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 β-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.

Lung cancer is one of the leading causes of cancer-related deaths worldwide. Despite the emergence of novel anticancer drugs, the 5-year survival rate for lung cancer remains less than 20% (1). A greater understanding of the molecular mechanisms underlying lung carcinogenesis is urgently needed, as is the development of novel strategies for controlling lung cancer. Tobacco smoking is the main cause of lung cancer (2, 3). However, the lung cancer incidence is only approximately 10% among lifetime tobacco smokers (4), suggesting that environmental factors are also important for lung cancer development. Among various factors implicated in the pathogenesis of lung cancer in never smokers (3), psychologic factors, including chronic stress and depression, may be potential causes of cancer development and progression, but its importance has been relatively underestimated.

Psychologic stress can be provoked by a myriad of social, physical, and emotional stressors and is known to trigger the activation of the sympathetic nervous system and the hypothalamus–pituitary–adrenal axis, which mediate the “fight-or-flight” and defeat-withdrawal responses, respectively, by releasing stress-related neurotransmitters and hormones, such as norepinephrine, epinephrine, and cortisol (5). Norepinephrine and epinephrine activate signal transduction pathways that control cell proliferation and survival by binding to adrenergic receptors (AR) that are expressed in many organs, including the lungs (6). Cortisol is known to regulate growth, metabolism, and immune reactions (5). Psychologic stress was associated with a higher incidence of lung cancer and poorer survival in lung cancer patients (7), and the use of an antidepressant reduced lung cancer risk in epidemiologic studies (8). Chronic stress also exacerbated the growth of human lung cancer cell xenograft tumors (9). These findings imply that chronic psychologic stress can be both a cause and a driver of lung carcinogenesis. However, controversial reports related to the role of stress in lung cancer have also been published (10, 11). Hence, additional studies are required to clarify the impact of chronic stress on lung cancer.

In the current study, we demonstrate that chronic stress exhibits a powerful impact in promoting carcinogen- or oncogene-induced lung tumor formation. Mechanistically, the norepinephrine-stimulated β-adrenergic receptor/protein kinase A (β-AR/PKA) pathway stimulated the exocytosis of insulin-like growth factor 2 (IGF2) via activating L-type voltage-dependent calcium channel (VDCC), leading to the sustained activation of the type I IGF receptor (IGF-1R) pathway, a key mechanism for cell transformation and survival. The β-AR/PKA pathway also induced long-term activation of IGF-1R through transcriptional upregulation of IGF2 expression. Finally, we confirmed that the chronic stress–induced events were significantly suppressed by treatment with antagonists against β-AR, L-type VDCC, or IGF-1R. These results provide direct evidence that chronic stress promotes lung tumor development and that β-AR, L-type VDCC, and IGF-1R are potential novel targets for lung cancer chemoprevention in individuals under chronic stress.

Additional or detailed methods are described in the Supplementary Materials and Methods.

Cell culture

Normal human bronchial epithelial (NHBE) cells were purchased from Lifeline Cell Technology in 2010. Human bronchial epithelial (HBE) cells and cells in which TP53 expression was knocked down using RNA interference (HBE/p53i) or carrying overexpressed mutant KRAS (HBE/Ras; ref. 12) were generously provided by Dr. John D. Minna (The University of Texas Southwestern Medical Center, Dallas, TX) in 2006. BEAS-2B and HB56B cells were kindly provided by Dr. A. Klein-Szanto (Fox Chase Cancer Center, Philadelphia, PA) and Dr. R Reddel (National Cancer Institute, Bethesda, MD), respectively, in 2004. These lung epithelial cells were cultured in K-SFM (Invitrogen) supplemented with 5 ng/mL recombinant EGF, 50 μg/mL bovine pituitary extracts, and antibiotics. Mouse embryonic fibroblast R and R+ cells were kindly provided by Dr. Renato Baserga (Columbia University, New York, NY) in 2005 and were cultured in DMEM supplemented with 10% FBS and antibiotics (all from Welgene). Cells were maintained at 37°C with 5% CO2 in a humidified atmosphere. BEAS-2B cells were authenticated at Genetic Resources Core Facility of Johns Hopkins University (Baltimore, MD) in 2010 and validated. Cells passed for fewer than 6 months after receipt or resuscitation of validated cells were used in this study.

Animal experiments

All animal experiments were performed using protocols approved by the Seoul National University Institutional Animal Care and Use Committee. Detailed procedures of animal experiments are described in Supplementary Materials and Methods.

Statistical analysis

The data are presented as the mean ± SD. All in vitro experiments were independently performed at least twice, and a representative result is presented. Statistical significance was analyzed using two-sided Student t tests or one-way ANOVA, followed by Dunnett post hoc test in Microsoft Excel 2013 (Microsoft Corp.) or in GraphPad Prism 6 (GraphPad Software Inc.), respectively. P values of less than 0.05 were considered statistically significant.

Chronic stress promotes lung tumorigenesis in mice

We investigated the impact of chronic stress on lung tumor development. Because sustained chronic stress is known to elevate the levels of catecholamines, including norepinephrine and epinephrine, and the corticosteroid cortisol (13), we assessed the effects of norepinephrine, epinephrine, and cortisol on the transformation of HBE cells. MTT assay revealed that a normal HBE cell line BEAS-2B had significantly increased viability in the absence of growth factors after exposure to norepinephrine (Supplementary Fig. S1A) or epinephrine (Supplementary Fig. S1B, left) at doses as low as 1 nmol/L. In contrast, cortisol treatment did not confer such benefit to BEAS-2B cells (Supplementary Fig. S1B, right). To further determine the biological significance, we assessed whether the cells were propagated during the norepinephrine exposure in the absence of growth factors. Trypan blue exclusion assay revealed significant increases in the viable cell number during exposure to norepinephrine up to 5 days (Fig. 1A), suggesting that norepinephrine stimulates proliferation of lung epithelial cells in the absence of growth factors. BEAS-2B cells also showed significantly increased abilities of anchorage-dependent and anchorage-independent colony formation after exposure to norepinephrine or epinephrine (Fig. 1A and Supplementary Fig. S1C).

Figure 1.

Promotion of carcinogen- and KrasG12D/+-driven lung tumorigenesis by exposure to chronic stress and norepinephrine. A, increases in cell number and anchorage-dependent and -independent colony formation by treatment with norepinephrine (NE) in lung epithelial cells (n = 4 or 5; mean ± SD). B, schematic diagram of the in vivo experiment. FVB mice were exposed to CUS for 4 months, and a carcinogen (U, urethane) was administrated 1 month after the initiation of stress. C, effects of CUS on the multiplicity, volume, and burden of murine lung tumors (n = 6; mean ± SD). D, schematic diagram of the in vivo experiment. Three-month-old FVB mice carrying the KrasG12D/+ transgene were exposed to CUS for 5 weeks. E, a representative IVIS Spectrum CT image showing lung tumor formation. Arrows, tumor formation. F, effects of CUS exposure on the burden of murine lung tumors (n = 4; mean ± SD). G, an increase in the level of norepinephrine was observed in serum obtained from the blood of CUS-exposed FVB mice, as determined using ELISA (n = 5, mean ± SD). H, the promotion of urethane-induced lung tumor formation by continuous exposure to norepinephrine via a micro-osmotic pump (n = 5; mean ± SD). Right, a representative image showing lung tumor formation. Scale bar, 100 μm. Statistical significance of difference was determined using a two-sided Student t test (A, F, and H) or one-way ANOVA (A, C, and G). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 1.

Promotion of carcinogen- and KrasG12D/+-driven lung tumorigenesis by exposure to chronic stress and norepinephrine. A, increases in cell number and anchorage-dependent and -independent colony formation by treatment with norepinephrine (NE) in lung epithelial cells (n = 4 or 5; mean ± SD). B, schematic diagram of the in vivo experiment. FVB mice were exposed to CUS for 4 months, and a carcinogen (U, urethane) was administrated 1 month after the initiation of stress. C, effects of CUS on the multiplicity, volume, and burden of murine lung tumors (n = 6; mean ± SD). D, schematic diagram of the in vivo experiment. Three-month-old FVB mice carrying the KrasG12D/+ transgene were exposed to CUS for 5 weeks. E, a representative IVIS Spectrum CT image showing lung tumor formation. Arrows, tumor formation. F, effects of CUS exposure on the burden of murine lung tumors (n = 4; mean ± SD). G, an increase in the level of norepinephrine was observed in serum obtained from the blood of CUS-exposed FVB mice, as determined using ELISA (n = 5, mean ± SD). H, the promotion of urethane-induced lung tumor formation by continuous exposure to norepinephrine via a micro-osmotic pump (n = 5; mean ± SD). Right, a representative image showing lung tumor formation. Scale bar, 100 μm. Statistical significance of difference was determined using a two-sided Student t test (A, F, and H) or one-way ANOVA (A, C, and G). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Because the anchorage-independent colony formation has been considered as an in vitro indicator of tumorigenic cell transformation (14), we next assessed the effects of chronic unpredictable stress (CUS; Supplementary Table S1), a well-established method for randomly applying various types of stress (15), on lung tumor formation in FVB mice. Exposure to CUS caused 100% lung tumor formation in mice in which tumors had been initiated by injection of a single dose of the carcinogen urethane (16). Figure 1B shows the treatment schedule for this experiment. The CUS significantly enhanced multiplicity, volume, and burden of the urethane-induced lung tumor formation (Fig. 1C). We further evaluated the effects of CUS on tumor development in mice carrying a mutation in the Kras gene (G12D) that corresponds to a frequent mutation found in human NSCLC (17). Figure 1D shows the treatment schedule for this experiment. As shown in Fig. 1E, the Tg mice exposed to CUS for 5 weeks revealed a clearly increased lung tumor formation relative to nonstressed mice. Postmortem analysis revealed 100% lung tumor incidence in the KrasG12D/+ Tg mice with significantly increased tumor burden in the CUS-exposed mice (Fig. 1F). We observed that exposure of mice to CUS increased the blood norepinephrine levels to 550 to 700 pg/mL (3.3–4.1 nmol/L), which was approximately 4 times higher than the norepinephrine levels observed in control mice (Fig. 1G). These concentrations are physiologically relevant to the plasma norepinephrine levels documented in stress-exposed individuals (18), suggesting that the physiologic changes experienced by the CUS-exposed mice recapitulate those observed in humans under chronic stress conditions. Importantly, mice in which a micro-osmotic pump containing norepinephrine was subcutaneously implanted to deliver norepinephrine at a controlled and continuous rate (a daily dose of 1 μmol/100 g body weight; ref. 19) exhibited significantly increased lung tumor burden when the initiation of lung tumors was induced by urethane injection (Fig. 1H). These data suggest that chronic stress promotes lung tumor development at least in part via the activities of norepinephrine and epinephrine. Because the lungs are strongly affected by norepinephrine due to the widespread distribution of sympathetic nerve terminals throughout the lungs (20), and because the lungs are a primary source of norepinephrine release into plasma (20), we chose norepinephrine for further studies.

Norepinephrine induces lung epithelial cell transformation by activating the IGF-1R pathway

We next investigated the mechanisms underlying norepinephrine-mediated lung epithelial cell transformation and lung tumor formation. On the basis of the role of deregulated receptor tyrosine kinase (RTK) signaling in carcinogenesis (21), the profiles of RTK activation were analyzed in norepinephrine-treated cells using an RTK array. A prominently greater IGF-1R phosphorylation was observed in norepinephrine-stimulated BEAS-2B cells than in control cells (Fig. 2A). Norepinephrine-mediated dose- and time-dependent increases in IGF-1R phosphorylation were confirmed in two different immortalized, normal HBE cell lines, including BEAS-2B (Fig. 2B) and HB56B (Fig. 2C; ref. 22), in primary cultured NHBE cells derived from large airways (Fig. 2D) and in a premalignant HBE cell line carrying siRNA-mediated loss of TP53 expression (HBE/p53i; Fig. 2E; ref. 12). As the antibody used in the Western blot analyses cannot distinguish IGF-IR and insulin receptor (IR), we confirmed the norepinephrine-mediated IGF-1R phosphorylation by performing immunoprecipitation analysis using IGF-1R- and IR-specific antibodies and Western blot analysis using anti-phosphotyrosine antibodies (Supplementary Fig. S2). We found very weak or undetectable levels of IR expression in BEAS-2B and HBE/p53i (23) cells, respectively. Therefore, the lack of IR phosphorylation in the lung epithelial cells after the norepinephrine exposure could be due to low levels of IR expression. Because norepinephrine is a known agonist for ARs and lung bronchus has shown higher expression of β-AR than α-AR (24), we assessed whether IGF-1R can be activated by β-AR agonists. Exposure to the nonselective β-AR agonist (epinephrine and isoproterenol), the β1-AR agonist (dobutamine), and the β2-AR agonist (metaproterenol) increased IGF-1R phosphorylation in HBE/p53i, BEAS-2B, and R+ cells (Fig. 2F and Supplementary Fig. S3). IGF-1R phosphorylation was also identified in vivo in lung epithelial lesions of FVB and KrasG12D/+ Tg mice that were exposed to CUS (Fig. 2G). The norepinephrine-induced IGF-1R phosphorylation, cell viability, and anchorage-dependent and -independent colony-forming abilities of BEAS-2B cells were significantly suppressed by the linsitinib treatment (Fig. 2H and Supplementary Fig. S4), suggesting that the β-AR pathway induces lung epithelial cell transformation through IGF-1R activation.

Figure 2.

Chronic stress- and norepinephrine-induced activation of IGF-1R in lung epithelial cells in vitro and in vivo. A, BEAS-2B cells were treated with either PBS (vehicle) or norepinephrine (NE; 10 μmol/L) for 15 minutes. Phospho-RTK array analysis was performed using lysates isolated from these cells. The corner spots (indicated as a blue box) correspond to the positive controls. B–E, Western blot analysis was performed using lysates isolated from BEAS-2B (B), HB56B (C), NHBE (D), and HBE/p53i (E) cells treated with various concentrations of norepinephrine for 15 minutes (B and E) or at a concentration of 10 μmol/L for the indicated time (B–E). F, Western blot analysis indicating a time-dependent increase of IGF-1R phosphorylation in BEAS-2B and HBE/p53i cells by treatment with β-AR agonists. Dobu, dobutamine; Meta, metaproterenol; Iso, isoproterenol. G, immunohistochemical analysis of phosphorylated IGF-1R in lung tissues. FVB mice were exposed to CUS for 4 weeks (left) or for 4 months. Con, control. A carcinogen (U, urethane) was administrated 1 month after the initiation of stress (middle). Right, three-month-old KrasG12D/+ Tg mice were exposed to CUS for 5 weeks; bottom, quantification of the pIGF-1R level was performed using InForm cell analysis software. Scale bar, 40 μm (top) and 10 μm (bottom). H, the inhibitory effect of linsitinib (Linsi) on the norepinephrine-induced acquisition of transformed phenotypes in vitro. The cell viability and anchorage-dependent and -independent colony formation of BEAS-2B cells that were treated with norepinephrine (10 μmol/L), linsitinib (0.5 μmol/L), or a combination of these agents are shown (n = 3 or 4; mean ± SD). Statistical significance of difference was determined using one-way ANOVA (G and H). ***, P < 0.001.

Figure 2.

Chronic stress- and norepinephrine-induced activation of IGF-1R in lung epithelial cells in vitro and in vivo. A, BEAS-2B cells were treated with either PBS (vehicle) or norepinephrine (NE; 10 μmol/L) for 15 minutes. Phospho-RTK array analysis was performed using lysates isolated from these cells. The corner spots (indicated as a blue box) correspond to the positive controls. B–E, Western blot analysis was performed using lysates isolated from BEAS-2B (B), HB56B (C), NHBE (D), and HBE/p53i (E) cells treated with various concentrations of norepinephrine for 15 minutes (B and E) or at a concentration of 10 μmol/L for the indicated time (B–E). F, Western blot analysis indicating a time-dependent increase of IGF-1R phosphorylation in BEAS-2B and HBE/p53i cells by treatment with β-AR agonists. Dobu, dobutamine; Meta, metaproterenol; Iso, isoproterenol. G, immunohistochemical analysis of phosphorylated IGF-1R in lung tissues. FVB mice were exposed to CUS for 4 weeks (left) or for 4 months. Con, control. A carcinogen (U, urethane) was administrated 1 month after the initiation of stress (middle). Right, three-month-old KrasG12D/+ Tg mice were exposed to CUS for 5 weeks; bottom, quantification of the pIGF-1R level was performed using InForm cell analysis software. Scale bar, 40 μm (top) and 10 μm (bottom). H, the inhibitory effect of linsitinib (Linsi) on the norepinephrine-induced acquisition of transformed phenotypes in vitro. The cell viability and anchorage-dependent and -independent colony formation of BEAS-2B cells that were treated with norepinephrine (10 μmol/L), linsitinib (0.5 μmol/L), or a combination of these agents are shown (n = 3 or 4; mean ± SD). Statistical significance of difference was determined using one-way ANOVA (G and H). ***, P < 0.001.

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Norepinephrine activates IGF-1R by inducing IGF2 secretion through the regulated secretory pathway

We next investigated the mechanisms underlying norepinephrine-mediated IGF-1R activation in lung epithelial cells. Given that the norepinephrine-induced IGF-1R phosphorylation occurred rapidly within 15 minutes of norepinephrine exposure without detectable changes in IGF-1R expression (Fig. 2B–E) and that IGFBPs regulate bioavailability of IGFs under certain conditions (25), we reasoned that ligands (IGF1 and IGF2) and IGF-binding proteins could have been involved in the rapid norepinephrine-induced IGF-1R activation. Hence, we first determined the effects of norepinephrine on transcription of IGF1, IGF2, and IGFBP3, the major IGF-binding protein responsible for more than 75% of IGF binding in circulation (26). Exposure to norepinephrine within 1 hour induced no significant change in the mRNA levels of IGF1, IGF2, and IGFBP3 (Fig. 3A, left). Similarly, extracellular IGF1 and IGFBP-3 protein levels in the conditioned media (CM) remained unchanged during the short-term exposure to norepinephrine (Fig. 3A, right, and Supplementary Fig. S5). In contrast, extracellular IGF2 levels were significantly increased as early as 15 minutes after the norepinephrine exposure. Live-cell time-lapse imaging (Fig. 3B) and Western blot (Fig. 3C) analyses of BEAS-2B cells, in which IGF2 overexpression was achieved by transient transfection with GFP-conjugated IGF2 (designated BEAS-2B/GFP-IGF2), also showed increased levels of GFP–IGF2 secretion upon exposure to norepinephrine. Moreover, when added to unexposed cells, the CM from norepinephrine-treated BEAS-2B and HBE/p53i cells induced a prominent IGF-1R phosphorylation (Fig. 3D). Moreover, treatment with a neutralizing antibody against IGF2 (Fig. 3E) or siRNA-mediated knockdown of IGF2 expression (Fig. 3F) attenuated norepinephrine-induced IGF-1R phosphorylation. The norepinephrine-induced GFP-IGF2 secretion and IGF-1R phosphorylation in BEAS-2B/GFP-IGF2 cells were suppressed by treatment with exocytosis inhibitors (nimodipine and Exo1; Fig. 3G) or by siRNA-mediated silencing of RAB27A expression (Supplementary Fig. S6), a small guanosine triphosphatase (GTPase) involved in the late stages of vesicle exocytosis (Fig. 3H; ref. 27). These findings indicated that norepinephrine-mediated regulated exocytosis of IGF2 was responsible for the short-term IGF-1R activation.

Figure 3.

Norepinephrine-induced IGF-1R phosphorylation is dependent on IGF2. A, C, and I, increases in IGF2 transcription and the IGF2 levels in the CM obtained from BEAS-2B or BEAS-2B/GFP-IGF2 cells treated with PBS (vehicle) or norepinephrine were determined by real-time PCR (A and I), ELISA (A and I), and Western blot (C) analyses. A Coomassie brilliant blue–stained gel (CB) is shown as a loading control. B, a time-lapse imaging analysis for norepinephrine-induced GFP-IGF2 secretion from BEAS-2B/GFP-IGF2 cells. Scale bar, 20 μm. D, BEAS-2B and HBE/p53i cells were treated with CM from BEAS-2B and HBE/p53i cells exposed to norepinephrine for 12 hours. Whole-cell lysates from these cells were subjected to Western blot analysis. E, after pretreatment with IGF2-neutralizing antibodies (10 μg/mL) for 1 hour, BEAS-2B and HBE/p53i cells were exposed to norepinephrine (NE) for 15 minutes. Cell lysates were analyzed using Western blot analysis. F, BEAS-2B and HBE/p53i cells were transfected with scrambled (Scr) or IGF2 siRNAs and then exposed to norepinephrine for the indicated times. Lysates from these cells were then subjected to Western blot analysis. G and H, BEAS-2B/GFP-IGF2 cells were pretreated with nimodipine (Nimo) or Exo1 for 3 hours (G) or were transfected with scrambled (Scr) or RAB27A siRNAs (H) and then stimulated with norepinephrine for 15 minutes. CM and whole-cell lysates from these cells were subjected to Western blot analysis. Statistical significance of difference was determined using a two-sided Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 3.

Norepinephrine-induced IGF-1R phosphorylation is dependent on IGF2. A, C, and I, increases in IGF2 transcription and the IGF2 levels in the CM obtained from BEAS-2B or BEAS-2B/GFP-IGF2 cells treated with PBS (vehicle) or norepinephrine were determined by real-time PCR (A and I), ELISA (A and I), and Western blot (C) analyses. A Coomassie brilliant blue–stained gel (CB) is shown as a loading control. B, a time-lapse imaging analysis for norepinephrine-induced GFP-IGF2 secretion from BEAS-2B/GFP-IGF2 cells. Scale bar, 20 μm. D, BEAS-2B and HBE/p53i cells were treated with CM from BEAS-2B and HBE/p53i cells exposed to norepinephrine for 12 hours. Whole-cell lysates from these cells were subjected to Western blot analysis. E, after pretreatment with IGF2-neutralizing antibodies (10 μg/mL) for 1 hour, BEAS-2B and HBE/p53i cells were exposed to norepinephrine (NE) for 15 minutes. Cell lysates were analyzed using Western blot analysis. F, BEAS-2B and HBE/p53i cells were transfected with scrambled (Scr) or IGF2 siRNAs and then exposed to norepinephrine for the indicated times. Lysates from these cells were then subjected to Western blot analysis. G and H, BEAS-2B/GFP-IGF2 cells were pretreated with nimodipine (Nimo) or Exo1 for 3 hours (G) or were transfected with scrambled (Scr) or RAB27A siRNAs (H) and then stimulated with norepinephrine for 15 minutes. CM and whole-cell lysates from these cells were subjected to Western blot analysis. Statistical significance of difference was determined using a two-sided Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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We further assessed the expression of IGF1, IGF2, and IGFBP3 during 12-hour exposure to norepinephrine. The long-term exposure to norepinephrine induced great increases in IGF2 transcription with moderate increases in IGF1 and IGFBP3 transcriptions (Fig. 3I, left). Furthermore, extracellular IGF2, but not IGF1 and IGFBP-3, levels were significantly increased during 12 hours of norepinephrine exposure (Fig. 3I, right, and Supplementary Fig. S7). Hence, transcriptional increase in ligands, especially IGF2, seemed to contribute to the norepinephrine-induced IGF-1R activation in a long-term period.

Norepinephrine-induced promotion of tumor development by activating the IGF2–IGF-1R pathway

We investigated whether IGF2 was a main driver for the norepinephrine-induced lung epithelial cell transformation and tumor development. We observed that two different IGF2-specific shRNAs (Supplementary Fig. S8) significantly suppressed norepinephrine-induced increases in viability (Fig. 4A and Supplementary Fig. S9) and colony-forming ability (Fig. 4B) of BEAS-2B cells. We then examined norepinephrine-induced IGF-1R phosphorylation in immortalized HBE cell derivatives that exhibited a range of endogenous IGF2 expression levels, including low (HBE), intermediate (HBE/Ras), and high (HBE/p53i) levels (23). When exposed to norepinephrine, these HBE cell derivatives showed corresponding results for IGF-1R phosphorylation, cell viability, and foci-forming ability (Fig. 4C). We then compared norepinephrine-induced lung tumor development in wild-type (WT) and lung-specific IGF1R Tg (IGF1RTg) mice. Urethane- and CUS-induced lung tumor development was enhanced in the IGF1RTg mice compared with the WT mice (Fig. 4D). The multiplicity, volume, and burden of lung tumors were also greater in the IGF1RTg (Fig. 4E) mice than in the WT mice following the administration of urethane and CUS exposure. Exposure to CUS induced prominent pIGF-1R expression in the lungs of IGF1RTg mice than in those of WT mice (Fig. 4F). Mice continuously exposed to norepinephrine by using a micro-osmotic pump also showed significantly increased lung tumor formation upon urethane injection (Fig. 4G and H) and pIGF-1R expression in lung tumors (Supplementary Fig. S10). These results suggest that chronic stress promotes lung cancer formation by activating the IGF2–IGF-1R signaling cascade.

Figure 4.

Crucial role of IGF2-mediated IGF-1R activation in promoting lung tumorigenesis in the presence of exposure to chronic stress or norepinephrine. A and B, suppression of norepinephrine (NE)-induced cell transformation, as determined by cell viability (A) and anchorage-dependent colony formation (B), by silencing IGF2 in BEAS-2B cells (n = 4; mean ± SD). C, left, Western blot analysis of lysates obtained from HBE, HBE/Ras, or HBE/p53i cells that were treated with either PBS (vehicle) or norepinephrine for the indicated times; middle and right, cell viability of and foci formation in HBE, HBE/Ras, and HBE/p53i cells treated with either PBS (vehicle) or norepinephrine at the indicated concentrations for 5 or 10 days. D, representative IVIS Spectrum CT images showing lung tumor formation in wild-type (WT; top) and IGF1RTg (bottom) mice. Arrows, tumor formation. U, urethane. E, increase in urethane-induced lung tumor formation in IGF1RTg mice exposed to CUS. The multiplicity, volume, and burden of murine lung tumors are presented as the mean ± SD (IGF-1RTg, n = 7; WT, n = 6). F, increase in IGF-1R phosphorylation by exposure to CUS, as determined by Western blot analysis. Densitometric analysis was performed using ImageJ software. G, increases in urethane-induced lung tumor formation in WT or IGF1RTg mice continuously exposed to norepinephrine using a micro-osmotic pump. The data are presented as the mean ± SD (IGF1RTg, n = 5; WT, n = 4). H, a representative image showing lung tumor formation from IGF1RTg mice shown in G. Statistical significance of difference was determined using a two-sided Student t test (A–C) and one-way ANOVA (E–G). *, P < 0.05; **, P < 0.01; ***, P < 0.001. NS, not significant.

Figure 4.

Crucial role of IGF2-mediated IGF-1R activation in promoting lung tumorigenesis in the presence of exposure to chronic stress or norepinephrine. A and B, suppression of norepinephrine (NE)-induced cell transformation, as determined by cell viability (A) and anchorage-dependent colony formation (B), by silencing IGF2 in BEAS-2B cells (n = 4; mean ± SD). C, left, Western blot analysis of lysates obtained from HBE, HBE/Ras, or HBE/p53i cells that were treated with either PBS (vehicle) or norepinephrine for the indicated times; middle and right, cell viability of and foci formation in HBE, HBE/Ras, and HBE/p53i cells treated with either PBS (vehicle) or norepinephrine at the indicated concentrations for 5 or 10 days. D, representative IVIS Spectrum CT images showing lung tumor formation in wild-type (WT; top) and IGF1RTg (bottom) mice. Arrows, tumor formation. U, urethane. E, increase in urethane-induced lung tumor formation in IGF1RTg mice exposed to CUS. The multiplicity, volume, and burden of murine lung tumors are presented as the mean ± SD (IGF-1RTg, n = 7; WT, n = 6). F, increase in IGF-1R phosphorylation by exposure to CUS, as determined by Western blot analysis. Densitometric analysis was performed using ImageJ software. G, increases in urethane-induced lung tumor formation in WT or IGF1RTg mice continuously exposed to norepinephrine using a micro-osmotic pump. The data are presented as the mean ± SD (IGF1RTg, n = 5; WT, n = 4). H, a representative image showing lung tumor formation from IGF1RTg mice shown in G. Statistical significance of difference was determined using a two-sided Student t test (A–C) and one-way ANOVA (E–G). *, P < 0.05; **, P < 0.01; ***, P < 0.001. NS, not significant.

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A β-AR signaling-mediated increase in intracellular calcium via VDCC activity is crucial for norepinephrine-induced secretion of IGF2 and IGF-1R phosphorylation

To identify the signaling effectors that are responsible for norepinephrine-induced production of IGF2, we investigated the effects of pharmacologic inhibitors of the β-AR signaling pathway, including atenolol (a β1-AR antagonist), ICI-118,551 (a β2-AR antagonist), propranolol (a nonselective β-AR antagonist), ESI-09 (an Epac inhibitor), H-89 (a PKA inhibitor), and gallein (a Gβγ subunit inhibitor) on norepinephrine-induced IGF-1R phosphorylation. Treatment with the β-AR antagonists effectively suppressed the norepinephrine-induced phosphorylation of IGF-1R and IGF2 levels in the CM in BEAS-2B cells (Fig. 5A). The norepinephrine-induced phosphorylation of IGF-1R was also sensitive to PKA and Epac inhibition but not to blockade of Gβγ (Fig. 5B). Hence, the norepinephrine-induced secretion of IGF2 and activation of IGF-1R appeared to be mediated by the activity of Gαs.

Figure 5.

Association with intracellular Ca2+ with the norepinephrine-induced secretion of IGF2 and activation of IGF-1R. A–C, H, BEAS-2B and BEAS-2B/GFP-IGF2 cells were pretreated with either β-AR antagonists [atenolol (Ate), ICI-118,551 (ICI), or propranolol (Pro); A], several inhibitors of β-AR downstream signaling (ESI-09, H-89, or gallein; B), Ca2+ chelators [intracellular, BAPTA-AM (BAPTA); extracellular, EGTA; C], and CCBs [amlodipine (Amlo) or nifedipine (Nife); H] for 3 hours before stimulation with norepinephrine (NE) for 15 minutes. The CM and lysates obtained from these cells were subjected to Western blot analysis. The Coomassie-stained gel is shown as a loading control (con) for CM. D, BEAS-2B and BEAS-2B/GFP-IGF2 cells were transfected with scrambled (Scr), CACNA1D, or CACNA1A siRNAs and then treated with norepinephrine for 15 minutes. CM and lysates obtained from these cells were subjected to Western blot analysis. E, a time-lapse imaging analysis for norepinephrine-induced GFP-IGF2 secretion from BEAS-2B/GFP-IGF2 cells. Cells were transfected with scrambled (Scr) or CACNA1D siRNAs and then stimulated with norepinephrine for 10 minutes. Scale bar, 20 μm. F, BEAS-2B cells were stimulated with norepinephrine for 15 minutes after pretreatment with H-89. IP, immunoprecipitation; WCL, whole-cell lysates. Cell lysates were immunoprecipitated using anti-CACNA1D antibodies, and then the precipitated proteins were analyzed by Western blot using anti-phosphoserine antibodies. G, top, after pretreatment with various indicated inhibitors or antagonists for 3 hours, BEAS-2B cells were stained with 2 μmol/L Fluo-4 AM. Cells were stimulated with norepinephrine for 5 minutes, and then fluorescence intensity was detected using fluorescence microscopy. Bottom, time-lapse imaging analysis for norepinephrine-induced GFP-IGF2 secretion. Scale bar, 100 μm (top) and 20 μm (bottom). I, representative images of confocal microscopy from BEAS-2B/GFP-IGF2 cells. Cells were pretreated with indicated inhibitors for 3 hours and then stimulated with norepinephrine. Scale bar, 10 μm. J, BEAS-2B cells were pretreated with propranolol or amlodipine for 3 hours and then further stimulated with norepinephrine for 15 minutes. The level of IGF2 in the CM was determined using ELISA (n = 3; mean ± SD). Statistical significance of difference was determined using one-way ANOVA. ***, P < 0.001.

Figure 5.

Association with intracellular Ca2+ with the norepinephrine-induced secretion of IGF2 and activation of IGF-1R. A–C, H, BEAS-2B and BEAS-2B/GFP-IGF2 cells were pretreated with either β-AR antagonists [atenolol (Ate), ICI-118,551 (ICI), or propranolol (Pro); A], several inhibitors of β-AR downstream signaling (ESI-09, H-89, or gallein; B), Ca2+ chelators [intracellular, BAPTA-AM (BAPTA); extracellular, EGTA; C], and CCBs [amlodipine (Amlo) or nifedipine (Nife); H] for 3 hours before stimulation with norepinephrine (NE) for 15 minutes. The CM and lysates obtained from these cells were subjected to Western blot analysis. The Coomassie-stained gel is shown as a loading control (con) for CM. D, BEAS-2B and BEAS-2B/GFP-IGF2 cells were transfected with scrambled (Scr), CACNA1D, or CACNA1A siRNAs and then treated with norepinephrine for 15 minutes. CM and lysates obtained from these cells were subjected to Western blot analysis. E, a time-lapse imaging analysis for norepinephrine-induced GFP-IGF2 secretion from BEAS-2B/GFP-IGF2 cells. Cells were transfected with scrambled (Scr) or CACNA1D siRNAs and then stimulated with norepinephrine for 10 minutes. Scale bar, 20 μm. F, BEAS-2B cells were stimulated with norepinephrine for 15 minutes after pretreatment with H-89. IP, immunoprecipitation; WCL, whole-cell lysates. Cell lysates were immunoprecipitated using anti-CACNA1D antibodies, and then the precipitated proteins were analyzed by Western blot using anti-phosphoserine antibodies. G, top, after pretreatment with various indicated inhibitors or antagonists for 3 hours, BEAS-2B cells were stained with 2 μmol/L Fluo-4 AM. Cells were stimulated with norepinephrine for 5 minutes, and then fluorescence intensity was detected using fluorescence microscopy. Bottom, time-lapse imaging analysis for norepinephrine-induced GFP-IGF2 secretion. Scale bar, 100 μm (top) and 20 μm (bottom). I, representative images of confocal microscopy from BEAS-2B/GFP-IGF2 cells. Cells were pretreated with indicated inhibitors for 3 hours and then stimulated with norepinephrine. Scale bar, 10 μm. J, BEAS-2B cells were pretreated with propranolol or amlodipine for 3 hours and then further stimulated with norepinephrine for 15 minutes. The level of IGF2 in the CM was determined using ELISA (n = 3; mean ± SD). Statistical significance of difference was determined using one-way ANOVA. ***, P < 0.001.

Close modal

Considering the previous findings that: (i) IGF2 colocalizes with insulin-secretory granules (28); (ii) VDCC-induced Ca2+ influx modulates insulin secretion (29); and (iii) activated PKA via GPCR signaling modulates VDCC activity through phosphorylation (30), we hypothesized that the activation of VDCC and the subsequent increase in intracellular Ca2+ may contribute to the norepinephrine-mediated secretion of IGF2 via exocytosis, leading to IGF-1R activation. Indeed, treatment with the intracellular (BAPTA-AM) and extracellular (EGTA) Ca2+ chelators abolished norepinephrine-induced IGF2 secretion and IGF-1R phosphorylation in BEAS-2B cells (Fig. 5C). VDCC is composed of five subunits, including α1, α2, δ, β, and γ subunits (31). Among various types of α1 subunit, the principal transmembrane subunit (31), both BEAS-2B and HBE/p53i cells were found to express the L- (Cav1.3) and P/Q- (Cav2.1) type VDCC. We then assessed the effects of inhibiting Cav1.3 (CACNA1D) and Cav2.1 (CACNA1A) using RNAi on the norepinephrine-induced IGF2 secretion. Depletion of CACNA1D, but not CACNA1A, in BEAS-2B cells attenuated the norepinephrine-induced IGF2 secretion and IGF-1R phosphorylation (Fig. 5D and E).

It has been shown that a PKA phosphorylates the α1D subunit of L-type VDCC at serines 1743 and 1816, leading to VDCC activation (i.e., opening; ref. 32). Indeed, norepinephrine induced phosphorylation of VDCC at Ser residue, and this effect was suppressed by treatment with H-89 (Fig. 5F). Moreover, treatment with propranolol, H-89, Ca2+ chelators (EGTA and BAPTA-AM), and L-type calcium channel blockers (CCBs; amlodipine and nifedipine) almost completely suppressed the norepinephrine-induced increases in intracellular Ca2+ level in BEAS-2B cells (Fig. 5G, top) and IGF2 secretion in BEAS-2B/GFP-IGF2 cells (Fig. 5G, bottom). Norepinephrine-induced activation of IGF-1R and secretion of IGF2 were attenuated by inhibitors of L-type VDCC (Fig. 5H). Confocal analysis also showed an obvious decrease in IGF2 fused to the cell membrane and in cellular periphery by treatment with propranolol, H-89, Ca2+ chelators (EGTA and BAPTA-AM), and CCBs (Fig. 5I). Treatment with propranolol or amlodipine almost completely blocked the norepinephrine-induced IGF2 secretion (Fig. 5J). These findings indicate that norepinephrine-induced β-AR signaling increases intracellular calcium levels by activating L-type VDCC, ultimately leading to increases in IGF2 secretion via exocytosis and the consequent phosphorylation of IGF-1R.

Blockade of β-AR and L-type VDCC suppresses malignant cell transformation and chronic stress–mediated promotion of lung tumorigenesis

We next confirmed that treatment with β-AR antagonists (atenolol, ICI 118,551, or propranolol; Fig. 6A) or CCBs (amlodipine or nifedipine; Fig. 6B) significantly suppressed norepinephrine-induced increases in HBE cell viability and anchorage-dependent and anchorage-independent colony formation. Notably, treatment with β-AR or L-type VDCC inhibitors reduced the lung tumor formation in CUS-exposed and urethane-treated mice (Supplementary Fig. S11). Tumor multiplicity, volume, and burden in mice exposed to chronic stress were also significantly decreased by administration of amlodipine or propranolol (Fig. 6C). A bronchoalveolar lavage fluid analysis indicated that blockade of β-AR or L-type VDCC significantly attenuated norepinephrine-induced IGF2 secretion in the lungs (Fig. 6D and Supplementary Fig. S12). Immunohistochemical analysis further confirmed the decreased levels of pIGF-1R in bronchial epithelial cells (Fig. 6E and F) and in lung tumors (Supplementary Fig. S13) obtained from mice that had been treated with propranolol or amlodipine. These results indicate that β-AR or the L-type VDCC are targets for preventing chronic stress–mediated lung tumorigenesis (Fig. 6G).

Figure 6.

Suppression of norepinephrine-induced in vitro lung epithelial cell transformation and chronic stress–induced promotion of in vivo lung tumorigenesis by blockade of β-AR or VDCC. A and B, suppression of norepinephrine (NE)-induced lung epithelial cell transformation by blockade of β-AR (A) and VDCC (B), as determined by cell viability and anchorage-dependent and -independent colony formation (n = 3 or 4, mean ± SD). ATE, atenolol; ICI, ICI-118,551; Pro, propranolol. C, the suppressive effect of blocking β-AR or VDCC on CUS-promoted lung tumor formation in vivo. Two weeks before the administration of urethane (U), IGF1RTg mice were orally administered with vehicle (sterile PBS), propranolol (80 mg/kg), or amlodipine (Amlo; 10 mg/kg) daily for 3.5 months. The effects of the indicated inhibitors on the incidence, multiplicity, volume, and burden of murine lung tumors are shown (the propranolol-treated group: n = 6; other groups: n = 7; expressed as the mean ± SD). D, relative IGF2 secretion in bronchoalveolar lavage fluids (BALF) from mice (n = 3 or 4) exposed to CUS for 4 weeks in the absence or presence of propranolol or amlodipine for 3.5 weeks. IGF2 levels in BALFs were evaluated by Western blot analysis, and the relative IGF2 secretion was determined by densitometric analysis using ImageJ software. A representative image of IGF2 blots is shown in Supplementary Fig. S12. E, a representative image of phosphorylated IGF-1R in murine lung tissues, as determined using immunohistochemical analysis. Scale bar, 10 μm. F, quantification of pIGF-1R levels in murine lung tissues using InForm cell analysis software. G, schematic model for the mechanism underlying chronic stress–induced promotion of lung tumorigenesis. Statistical significance of difference was determined using a two-sided Student t test (A and B) or one-way ANOVA (C, D, and F). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Figure 6.

Suppression of norepinephrine-induced in vitro lung epithelial cell transformation and chronic stress–induced promotion of in vivo lung tumorigenesis by blockade of β-AR or VDCC. A and B, suppression of norepinephrine (NE)-induced lung epithelial cell transformation by blockade of β-AR (A) and VDCC (B), as determined by cell viability and anchorage-dependent and -independent colony formation (n = 3 or 4, mean ± SD). ATE, atenolol; ICI, ICI-118,551; Pro, propranolol. C, the suppressive effect of blocking β-AR or VDCC on CUS-promoted lung tumor formation in vivo. Two weeks before the administration of urethane (U), IGF1RTg mice were orally administered with vehicle (sterile PBS), propranolol (80 mg/kg), or amlodipine (Amlo; 10 mg/kg) daily for 3.5 months. The effects of the indicated inhibitors on the incidence, multiplicity, volume, and burden of murine lung tumors are shown (the propranolol-treated group: n = 6; other groups: n = 7; expressed as the mean ± SD). D, relative IGF2 secretion in bronchoalveolar lavage fluids (BALF) from mice (n = 3 or 4) exposed to CUS for 4 weeks in the absence or presence of propranolol or amlodipine for 3.5 weeks. IGF2 levels in BALFs were evaluated by Western blot analysis, and the relative IGF2 secretion was determined by densitometric analysis using ImageJ software. A representative image of IGF2 blots is shown in Supplementary Fig. S12. E, a representative image of phosphorylated IGF-1R in murine lung tissues, as determined using immunohistochemical analysis. Scale bar, 10 μm. F, quantification of pIGF-1R levels in murine lung tissues using InForm cell analysis software. G, schematic model for the mechanism underlying chronic stress–induced promotion of lung tumorigenesis. Statistical significance of difference was determined using a two-sided Student t test (A and B) or one-way ANOVA (C, D, and F). *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Close modal

Chronic stress has been implicated in the onset and progression of various cancers, including lung cancer (6, 9, 13, 33–35). Lung cancer patients with severe psychologic distress at the time of diagnosis had frequently experienced earlier psychologic stress (36), and psychologic distress was found to be a significant predictor of lung cancer mortality (37). However, the role of chronic stress in lung tumorigenesis remains controversial, and it has not been clearly defined in animal models. Moreover, few studies have been conducted to investigate the mechanisms underlying stress-mediated changes in lung epithelial cells during tumor formation. Therefore, a better understanding of the mechanisms underlying the chronic stress–induced initiation and progression of lung cancer may provide new strategies for controlling lung cancer. The results presented here provide the first direct preclinical evidence for the role of chronic stress in lung cancer development and the mechanisms by which chronic stress promotes carcinogen- and oncogene-initiated lung tumor development.

In the current study, we show that CUS increased circulating norepinephrine levels to physiologically relevant levels and greatly promoted the urethane- or KrasG12D/+-induced development of lung tumors. Previous studies have shown increased levels of circulating catecholamines and corticosteroids, resulting in their high concentrations during stress (5, 38). In this study, we show that catecholamines, but not corticosteroids, induce transformation of lung epithelial cells. Notably, implantation of mice with an osmotic pump that releases physiologically relevant doses of norepinephrine promoted lung tumor formation. Therefore, chronic stress appears to be an environmental factor that promotes lung tumorigenesis via the production of catecholamines, especially norepinephrine. The mechanisms by which chronic stress promotes lung tumorigenesis are largely unknown. A wide variety of studies have suggested that chronic stress promotes the initiation and progression of cancer by impairing immune functions that are relevant to immune surveillance. These impairments can include altered cytokine production, cytotoxicity in natural killer (NK) cells, and T-cell mitogenesis (38). However, chronic stress was found to promote tumor growth in immunodeficient nude mice carrying defects in immune functions (6, 9, 33–35). Hence, additional mechanisms, such as somatic mutations, genomic instability, and inhibited apoptosis, have been proposed to explain the effects of chronic stress on tumor development (5, 35). Our study provides direct evidence that the norepinephrine-induced activation of IGF-1R in lung epithelial cells is a biochemical event that promotes lung carcinogenesis. Considering the role of the IGF-1R pathway in cell transformation and survival (39, 40), chronic stress seems to provide cancer-initiated lung epithelial cells with survival potential by activating IGF-1R signaling and thereby accelerating lung tumor formation. Indeed, CUS-induced lung tumor formation was significantly attenuated by inactivation of the IGF-1R pathway.

On the basis of our subsequent findings, indicating that norepinephrine-induced exocytosis of IGF2 was responsible for the norepinephrine-mediated immediate activation of IGF-1R, we extended our investigation to assess the mechanisms by which norepinephrine regulates the secretion of IGF2. Our results indicate that (i) the norepinephrine-induced activation of PKA led to the phosphorylation of VDCC and increased intracellular Ca2+ levels, which have a crucial role in mediating exocytosis; (ii) treatment with antagonists of norepinephrine/β-AR/PKA signaling or CCBs effectively suppressed the norepinephrine-induced upregulation of intracellular calcium levels and the secretion of IGF2; and (iii) inhibitors of exocytosis and Ca2+ chelators almost completely suppressed norepinephrine-induced IGF2 secretion. Recent studies have shown (i) an association between IGF2 and insulin secretory granules (28) and (ii) a role for Epac2 in insulin secretion (41). These results suggest that norepinephrine/β-AR/PKA/VDCC–mediated Ca2+ influx stimulates secretagogue-induced exocytosis of IGF2. IGFBPs have been known to regulate IGF bioavailability under certain conditions (25). IGFBP-3 action is antagonized by proteolysis, and various proteases, such as kallikrein-like serine proteases, cathepsins, and matrix metalloproteinases, are known to cleave IGFBP-3 into small fragments with reduced affinity for IGFs (26). Therefore, regulation of IGFBPs was postulated to confer the norepinephrine-induced increase in the extracellular IGF2 levels. However, intracellular and extracellular IGFBP-3 levels remained unchanged during 12 hours of norepinephrine exposure, when IGF-1R phosphorylation and extracellular IGF2 were obviously increased. Together with our findings showing norepinephrine-induced upregulation of IGF2 mRNA levels after a long-term exposure, the β-AR signaling seemed to activate the IGF-1R signaling pathway transiently by stimulating IGF2 exocytosis via the regulated pathway and chronically by stimulating transcription program for IGF2 expression.

We have demonstrated lung tumor development in mice with a lung-specific IGF overexpression (23). Hence, increases in IGF2 secretion from lung epithelial cells could have contributed to the CUS-induced lung tumor development. The keys to the validation of the hypothesis are our data: (i) norepinephrine-induced IGF-1R activation and transformed phenotypes in lung epithelial cells were suppressed by blocking the activity of IGF2 and IGF-1R activation; and (ii) lung tumor development was augmented upon exposure to CUS or norepinephrine in transgenic mice with lung-specific IGF-1R expression. Several prospective epidemiologic studies have implicated circulating IGFs as risk factors for developing diverse human cancer (39, 40). However, the evidence supporting a role for circulating IGFs in lung cancer is circumstantial (42). We previously reported that IGF overexpression and IGF-1R activation in airway epithelial cells are early biochemical events in human lung carcinogenesis (23). Therefore, pathways involved in the norepinephrine-induced secretion of tissue-derived IGFs, rather than those involved in increasing circulating IGFs, would be attractive targets for preventing chronic stress–induced lung cancer.

Collectively, the results of the current study support the existence of a novel mechanism that defines the chronic stress–mediated development of lung tumors (Fig. 6G). We show that the norepinephrine-induced activation of β-AR signaling stimulates VDCC-mediated Ca2+ influx, thereby causing IGF-1R activation via the secretion of IGF2 by airway epithelial cells. The activation of this β-AR–PKA–VDCC–IGF-1R transactivation pathway provides premalignant lung epithelial cells with survival potential when experiencing genetic insult by carcinogenic or oncogenic activation, leading to the formation of lung tumors. Modulation of β-AR signaling activation is a common signaling node between norepinephrine and the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (43). Therefore, it is possible that chronic stress is a synergistic driver of tobacco smoking–mediated lung carcinogenesis, and our proposed approaches may be applicable to both smokers and nonsmokers. In accordance with our results, previous studies have suggested the use of β-blockers as an adjuvant anticancer therapy (6). Moreover, the use of β-blockers has been shown to reduce mortality in patients with several types of cancers, such as lung and breast cancers (44, 45). Our results also provide preclinical evidence that supports the use of CCBs to prevent lung cancer in nonsmokers who are under chronic psychologic stress. β-Blockers and CCBs have been widely used as antihypertensive agents. Hence, their efficacy and safety have already been determined, and their use may be directly applicable to further clinical investigations. In this context, substantial attention should be paid to the use of these drugs for lung cancer prevention. Further studies are warranted to evaluate the clinical utility of using antihypertensive drugs to prevent lung cancer in smokers and nonsmokers experiencing chronic psychologic stress.

No potential conflicts of interest were disclosed.

Conception and design: H.-J. Jang, H.-Y. Lee

Development of methodology: H.-J. Jang, H.-J. Boo, H.-Y. Lee

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H.-J. Jang, H.J. Lee, H.-Y. Min, H.-Y. Lee

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H.-J. Jang, H.-Y. Lee

Writing, review, and/or revision of the manuscript: H.-Y. Lee

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H.-J. Boo, H.J. Lee, H.-Y. Min, H.-Y. Lee

Study supervision: H.-Y. Lee

This work was supported by grants from the National Research Foundation of Korea (NRF), the Ministry of Science, ICT & Future Planning (MSIP), and the Republic of Korea (nos. NRF-2011-0017639 and NRF-2016R1A3B1908631).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data