Tobacco-induced hyperglycemia promotes lung cancer progression via cancer cell-macrophage interaction through paracrine IGF2/IR/NPM1-driven PD-L1 expression.

Tobacco smoking (TS) is implicated in lung cancer (LC) progression through the development of metabolic syndrome. However, direct evidence linking metabolic syndrome to TS-mediated LC progression remains to be established. Our findings demonstrate that 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and benzo[a]pyrene (NNK and BaP; NB), components of tobacco smoke, induce metabolic syndrome characteristics, particularly hyperglycemia, promoting lung cancer progression in male C57BL/6 J mice. NB enhances glucose uptake in tumor-associated macrophages by increasing the expression and surface localization of glucose transporter (GLUT) 1 and 3, thereby leading to transcriptional upregulation of insulin-like growth factor 2 (IGF2), which subsequently activates insulin receptor (IR) in LC cells in a paracrine manner, promoting its nuclear import. Nuclear IR binds to nucleophosmin (NPM1), resulting in IR/NPM1-mediated activation of the CD274 promoter and expression of programmed death ligand-1 (PD-L1). Restricting glycolysis, depleting macrophages, or blocking PD-L1 inhibits NB-mediated LC progression. Analysis of patient tissues and public databases reveals elevated levels of IGF2 and GLUT1 in tumor-associated macrophages, as well as tumoral PD-L1 and phosphorylated insulin-like growth factor 1 receptor/insulin receptor (pIGF-1R/IR) expression, suggesting potential poor prognostic biomarkers for LC patients. Our data indicate that paracrine IGF2/IR/NPM1/PD-L1 signaling, facilitated by NB-induced dysregulation of glucose levels and metabolic reprogramming of macrophages, contributes to TS-mediated LC progression.

Adult height in girls with central precocious puberty without gonadotropin-releasing hormone agonist treatment: a retrospective case-control study.

BACKGROUND: The primary aim of this study was to investigate the final adult height (FAH) of girls diagnosed with central precocious puberty (CPP) who were untreated. METHODS: We retrospectively analyzed the medical records of 36 girls diagnosed with CPP between 8 and 9 years of age who did not receive treatment, and 206 girls diagnosed with CPP within the same age range who received gonadotropin-releasing hormone (GnRH) agonist treatment. Midparental height (MPH), predicted adult height (PAH) obtained using height and bone age (BA) at the time of diagnosis (PAH for BA), and PAH obtained using the Bayley-Pinneau method (PAH by BP) were calculated. Additionally, height at the time of growth completion was compared with the predicted height. RESULTS: The FAHs were 160.71±4.56 cm in the untreated group and 159.31±4.26 cm in the treated group. In the untreated group, the FAH was 0.99±4.50 cm shorter than the MPH but 4.29±3.33 cm and 3.46±3.93 cm greater than the PAH for BA and PAH by BP, respectively. CONCLUSION: In children diagnosed with CPP between 8 and 9 years of age who were untreated, FAH was greater than PAH for BA and PAH by BP at the time of diagnosis, indicating that the prognosis of FAH was not poor. Therefore, for girls diagnosed with CPP, it is recommended to consider various conditions, such as pubertal onset, height at diagnosis, BA, peak luteinizing hormone level, predicted height, and speed of puberty, when deciding whether to administer GnRH agonists.

Effect of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol and benzo[a]pyrene exposure on the development of metabolic syndrome in mice.

AIM: The prevalence of metabolic syndrome (MetS), a cluster of serious medical conditions that raise the risk of lung cancer, has increased worldwide. Tobacco smoking (TS) potentially increases the risk of developing MetS. Despite the potential association of MetS with lung cancer, preclinical models that mimic human diseases, including TS-induced MetS, are limited. Here we evaluated the impact of exposure to tobacco smoke condensate (TSC) and two representative tobacco carcinogens, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNK) and benzo[a]pyrene (BaP), on MetS development in mice. MATERIALS AND METHODS: FVB/N or C57BL/6 mice were exposed to vehicle, TSC, or NNK and BaP (NB) twice weekly for 5 months. The serum levels of total cholesterol (TCHO), triglycerides, high-density lipoprotein (HDL), blood glucose, and metabolites, along with glucose tolerance and body weight, were measured. KEY FINDINGS: Compared with those of vehicle-treated mice, mice with TSC or NB exposure displayed major phenotypes associated with MetS, including increased serum levels of TCHO, triglycerides, and fasting and basal blood glucose and decreased glucose tolerance, and serum levels of HDL. These MetS-associated changes were found in both FVB/N and C57BL/6 mice that were susceptible or resistant to carcinogen-induced tumorigenesis, respectively, indicating that tumor formation is not involved in the TSC- or NB-mediated MetS. Moreover, oleic acid and palmitoleic acid, which are known to be associated with MetS, were significantly upregulated in the serum of TSC- or NB-treated mice compared with those in vehicle-treated mice. SIGNIFICANCE: Both TSC and NB caused detrimental health problems, leading to the development of MetS in experimental mice.

Targeting epidermal growth factor receptor in paclitaxel-resistant human breast and lung cancer cells with upregulated glucose-6-phosphate dehydrogenase.

BACKGROUND: Chemoresistance is a major obstacle to the successful treatment of triple-negative breast cancer (TNBC) and non-small-cell lung cancer (NSCLC). Therapeutic strategies to overcome chemoresistance are necessary to improve the prognosis of patients with these cancers. METHODS: Paclitaxel-resistant TNBC and NSCLC sublines were generated through continuous paclitaxel treatment over 6 months. The mechanistic investigation was conducted using MTT assay, LC/MS-based metabolite analysis, flow cytometry, western blot analysis, real-time PCR and tumour xenograft experiments. RESULTS: Glucose-6-phosphate dehydrogenase (G6PD) expression along with an increase in 3-phosphoglycerates and ribulose-5-phosphate production was upregulated in paclitaxel-resistant cells. Blockade of G6PD decreased viability of paclitaxel-resistant cells in vitro and the growth of paclitaxel-resistant MDA/R xenograft tumours in vivo. Mechanistically, activation of the epidermal growth factor receptor (EGFR)/Akt pathway mediates G6PD expression and G6PD-induced cell survival. Blockade of the EGFR pathway inhibited G6PD expression and sensitised those paclitaxel-resistant cells to paclitaxel treatment in vitro and in vivo. Analysis of publicly available datasets revealed an association between G6PD and unfavourable clinical outcomes in patients with breast or lung cancer. CONCLUSIONS: EGFR signaling-mediated G6PD expression plays a pivotal role in paclitaxel resistance, highlighting the potential of targeting EGFR to overcome paclitaxel resistance in TNBC and NSCLC cells overexpressing G6PD.

Ninjurin1 drives lung tumor formation and progression by potentiating Wnt/ß-Catenin signaling through Frizzled2-LRP6 assembly.

BACKGROUND: Cancer stem-like cells (CSCs) play a pivotal role in lung tumor formation and progression. Nerve injury-induced protein 1 (Ninjurin1, Ninj1) has been implicated in lung cancer; however, the pathological role of Ninj1 in the context of lung tumorigenesis remains largely unknown. METHODS: The role of Ninj1 in the survival of non-small cell lung cancer (NSCLC) CSCs within microenvironments exhibiting hazardous conditions was assessed by utilizing patient tissues and transgenic mouse models where Ninj1 repression and oncogenic KrasG12D/+ or carcinogen-induced genetic changes were induced in putative pulmonary stem cells (SCs). Additionally, NSCLC cell lines and primary cultures of patient-derived tumors, particularly Ninj1high and Ninj1low subpopulations and those with gain- or loss-of-Ninj1 expression, and also publicly available data were all used to assess the role of Ninj1 in lung tumorigenesis. RESULTS: Ninj1 expression is elevated in various human NSCLC cell lines and tumors, and elevated expression of this protein can serve as a biomarker for poor prognosis in patients with NSCLC. Elevated Ninj1 expression in pulmonary SCs with oncogenic changes promotes lung tumor growth in mice. Ninj1high subpopulations within NSCLC cell lines, patient-derived tumors, and NSCLC cells with gain-of-Ninj1 expression exhibited CSC-associated phenotypes and significantly enhanced survival capacities in vitro and in vivo in the presence of various cell death inducers. Mechanistically, Ninj1 forms an assembly with lipoprotein receptor-related protein 6 (LRP6) through its extracellular N-terminal domain and recruits Frizzled2 (FZD2) and various downstream signaling mediators, ultimately resulting in transcriptional upregulation of target genes of the LRP6/ß-catenin signaling pathway. CONCLUSIONS: Ninj1 may act as a driver of lung tumor formation and progression by protecting NSCLC CSCs from hostile microenvironments through ligand-independent activation of LRP6/ß-catenin signaling.

In vitro modeling for inherited neurological diseases using induced pluripotent stem cells: from 2D to organoid.

Stem cells are characterized by self-renewal and by their ability to differentiate into cells of various organs. With massive progress in 2D and 3D cell culture techniques, in vitro generation of various types of such organoids from patient-derived stem cells is now possible. As in vitro differentiation protocols are usually made to resemble human developmental processes, organogenesis of patient-derived stem cells can provide key information regarding a range of developmental diseases. Human stem cell-based in vitro modeling as opposed to using animal models can particularly benefit the evaluation of neurological diseases because of significant differences in structure and developmental processes between the human and the animal brain. This review focuses on stem cell-based in vitro modeling of neurodevelopmental disorders, more specifically, the fundamentals and technical advancements in monolayer neuron and brain organoid cultures. Furthermore, we discuss the drawbacks of the conventional culture method and explore the advanced, cutting edge 3D organoid models for several neurodevelopmental diseases, including genetic diseases such as Down syndrome, Rett syndrome, and Miller-Dieker syndrome, as well as brain malformations like macrocephaly and microcephaly. Finally, we discuss the limitations of the current organoid techniques and some potential solutions that pave the way for accurate modeling of neurological disorders in a dish.

Potent Anticancer Effect of the Natural Steroidal Saponin Gracillin Is Produced by Inhibiting Glycolysis and Oxidative Phosphorylation-Mediated Bioenergetics.

Metabolic rewiring to utilize aerobic glycolysis is a hallmark of cancer. However, recent findings suggest the role of mitochondria in energy generation in cancer cells and the metabolic switch to oxidative phosphorylation (OXPHOS) in response to the blockade of glycolysis. We previously demonstrated that the antitumor effect of gracillin occurs through the inhibition of mitochondrial complex II-mediated energy production. Here, we investigated the potential of gracillin as an anticancer agent targeting both glycolysis and OXPHOS in breast and lung cancer cells. Along with the reduction in adenosine triphosphate (ATP) production, gracillin markedly suppresses the production of several glycolysis-associated metabolites. A docking analysis and enzyme assay suggested phosphoglycerate kinase 1 (PGK1) is a potential target for the antiglycolytic effect of gracillin. Gracillin reduced the viability and colony formation ability of breast cancer cells by inducing apoptosis. Gracillin displayed efficacious antitumor effects in mice bearing breast cancer cell line or breast cancer patient-derived tumor xenografts with no overt changes in body weight. An analysis of publicly available datasets further suggested that PGK1 expression is associated with metastasis status and poor prognosis in patients with breast cancer. These results suggest that gracillin is a natural anticancer agent that inhibits both glycolysis and mitochondria-mediated bioenergetics.

The Interplay between Slow-Cycling, Chemoresistant Cancer Cells and Fibroblasts Creates a Proinflammatory Niche for Tumor Progression.

Quiescent cancer cells are believed to cause cancer progression after chemotherapy through unknown mechanisms. We show here that human non-small cell lung cancer (NSCLC) cell line-derived, quiescent-like, slow-cycling cancer cells (SCC) and residual patient-derived xenograft (PDX) tumors after chemotherapy experience activating transcription factor 6 (ATF6)-mediated upregulation of various cytokines, which acts in a paracrine manner to recruit fibroblasts. Cancer-associated fibroblasts (CAF) underwent transcriptional upregulation of COX2 and type I collagen (Col-I), which subsequently triggered a slow-to-active cycling switch in SCC through prostaglandin E2 (PGE2)- and integrin/Src-mediated signaling pathways, leading to cancer progression. Both antagonism of ATF6 and cotargeting of Src/COX2 effectively suppressed cytokine production and slow-to-active cell cycling transition in SCC, withholding cancer progression. Expression of COX2 and Col-I and activation of Src were observed in patients with NSCLC who progressed while receiving chemotherapy. Public data analysis revealed significant association between COL1A1 and SRC expression and NSCLC relapse. Overall, these findings indicate that a proinflammatory niche created by the interplay between SCC and CAF triggers tumor progression. SIGNIFICANCE: Cotargeting COX2 and Src may be an effective strategy to prevent cancer progression after chemotherapy.

The natural compound gracillin exerts potent antitumor activity by targeting mitochondrial complex II.

Mitochondria play a pivotal role in cancer bioenergetics and are considered a potential target for anticancer therapy. Considering the limited efficacy and toxicity of currently available mitochondria-targeting agents, it is necessary to develop effective mitochondria-targeting anticancer drugs. By screening a large chemical library consisting of natural products with diverse chemical entities, we identified gracillin, a steroidal saponin, as a mitochondria-targeting antitumor drug. Gracillin displayed broad-spectrum inhibitory effects on the viability of a large panel of human cancer cell lines, including those carrying acquired resistance to chemotherapy or EGFR-targeting drugs, by inducing apoptosis. We show that gracillin attenuates mitochondria-mediated cellular bioenergetics by suppressing ATP synthesis and by producing reactive oxygen species (ROS). Mechanistically, gracillin disrupts complex II (CII) function by abrogating succinate dehydrogenase (SDH) activity without affecting the succinate:ubiquinone reductase. The gracillin-induced cell death was potentiated by 3-nitropropionic acid (3-NPA) or thenoyltrifluoroacetone (TTFA), which inhibit CII by binding to the active site of SDHA or to the ubiquinone-binding site, respectively. Finally, we show that gracillin effectively suppressed the mutant-Kras-driven lung tumorigenesis and the growth of xenograft tumors derived from cell lines or patient tissues. Gracillin displayed no obvious pathophysiological features in mice. Collectively, gracillin has potential as a CII-targeting antitumor drug.

The clinical characteristics and neurodevelopmental outcome of preterm infants with persistent periventricular echogenicity.

PURPOSE: Periventricular echogenicity (PVE) presents as diffuse echo dense lesions of the periventricular white matter on cranial ultrasonography. Beyond two weeks of life, it is considered as prolonged or persistent PVE. The aim of our study was to investigate the clinical characteristics of preterm infants with persistent PVE beyond 2 weeks after birth and to determine whether these infants had an adverse neurodevelopmental outcome. METHODS: The medical records of preterm infants who were born at < 34 weeks of gestation and admitted to Pusan National University Hospital between 2009 and 2014 were reviewed. A total of 28 preterm infants with persistent PVE were enrolled. Sixty compatible infants closely matched for gestational age and birth weight to infants with PVE were selected as the control group. Clinical data, including maternal, perinatal and neonatal characteristics, were analyzed. We compared the Bayley Scales of Infant Development-III at 12 months' corrected age. RESULTS: The mean gestational age and birth weight were 31 + 3 (range, 29 + 2-33 + 6) weeks and 1523 (range, 911-2210) g, respectively, in the persistent PVE group. In the control group, the mean gestational age was 31 + 4 (range, 29 + 2-33 + 6) weeks and the mean birth weight was 1537 (range, 840-2100) g. There was no significant difference between the persistent PVE group and the control group, except for a significantly higher incidence of late sepsis in the persistent PVE group (p = 0.001). The results of Bayley test at 12 months of corrected age were available for 24 infants in the persistent PVE group and for 26 infants in the control group. A motor score of 86 (range, 78-95) versus 88 (range, 79-100), a language composite score of 88 (range, 78-97) versus 89 (range, 80-105), and a cognitive score of 90 (range, 81-100) versus 92 (range, 85-105) were observed in the persistent PVE group and the control group, respectively. No difference was detected in any scores between the two groups. CONCLUSION: The clinical characteristics and neurodevelopmental outcomes of preterm infants with persistent PVE were not different from those of infants with normal findings. Our study supports the concept that persistent PVE without cystic change may be a benign finding.

Smoking-associated lung cancer prevention by blockade of the beta-adrenergic receptor-mediated insulin-like growth factor receptor activation.

Activation of receptor tyrosine kinases (RTKs) is associated with carcinogenesis, but its contribution to smoking-associated lung carcinogenesis is poorly understood. Here we show that a tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced insulin-like growth factor 1 receptor (IGF-1R) activation via ß-adrenergic receptor (ß-AR) is crucial for smoking-associated lung carcinogenesis. Treatment with NNK stimulated the IGF-1R signaling pathway in a time- and dose-dependent manner, which was suppressed by pharmacological or genomic blockade of ß-AR and the downstream signaling including a Gßγ subunit of ß-AR and phospholipase C (PLC). Consistently, ß-AR agonists led to increased IGF-1R phosphorylation. The increase in IGF2 transcription via ß-AR, signal transducer and activator of transcription 3 (STAT3), and nuclear factor-kappa B (NF-κB) was associated with NNK-induced IGF-1R activation. Finally, treatment with ß-AR antagonists suppressed the acquisition of transformed phenotypes in lung epithelial cells and lung tumor formation in mice. These results suggest that blocking ß-AR-mediated IGF-1R activation can be an effective strategy for lung cancer prevention in smokers.

Chronic Stress Facilitates Lung Tumorigenesis by Promoting Exocytosis of IGF2 in Lung Epithelial Cells.

Molecular insights into how chronic stress affects lung tumorigenesis may offer new routes to chemoprevention. In this study, we show that chronic stress in mice chemically or genetically initiated for lung cancer leads to the release of norepinephrine and other catecholamines, thereby promoting lung tumorigenesis. Mechanistically, norepinephrine induced phosphorylation of L-type voltage-dependent calcium channels (VDCC) through the ß-adrenergic receptor-PKA pathway. VDCC triggered calcium mobilization, thereby inducing activation of IGF-1R via exocytosis of insulin-like growth factor 2 (IGF2). Mice expressing lung-specific IGF-1R exhibited accelerated lung tumor development in response to chronic stress. Notably, clinically approved antihypertensive drugs that block L-type VDCC prevented the effects of chronic stress or norepinephrine on the IGF2/IGF-1R signaling cascade, along with transformation of lung epithelial cells and lung tumor formation. Overall, our results identify an actionable mechanism to limit the effects of chronic stress on lung tumorigenesis. Cancer Res; 76(22); 6607-19. ©2016 AACR.

The tobacco-specific carcinogen-operated calcium channel promotes lung tumorigenesis via IGF2 exocytosis in lung epithelial cells.

Nicotinic acetylcholine receptors (nAChRs) binding to the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces Ca2+ signalling, a mechanism that is implicated in various human cancers. In this study, we investigated the role of NNK-mediated Ca2+ signalling in lung cancer formation. We show significant overexpression of insulin-like growth factors (IGFs) in association with IGF-1R activation in human preneoplastic lung lesions in smokers. NNK induces voltage-dependent calcium channel (VDCC)-intervened calcium influx in airway epithelial cells, resulting in a rapid IGF2 secretion via the regulated pathway and thus IGF-1R activation. Silencing nAChR, α1 subunit of L-type VDCC, or various vesicular trafficking curators, including synaptotagmins and Rabs, or blockade of nAChR/VDCC-mediated Ca2+ influx significantly suppresses NNK-induced IGF2 exocytosis, transformation and tumorigenesis of lung epithelial cells. Publicly available database reveals inverse correlation between use of calcium channel blockers and lung cancer diagnosis. Our data indicate that NNK disrupts the regulated pathway of IGF2 exocytosis and promotes lung tumorigenesis.

Targeting the insulin-like growth factor receptor and Src signaling network for the treatment of non-small cell lung cancer.

BACKGROUND: Therapeutic interventions in the insulin-like growth factor receptor (IGF-1R) pathway were expected to provide clinical benefits; however, IGF-1R tyrosine kinase inhibitors (TKIs) have shown limited antitumor efficacy, and the mechanisms conveying resistance to these agents remain elusive. METHODS: The expression and activation of the IGF-1R and Src were assessed via the analysis of a publicly available dataset, as well as immunohistochemistry, Western blotting, RT-PCR, and in vitro kinase assays. The efficacy of IGF-1R TKIs alone or in combination with Src inhibitors was analyzed using MTT assays, colony formation assays, flow cytometric analysis, and xenograft tumor models. RESULTS: The co-activation of IGF-1R and Src was observed in multiple human NSCLC cell lines as well as in a tissue microarray (n = 353). The IGF-1R and Src proteins mutually phosphorylate on their autophosphorylation sites. In high-pSrc-expressing NSCLC cells, linsitinib treatment initially inactivated the IGF-1R pathway but led a Src-dependent reactivation of downstream effectors. In low-pSrc-expressing NSCLC cells, linsitinib treatment decreased the turnover of the IGF-1R and Src proteins, ultimately amplifying the reciprocal co-activation of IGF-1R and Src. Co-targeting IGF-1R and Src significantly suppressed the proliferation and tumor growth of both high-pSrc-expressing and low-pSrc-expressing NSCLC cells in vitro and in vivo and the growth of patient-derived tissues in vivo. CONCLUSIONS: Reciprocal activation between Src and IGF-1R occurs in NSCLC. Src causes IGF-1R TKI resistance by acting as a key downstream modulator of the cross-talk between multiple membrane receptors. Targeting Src is a clinically applicable strategy to overcome resistance to IGF-1R TKIs.