clearly inhibited by ROR1 overexpression in both steady state and oxidative stress-elicited conditions in MSTO-211H cells. In addition, we found that ROR1 was physically associated with ASK1 at the C-terminal serine threonine-rich domain of ROR1. Furthermore, ROR1 kinase activity was shown to be required to repress the ASK1-p38 axis and oxidative stress-induced cell death. The present findings thus support our notion that ROR1 sustains lung adenocarcinoma survival, at least in part, through direct physical interaction with ASK1 and consequential repression of the pro-apoptotic ASK1-p38 axis in a ROR1 kinase activity-dependent manner.

miR-342-3p regulates MYC transcriptional activity via direct repression of E2F1 in human lung cancer.

Accumulating evidence indicates that altered miRNA expression is crucially involved in lung cancer development, though scant information is available regarding how MYC, an archetypical oncogene, is regulated by miRNAs, especially via a mechanism involving MYC cofactors. In this study, we attempted to identify miRNAs involved in regulation of MYC transcriptional activity in lung cancer. To this end, we utilized an integrative approach with combinatorial usage of miRNA and mRNA expression profile datasets of patient tumor tissues, as well as those of MYC-inducible cell lines in vitro. In addition to miRNAs previously reported to be directly regulated by MYC, including let-7 and miR-17-92, our strategy also helped to identify miR-342-3p as capable of indirectly regulating MYC activity via direct repression of E2F1, a MYC-cooperating molecule. Furthermore, miR-342-3p module activity, which we defined as a gene set reflecting the experimentally substantiated influence of miR-342-3p on mRNA expression, was found to be inversely correlated with MYC activity reflected by MYC module activity in three independent datasets of lung adenocarcinoma patients obtained from the Director's Challenge Consortium of the United States (P = 1.94 × 10(-73)), the National Cancer Center of Japan (P = 9.05 × 10(-34)) and the present study (P = 1.17 × 10(-19)). Our integrative approach appears to be useful to elucidate inter-regulatory relationships between miRNAs and protein coding genes of interest, even those present in patient tumor tissues, which remains a challenge to better understand the pathogenesis of this devastating disease.

Lung adenocarcinoma subtypes definable by lung development-related miRNA expression profiles in association with clinicopathologic features.

Accumulation of genetic and epigenetic changes alters regulation of a web of interconnected genes including microRNAs (miRNAs), which confer hallmark capabilities and characteristic cancer features. In this study, the miRNA and messenger RNA expression profiles of 126 non-small cell lung cancer specimens were analyzed, with special attention given to the diversity of lung adenocarcinomas. Of those, 76 adenocarcinomas were classified into two major subtypes, developing lung-like and adult lung-like, based on their distinctive miRNA expression profiles resembling those of either developing or adult lungs, respectively. A systems biology-based approach using a Bayesian network and non-parametric regression was employed to estimate the gene regulatory circuitry functioning in patient tumors in order to identify subnetworks enriched for genes with differential expression between the two major subtypes. miR-30d and miR-195, identified as hub genes in such subnetworks, had lower levels of expression in the developing lung-like subtype, whereas introduction of miR-30d or miR-195 into the lung cancer cell lines evoked shifts of messenger RNA expression profiles toward the adult lung-like subtype. Conversely, the influence of miR-30d and miR-195 was significantly different between the developing lung-like and adult lung-like subtypes in our analysis of the patient data set. In addition, RRM2, a child gene of the miR-30d-centered subnetwork, was found to be a direct target of miR-30d. Together, our findings reveal the existence of two miRNA expression profile-defined lung adenocarcinoma subtypes with distinctive clinicopathologic features and also suggest the usefulness of a systems biology-based approach to gain insight into the altered regulatory circuitry involved in cancer development.

Aven-dependent activation of ATM following DNA damage.

BACKGROUND: In response to DNA damage, cells undergo either cell-cycle arrest or apoptosis, depending on the extent of damage and the cell's capacity for DNA repair. Cell-cycle arrest induced by double-stranded DNA breaks depends on activation of the ataxia-telangiectasia (ATM) protein kinase, which phosphorylates cell-cycle effectors such as Chk2 and p53 to inhibit cell-cycle progression. ATM is recruited to double-stranded DNA breaks by a complex of sensor proteins, including Mre11/Rad50/Nbs1, resulting in autophosphorylation, monomerization, and activation of ATM kinase. RESULTS: In characterizing Aven protein, a previously reported apoptotic inhibitor, we have found that Aven can function as an ATM activator to inhibit G2/M progression. Aven bound to ATM and Aven overexpressed in cycling Xenopus egg extracts prevented mitotic entry and induced phosphorylation of ATM and its substrates. Immunodepletion of endogenous Aven allowed mitotic entry even in the presence of damaged DNA, and RNAi-mediated knockdown of Aven in human cells prevented autophosphorylation of ATM at an activating site (S1981) in response to DNA damage. Interestingly, Aven is also a substrate of the ATM kinase. Mutation of ATM-mediated phosphorylation sites on Aven reduced its ability to activate ATM, suggesting that Aven activation of ATM after DNA damage is enhanced by ATM-mediated Aven phosphorylation. CONCLUSIONS: These results identify Aven as a new ATM activator and describe a positive feedback loop operating between Aven and ATM. In aggregate, these findings place Aven, a known apoptotic inhibitor, as a critical transducer of the DNA-damage signal.

TAK1 MAPK kinase kinase mediates transforming growth factor-beta signaling by targeting SnoN oncoprotein for degradation.

Transforming growth factor-beta (TGF-beta) regulates a variety of physiologic processes through essential intracellular mediators Smads. The SnoN oncoprotein is an inhibitor of TGF-beta signaling. SnoN recruits transcriptional repressor complex to block Smad-dependent transcriptional activation of TGF-beta-responsive genes. Following TGF-beta stimulation, SnoN is rapidly degraded, thereby allowing the activation of TGF-beta target genes. Here, we report the role of TAK1 as a SnoN protein kinase. TAK1 interacted with and phosphorylated SnoN, and this phosphorylation regulated the stability of SnoN. Inactivation of TAK1 prevented TGF-beta-induced SnoN degradation and impaired induction of the TGF-beta-responsive genes. These data suggest that TAK1 modulates TGF-beta-dependent cellular responses by targeting SnoN for degradation.

Protein phosphatase 6 down-regulates TAK1 kinase activation in the IL-1 signaling pathway.

TAK1 (transforming growth factor beta-activated kinase 1) is a serine/threonine kinase that is a mitogen-activated protein kinase kinase kinase and an essential intracellular signaling component in inflammatory signaling pathways. Upon stimulation of cells with inflammatory cytokines, TAK1 binds proteins that stimulate autophosphorylation within its activation loop and is thereby catalytically activated. This activation is transient; it peaks within a couple of minutes and is subsequently down-regulated rapidly to basal levels. The mechanism of down-regulation of TAK1 has not yet been elucidated. In this study, we found that toxin inhibition of type 2A protein phosphatases greatly enhances interleukin 1 (IL-1)-dependent phosphorylation of Thr-187 in the TAK1 activation loop as well as the catalytic activity of TAK1. From proteomic analysis of TAK1-binding proteins, we identified protein phosphatase 6 (PP6), a type-2A phosphatase, and demonstrated that PP6 associated with and inactivated TAK1 by dephosphorylation of Thr-187. Ectopic and endogenous PP6 co-precipitated with TAK1, and expression of PP6 reduced IL-1 activation of TAK1 but did not affect osmotic activation of MLK3, another MAPKKK. Reduction of PP6 expression by small interfering RNA enhances IL-1-induced phosphorylation of Thr-187 in TAK1. Enhancement occurred without change in levels of PP2A showing specificity for PP6. Our results demonstrate that PP6 specifically down-regulates TAK1 through dephosphorylation of Thr-187 in the activation loop, which is likely important for suppressing inflammatory responses via TAK1 signaling pathways.

TAK1 is a component of the Epstein-Barr virus LMP1 complex and is essential for activation of JNK but not of NF-kappaB.

Epstein-Barr virus latent membrane protein 1 (LMP1) activates NF-kappaB and c-Jun N-terminal kinase (JNK), which is essential for LMP1 oncogenic activity. Genetic analysis has revealed that tumor necrosis factor receptor-associated factor 6 (TRAF6) is an indispensable intermediate of LMP1 signaling leading to activation of both NF-kappaB and JNK. However, the mechanism by which LMP1 engages TRAF6 for activation of NF-kappaB and JNK is not well understood. Here we demonstrate that TAK1 mitogen-activated protein kinase kinase kinase and TAK1-binding protein 2 (TAB2), together with TRAF6, are recruited to LMP1 through its N-terminal transmembrane region. The C-terminal cytoplasmic region of LMP1 facilitates the assembly of this complex and enhances activation of JNK. In contrast, IkappaB kinase gamma is recruited through the C-terminal cytoplasmic region and this is essential for activation of NF-kappaB. Furthermore, we found that ablation of TAK1 resulted in the loss of LMP1-induced activation of JNK but not of NF-kappaB. These results suggest that an LMP1-associated complex containing TRAF6, TAB2, and TAK1 plays an essential role in the activation of JNK. However, TAK1 is not an exclusive intermediate for NF-kappaB activation in LMP1 signaling.

A resorcylic acid lactone, 5Z-7-oxozeaenol, prevents inflammation by inhibiting the catalytic activity of TAK1 MAPK kinase kinase.

TAK1, a member of the mitogen-activated kinase kinase kinase (MAPKKK) family, participates in proinflammatory cellular signaling pathways by activating JNK/p38 MAPKs and NF-kappaB. To identify drugs that prevent inflammation, we screened inhibitors of TAK1 catalytic activity. We identified a natural resorcylic lactone of fungal origin, 5Z-7-oxozeaenol, as a highly potent inhibitor of TAK1. This compound did not effectively inhibit the catalytic activities of the MEKK1 or ASK1 MAPKKKs, suggesting that 5Z-7-oxozeaenol is a selective inhibitor of TAK1. In cell culture, 5Z-7-oxozeaenol blocked interleukin-1-induced activation of TAK1, JNK/p38 MAPK, IkappaB kinases, and NF-kappaB, resulting in inhibition of cyclooxgenase-2 production. Furthermore, in vivo 5Z-7-oxozeaenol was able to inhibit picryl chloride-induced ear swelling. Thus, 5Z-7-oxozeaenol blocks proinflammatory signaling by selectively inhibiting TAK1 MAPKKK.