Focal adhesion kinase-YAP signaling axis drives drug-tolerant persister cells and residual disease in lung cancer.

Targeted therapy is effective in many tumor types including lung cancer, the leading cause of cancer mortality. Paradigm defining examples are targeted therapies directed against non-small cell lung cancer (NSCLC) subtypes with oncogenic alterations in EGFR, ALK and KRAS. The success of targeted therapy is limited by drug-tolerant persister cells (DTPs) which withstand and adapt to treatment and comprise the residual disease state that is typical during treatment with clinical targeted therapies. Here, we integrate studies in patient-derived and immunocompetent lung cancer models and clinical specimens obtained from patients on targeted therapy to uncover a focal adhesion kinase (FAK)-YAP signaling axis that promotes residual disease during oncogenic EGFR-, ALK-, and KRAS-targeted therapies. FAK-YAP signaling inhibition combined with the primary targeted therapy suppressed residual drug-tolerant cells and enhanced tumor responses. This study unveils a FAK-YAP signaling module that promotes residual disease in lung cancer and mechanism-based therapeutic strategies to improve tumor response.

3-Phosphoinositide-dependent kinase 1 drives acquired resistance to osimertinib.

Osimertinib sensitive and resistant NSCLC NCI-H1975 clones are used to model osimertinib acquired resistance in humanized and non-humanized mice and delineate potential resistance mechanisms. No new EGFR mutations or loss of the EGFR T790M mutation are found in resistant clones. Resistant tumors grown under continuous osimertinib pressure both in humanized and non-humanized mice show aggressive tumor regrowth which is significantly less sensitive to osimertinib as compared with parental tumors. 3-phosphoinositide-dependent kinase 1 (PDK1) is identified as a potential driver of osimertinib acquired resistance, and its selective inhibition by BX795 and CRISPR gene knock out, sensitizes resistant clones. In-vivo inhibition of PDK1 enhances the osimertinib sensitivity against osimertinib resistant xenograft and a patient derived xenograft (PDX) tumors. PDK1 knock-out dysregulates PI3K/Akt/mTOR signaling, promotes cell cycle arrest at the G1 phase. Yes-associated protein (YAP) and active-YAP are upregulated in resistant tumors, and PDK1 knock-out inhibits nuclear translocation of YAP. Higher expression of PDK1 and an association between PDK1 and YAP are found in patients with progressive disease following osimertinib treatment. PDK1 is a central upstream regulator of two critical drug resistance pathways: PI3K/AKT/mTOR and YAP.

TUSC2 immunogene enhances efficacy of chemo-immuno combination on KRAS/LKB1 mutant NSCLC in humanized mouse model.

KRAS/LKB1 (STK11) NSCLC metastatic tumors are intrinsically resistant to anti-PD-1 or PD-L1 immunotherapy. In this study, we use a humanized mouse model to show that while carboplatin plus pembrolizumab reduce tumor growth moderately and transiently, the addition of the tumor suppressor gene TUSC2, delivered systemically in nanovesicles, to this combination, eradicates tumors in the majority of animals. Immunoprofiling of the tumor microenvironment shows the addition of TUSC2 mediates: (a) significant infiltration of reconstituted human functional cytotoxic T cells, natural killer cells, and dendritic cells; (b) induction of antigen-specific T cell responses; (c) enrichment of functional central and memory effector T cells; and (d) decreased levels of PD-1+ T cells, myeloid-derived suppressor cells, Tregs, and M2 tumor associated macrophages. Depletion studies show the presence of functional central and memory effector T cells are required for the efficacy. TUSC2 sensitizes KRAS/LKB1 tumors to carboplatin plus pembrolizumab through modulation of the immune contexture towards a pro-immune tumor microenvironment.

Therapeutic targeting of the PI4K2A/PKR lysosome network is critical for misfolded protein clearance and survival in cancer cells.

The role of RNA-dependent protein kinase R (PKR) and its association with misfolded protein expression in cancer cells are unclear. Herein we report that PKR regulates misfolded protein clearance by preventing it release through exosomes and promoting lysosomal degradation of misfolded prion proteins in cancer cells. We demonstrated that PKR contributes to the lysosome function and regulates misfolded prion protein clearance. We hypothesized that PKR-associated lysosome function is critical for cancer but not normal cell survival, representing an effective approach for highly targeted cancer therapy. In screening a compound library, we identified two PKR-associated compounds 1 and 2 (Pac 1 and 2) did not affect normal cells but selectively induced cell death in cancer cells depending on their PKR expression status. Pac 1 significantly inhibited the growth of human lung and breast xenograft tumors in mice with no toxicity. Pac 1 binds to PI4K2A and disrupts the PKR/PI4K2A-associated lysosome complex, contributing to destabilization of cancer cell lysosomes and triggering cell death. We observed that PKR and PI4K2A play significant prognostic roles in breast cancer patients. These results demonstrate that targeting of a PI4K2A/PKR lysosome complex may be an effective approach for cancer therapy.

An Improved Patient-Derived Xenograft Humanized Mouse Model for Evaluation of Lung Cancer Immune Responses.

Human tumor xenograft models do not replicate the human immune system and tumor microenvironment. We developed an improved humanized mouse model, derived from fresh cord blood CD34+ stem cells (CD34+ HSC), and combined it with lung cancer cell line-derived human xenografts or patient-derived xenografts (Hu-PDX). Fresh CD34+ HSCs could reconstitute detectable mature human leukocytes (hCD45+) in mice at four weeks without the onset of graft-versus-host disease (GVHD). Repopulated human T cells, B cells, natural killer (NK) cells, dendritic cells (DC), and myeloid-derived suppressor cells (MDSC) increased in peripheral blood, spleen, and bone marrow over time. Although cultured CD34+ HSCs labeled with luciferase could be detected in mice, the cultured HSCs did not develop into mature human immune cells by four weeks, unlike fresh CD34+ HSCs. Ex vivo, reconstituted T cells, obtained from the tumor-bearing humanized mice, secreted IFNγ upon treatment with phorbol myristate acetate (PMA) or exposure to human A549 lung tumor cells and mediated antigen-specific CTL responses, indicating functional activity. Growth of engrafted PDXs and tumor xenografts was not dependent on the human leukocyte antigen status of the donor. Treatment with the anti-PD-1 checkpoint inhibitors pembrolizumab or nivolumab inhibited tumor growth in humanized mice significantly, and correlated with an increased number of CTLs and decreased MDSCs, regardless of the donor HLA type. In conclusion, fresh CD34+HSCs are more effective than their expanded counterparts in humanizing mice, and do so in a shorter time. The Hu-PDX model provides an improved platform for evaluation of immunotherapy.

TUSC2 Immunogene Therapy Synergizes with Anti-PD-1 through Enhanced Proliferation and Infiltration of Natural Killer Cells in Syngeneic Kras-Mutant Mouse Lung Cancer Models.

Expression of the multikinase inhibitor encoded by the tumor suppressor gene TUSC2 (also known as FUS1) is lost or decreased in non-small cell lung carcinoma (NSCLC). TUSC2 delivered systemically by nanovesicles has mediated tumor regression in clinical trials. Because of the role of TUSC2 in regulating immune cells, we assessed TUSC2 efficacy on antitumor immune responses alone and in combination with anti-PD-1 in two Kras-mutant syngeneic mouse lung cancer models. TUSC2 alone significantly reduced tumor growth and prolonged survival compared with anti-PD-1. When combined, this effect was significantly enhanced, and correlated with a pronounced increases in circulating and splenic natural killer (NK) cells and CD8+ T cells, and a decrease in regulatory T cells (Tregs), myeloid-derived suppressor cells (MDSCs), and T-cell checkpoint receptors PD-1, CTLA-4, and TIM-3. TUSC2 combined with anti-PD-1 induced tumor infiltrating more than NK and CD8+ T cells and fewer MDSCs and Tregs than each agent alone, both in subcutaneous tumor and in lung metastases. NK-cell depletion abrogated the antitumor effect and Th1-mediated immune response of this combination, indicating that NK cells mediate TUSC2/anti-PD-1 synergy. Release of IL15 and IL18 cytokines and expression of the IL15Rα chain and IL18R1 were associated with NK-cell activation by TUSC2. Immune response-related gene expression in the tumor microenvironment was altered by combination treatment. These data provide a rationale for immunogene therapy combined with immune checkpoint blockade in the treatment of NSCLC. Cancer Immunol Res; 6(2); 163-77. ©2018 AACR.

Serine Proteases Enhance Immunogenic Antigen Presentation on Lung Cancer Cells.

Immunotherapies targeting immune checkpoints have proven efficacious in reducing the burden of lung cancer in patients; however, the antigenic targets of these reinvigorated T cells remain poorly defined. Lung cancer tumors contain tumor-associated macrophages (TAM) and neutrophils, which release the serine proteases neutrophil elastase (NE) and proteinase 3 (P3) into the tumor microenvironment. NE and P3 shape the antitumor adaptive immune response in breast cancer and melanoma. In this report, we demonstrate that lung cancer cells cross-presented the tumor-associated antigen PR1, derived from NE and P3. Additionally, NE and P3 enhanced the expression of human leukocyte antigen (HLA) class I molecules on lung cancer cells and induced unique, endogenous peptides in the immunopeptidome, as detected with mass spectrometry sequencing. Lung cancer patient tissues with high intratumoral TAMs were enriched for MHC class I genes and T-cell markers, and patients with high TAM and cytotoxic T lymphocyte (CTL) infiltration had improved overall survival. We confirmed the immunogenicity of unique, endogenous peptides with cytotoxicity assays against lung cancer cell lines, using CTLs from healthy donors that had been expanded against select peptides. Finally, CTLs specific for serine proteases-induced endogenous peptides were detected in lung cancer patients using peptide/HLA-A2 tetramers and were elevated in tumor-infiltrating lymphocytes. Thus, serine proteases in the tumor microenvironment of lung cancers promote the presentation of HLA class I immunogenic peptides that are expressed by lung cancer cells, thereby increasing the antigen repertoire that can be targeted in lung cancer. Cancer Immunol Res; 5(4); 319-29. ©2017 AACR.

TUSC2 downregulates PD-L1 expression in non-small cell lung cancer (NSCLC).

Expression of the TUSC2 tumor-suppressor gene in TUSC2-deficient NSCLC cells decreased PD-L1 expression and inhibited mTOR activity. Overexpressing TUSC2 or treatment with rapamycin resulted in similar inhibition of PD-L1 expression. Both TUSC2 and rapamycin decreased p70 and SK6 phosphorylation, suggesting that TUSC2 and rapamycin share the same mTOR target. Microarray mRNA expression analysis using TUSC2-inducible H1299 showed that genes that negatively regulate the mTOR pathway were significantly upregulated by TUSC2 compared with control. The presence of IFN-γ significantly increased PD-L1 expression in lung cancer cell lines, but overexpressing TUSC2 in these cell lines prevented PD-L1 from increasing in the presence of IFN-γ. Taken together, these findings show that TUSC2 can decrease PD-L1 expression in lung cancer cells. This ability to modify the tumor microenvironment suggests that TUSC2 could be added to checkpoint inhibitors to improve the treatment of lung cancer.

TUSC2(FUS1)-erlotinib Induced Vulnerabilities in Epidermal Growth Factor Receptor(EGFR) Wildtype Non-small Cell Lung Cancer(NSCLC) Targeted by the Repurposed Drug Auranofin.

Expression of the TUSC2/FUS1 tumor suppressor gene in TUSC2 deficient EGFR wildtype lung cancer cells increased sensitivity to erlotinib. Microarray mRNA expression analysis of TUSC2 inducible lung cancer cells treated with erlotinib uncovered defects in the response to oxidative stress suggesting that increasing reactive oxygen species (ROS) would enhance therapeutic efficacy. Addition of the thioredoxin reductase 1 inhibitor (TXNRD1) auranofin (AF) to NSCLC cells treated with combination of TUSC2 forced expression with erlotinib increased tumor cell apoptosis and inhibited colony formation. TXNRD1 overexpression rescued tumors from AF-TUSC2-erlotinib induced apoptosis. Neutralizing ROS with nordihydroguaiaretic acid (NDGA) abrogated cell death induced by AF-TUSC2-erlotinib, indicating a regulatory role for ROS in the efficacy of the three drug combination. Isobologram-based statistical analysis of this combination demonstrated superior synergism, compared with each individual treatment at lower concentrations. In NSCLC tumor xenografts, tumor growth was markedly inhibited and animal survival was prolonged over controls by AF-TUSC2-erlotinib. Microarray mRNA expression analysis uncovered oxidative stress and DNA damage gene signatures significantly upregulated by AF-TUSC2-erlotinib compared to TUSC2-erlotinib. Pathway analysis showed the highest positive z-score for the NRF2-mediated oxidative stress response. Taken together these findings show that the combination of TUSC2-erlotinib induces additional novel vulnerabilities that can be targeted with AF.

Auranofin-mediated inhibition of PI3K/AKT/mTOR axis and anticancer activity in non-small cell lung cancer cells.

Auranofin, a gold complex that has been used to treat rheumatoid arthritis in clinics and has documented pharmacokinetic and safety profiles in humans, has recently been investigated for its anticancer activity in leukemia and some solid cancers. However, auranofin's single agent activity in lung cancer is not well characterized. To determine whether auranofin has single agent activity in lung cancer, we evaluated auranofin's activity in a panel of 10 non-small cell lung cancer (NSCLC) cell lines. Cell viability analysis revealed that auranofin induced growth inhibition in a subset of NSCLC cell lines with a half maximal inhibitory concentration (IC50) below 1.0 µM. Treatment with auranofin elicited apoptosis and necroptosis in auranofin-sensitive cell lines. Moreover, the susceptibility of NSCLC cells to auranofin was inversely correlated with TXNRD1 expression in the cells. Transient transfection of the TXNRD1-expressing plasmid in auranofin-sensitive Calu3 cells resulted in partial resistance, indicating that high TXNRD level is one of causal factors for resistance to auranofin. Further mechanistic characterization with proteomic analysis revealed that auranofin inhibits expression and/or phosphorylation of multiple key nodes in the PI3K/AKT/mTOR pathway, including S6, 4EBP1, Rictor, p70S6K, mTOR, TSC2, AKT and GSK3. Ectopic expression of TXNRD1 partially reversed auranofin-mediated PI3K/AKT/mTOR inhibition, suggesting that TXNRD1 may participate in the regulation of PI3K/AKT/mTOR pathway. Administration of auranofin to mice with xenograft tumors derived from NSCLC cells significantly suppressed tumor growth without inducing obvious toxic effects. Our results demonstrated feasibility of repurposing auranofin for treatment of lung cancer.

Exogenous Restoration of TUSC2 Expression Induces Responsiveness to Erlotinib in Wildtype Epidermal Growth Factor Receptor (EGFR) Lung Cancer Cells through Context Specific Pathways Resulting in Enhanced Therapeutic Efficacy.

Expression of the tumor suppressor gene TUSC2 is reduced or absent in most lung cancers and is associated with worse overall survival. In this study, we restored TUSC2 gene expression in several wild type EGFR non-small cell lung cancer (NSCLC) cell lines resistant to the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib and analyzed their sensitivity to erlotinib in vitro and in vivo. A significant inhibition of cell growth and colony formation was observed with TUSC2 transient and stable expression. TUSC2-erlotinib cooperativity in vitro could be reproduced in vivo in subcutaneous tumor growth and lung metastasis formation lung cancer xenograft mouse models. Combination treatment with intravenous TUSC2 nanovesicles and erlotinib synergistically inhibited tumor growth and metastasis, and increased apoptotic activity. High-throughput qRT-PCR array analysis enabling multi-parallel expression profile analysis of eighty six receptor and non-receptor tyrosine kinase genes revealed a significant decrease of FGFR2 expression level, suggesting a potential role of FGFR2 in TUSC2-enhanced sensitivity to erlotinib. Western blots showed inhibition of FGFR2 by TUSC2 transient transfection, and marked increase of PARP, an apoptotic marker, cleavage level after TUSC2-erlotinb combined treatment. Suppression of FGFR2 by AZD4547 or gene knockdown enhanced sensitivity to erlotinib in some but not all tested cell lines. TUSC2 inhibits mTOR activation and the latter cell lines were responsive to the mTOR inhibitor rapamycin combined with erlotinib. These results suggest that TUSC2 restoration in wild type EGFR NSCLC may overcome erlotinib resistance, and identify FGFR2 and mTOR as critical regulators of this activity in varying cellular contexts. The therapeutic activity of TUSC2 could extend the use of erlotinib to lung cancer patients with wildtype EGFR.

The tumor suppressor gene TUSC2 (FUS1) sensitizes NSCLC to the AKT inhibitor MK2206 in LKB1-dependent manner.

TUSC2-defective gene expression is detected in the majority of lung cancers and is associated with worse overall survival. We analyzed the effects of TUSC2 re-expression on tumor cell sensitivity to the AKT inhibitor, MK2206, and explored their mutual signaling connections, in vitro and in vivo. TUSC2 transient expression in three LKB1-defective non-small cell lung cancer (NSCLC) cell lines combined with MK2206 treatment resulted in increased repression of cell viability and colony formation, and increased apoptotic activity. In contrast, TUSC2 did not affect the response to MK2206 treatment for two LKB1-wild type NSCLC cell lines. In vivo, TUSC2 systemic delivery, by nanoparticle gene transfer, combined with MK2206 treatment markedly inhibited growth of tumors in a human LKB1-defective H322 lung cancer xenograft mouse model. Biochemical analysis showed that TUSC2 transient expression in LKB1-defective NSCLC cells significantly stimulated AMP-activated protein kinase (AMPK) phosphorylation and enzymatic activity. More importantly, AMPK gene knockdown abrogated TUSC2-MK2206 cooperation, as evidenced by reduced sensitivity to the combined treatment. Together, TUSC2 re-expression and MK2206 treatment was more effective in inhibiting the phosphorylation and kinase activities of AKT and mTOR proteins than either single agent alone. In conclusion, these findings support the hypothesis that TUSC2 expression status is a biological variable that potentiates MK2206 sensitivity in LKB1-defective NSCLC cells, and identifies the AMPK/AKT/mTOR signaling axis as an important regulator of this activity.

KEAP1-dependent synthetic lethality induced by AKT and TXNRD1 inhibitors in lung cancer.

Intrinsic resistance to agents targeting phosphoinositide 3-kinase (PI3K)/AKT pathway is one of the major challenges in cancer treatment with such agents. The objective of this study is to identify the genes or pathways that can be targeted to overcome the resistance of non-small cell lung carcinoma (NSCLC) to the AKT inhibitor MK2206, which is currently being evaluated in phase I and II clinical trials. Using a genome-wide siRNA library screening and biologic characterization, we identified that inhibition of thioredoxin reductase-1 (TXNRD1), one of the key antioxidant enzymes, with siRNAs or its inhibitor, auranofin, sensitized NSCLC cells to MK2206 treatment in vitro and in vivo. We found that simultaneous inhibition of TXNRD1 and AKT pathways induced robust reactive oxygen species production, which was involved in c-jun-NH2-kinase (JNK; MAPK8) activation and cell apoptosis. Furthermore, we found that the synthetic lethality interaction between the TXNRD1 and AKT pathways occurred through the KEAP1/NRF2 cellular antioxidant pathway. Finally, we found that synthetic lethality induced by TXNRD1 and AKT inhibitors relied on wild-type KEAP1 function. Our study indicates that targeting the interaction between AKT and TXNRD1 antioxidant pathways with MK2206 and auranofin, a U.S. Food and Drug Administration-approved drug, is a rational strategy to treat lung cancer and that KEAP1 mutation status may offer a predicative biomarker for such combination approaches.

Gamma-aminobutyric acid, a potential tumor suppressor for small airway-derived lung adenocarcinoma.

Pulmonary adenocarcinoma (PAC) is the leading type of lung cancer in smokers and non-smokers that arises in most cases from small airway epithelial cells. PAC has a high mortality due to its aggressive behavior and resistance to cancer therapeutics. We have shown previously that the proliferation of human PAC cells NCI-H322 and immortalized human small airway epithelial cells HPL1D is stimulated by cyclic adenosine monophosphate (cAMP)/protein kinase A-dependent phosphorylation of cyclic adenosine monophosphate response element-binding (CREB) protein and transactivation of the epidermal growth factor receptor and that this pathway is activated by beta-1-adrenoreceptors (beta(1)-ARs) and the non-genomic estrogen receptor beta. Our current in vitro studies with HPL1D and NCI-H322 cells showed that signaling via the gamma-amino butyric acid receptor (GABA(B)R) strongly inhibited base level and isoproterenol-induced cAMP, p-CREB, cyclic adenosine monophosphate response element-luciferase activity and p-extracellular regulated kinase-1 (ERK1)/2 and effectively blocked DNA synthesis and cell migration. The inhibitory effects of gamma-amino butyric acid (GABA) were disinhibited by the GABA(B)R antagonist CGP-35348 or GABA(B)R knockdown. Immunohistochemical investigation of hamster lungs showed significant underexpression of GABA in animals with small airway-derived PACs induced by the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). These findings suggest that GABA may have tumor suppressor function in small airway epithelia and the PACs derived from them and that downregulation of GABA by NNK may contribute to the development of this cancer in smokers. Our findings suggest that marker-guided treatment with GABA or a GABA(B)R agonist of individuals with downregulated pulmonary GABA may provide a novel targeted approach for the prevention of PAC in smokers.

GABA B receptor is a novel drug target for pancreatic cancer.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death. Smoking, diabetes, and pancreatitis are risk factors. It has been shown that the growth of PDAC and pancreatic duct epithelial cells is regulated by beta-adrenoreceptors (beta-ARs). The activity of beta-ARs in the central nervous system is counteracted by gamma-aminobutyric acid (GABA) via GABA B receptor-mediated inhibition of adenylyl cyclase. The aim of the study was to investigate if GABA B R inhibits beta-AR signaling in PDAC and pancreatic duct epithelial cells, thus blocking driving forces of cancer progression, such as cell proliferation and cell migration. METHODS: Intracellular cAMP was measured by immunoassays, DNA synthesis by BrdU incorporation assays, activation of ERK1/2 by ERK activation assays, and Western blots and metastatic potential by cell migration assays in the human PDAC cell lines PANC-1 and BXPC-3 and immortalized human pancreatic duct epithelial cells HPDE6-C7. The expression of norepinephrine, PKAR IIalpha, and GABA in PDAC microarrays was assessed by immunohistochemistry. RESULTS.: Stimulation of the GABA B R by GABA or baclofen inhibited isoproterenol-induced cAMP signaling below base levels. ERK1/2 activity in response to isoproterenol was blocked by GABA, an effect enhanced by transient overexpression of the GABA B R and abolished by GABA B R knockdown. DNA synthesis and cell migration were stimulated by isoproterenol, responses blocked by GABA and baclofen. Norepinephrine and PKAR IIalpha were overexpressed while GABA was underexpressed in human PDAC tissue arrays. CONCLUSIONS: The data suggest the stimulation of GABA B R signaling as a novel target for the treatment and prevention of pancreatic cancer.

Low concentrations of beta-carotene stimulate the proliferation of human pancreatic duct epithelial cells in a PKA-dependent manner.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is among the most common causes of cancer death. Preclinical and clinical studies on the preventive effects of beta-carotene or other retinoids have used dietary supplements that yielded high systemic concentrations (1-50 microM). While some of the preclinical data suggested cancer preventive effects of these agents, they have disappointed in clinical investigations. MATERIALS AND METHODS: The effects of low concentrations (10 fM-200 nM)of beta-carotene on the proliferation, intracellular cAMP levels, PKA activation status and phosphorylation of EGFR-specific tyrosine kinases and ERK1/2 in immortalized human pancreatic duct epithelial cells was investigated. RESULTS: Our data show significant concentration-dependent and PKA-dependent stimulation of all measured endpoints. Similar responses were achieved with forskolin. Our data indicate that low concentrations of beta-carotene stimulate the proliferation of the putative origin of PDAC, pancreatic duct epithelial cells via cAMP and PKA-dependent transactivation of the EGFR pathway. This could potentially have promoting effects on the development of PDAC.

Nongenomic beta estrogen receptors enhance beta1 adrenergic signaling induced by the nicotine-derived carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in human small airway epithelial cells.

Women are at higher risk for the development of lung adenocarcinoma than men; however, the mechanisms responsible for this are poorly understood. In lung adenocarcinoma cells, the estrogen receptor beta (ERbeta) is the predominating form. We found that 17beta-estradiol enhanced proliferation of the putative cells of origin of lung adenocarcinoma, small airway epithelial cells (HPLD1), in response to the nicotine-derived nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Reverse-phase protein microarrays combined with Western blotting revealed that NNK induced phosphorylation of ERbeta, an effect that involved stimulation of the adrenergic receptors beta1 (beta1AR). In transiently transfected cells, beta1AR coprecipitated with ERbeta, which increased with NNK treatment. ERbeta enhanced NNK-induced cyclic AMP accumulation as well as Galphai-mediated mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 activation. Coexpression of beta1AR and ERbeta activated NNK-mediated ERK1/2 cooperatively. ERbeta gene knockdown, as well as coexpression of the dominant negative Ras and Raf, reduced stimulation of ERK1/2 by NNK. Whereas NNK phosphorylated Akt at Thr(308) and Ser(473), ERbeta had no effect on this activity. Luciferase reporter assays showed that, in response to NNK, ERbeta stimulated transcription of serum responsive element (SRE) but had a very small effect on the activity of estrogen responsive element (ERE). Together, the phosphorylation of ERbeta, the dependence on Galphai proteins, the activation of ERK1/2, and the preferential targeting of SRE over the classic ERE pathway support a role for nongenomic ERbeta in the development of smoking-associated lung cancer. This novel cooperation between beta1AR and ERbeta signaling may contribute to the prominence of lung adenocarcinoma in women.

Pulmonary fibroblasts stimulate the proliferation of cell lines from human lung adenocarcinomas.

Human lung cancer cell lines are widely used to test anticancer drugs. These in-vitro tests, however, preclude the detection of responses to paracrine factors from surrounding stroma. We have cocultured pulmonary fibroblasts CCD-19Lu, from a healthy donor, or HLF-A, from a patient with epidermoid carcinoma of the lung, with two human pulmonary adenocarcinoma cell lines to test the hypothesis that the fibroblasts stimulate the growth of the tumor cells. Both fibroblast cell lines significantly increased the proliferation of the pulmonary adenocarcinoma cell lines in 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide assays, with HLF-A fibroblasts yielding the most pronounced responses. The proliferation of the pulmonary adenocarcinoma cell lines in coculture with fibroblasts was blocked by antibodies against the transforming growth factor-alpha and amphiregulin. In addition, reverse transcription-polymerase chain reaction showed expression of mRNA for amphiregulin and transforming growth factor-alpha in all cell lines, whereas mRNA for the epidermal growth factor was detected only in pulmonary adenocarcinoma cell lines. Western blot analysis revealed that medium containing growth factors released by each fibroblast cell line activated extracellular signal-regulated kinase 1/2 in the both tested pulmonary adenocarcinoma cell lines, but activated Akt kinase only in A549 cells. Assessment of protein levels for cyclin D1 and cyclin E by Western blots demonstrated pronounced increases of both proteins in each pulmonary adenocarcinoma cell line, whereas protein levels for cyclin-dependent kinase inhibitor p21 remained unchanged. Immunocytochemical analysis showed positive immunoreactivity for P-extracellular signal-regulated kinase 1/2, cyclin D1 and cyclin E in pulmonary adenocarcinoma cells cocultured with fibroblasts or exposed to fibroblast-conditioned media. Our data suggest that the growth of pulmonary adenocarcinoma is stimulated by amphiregulin and transforming growth factor-alpha released from pulmonary fibroblasts. This may contribute to the disappointing clinical responses to anticancer drugs, which have shown promise in tests with lung cancer cell lines.

Keratin expression in schwannoma; a study of 115 retroperitoneal and 22 peripheral schwannomas.

Schwannomas have been variably observed to be glial fibrillary acid protein (GFAP) and occasionally keratin positive, with antibodies reacting with multiple keratins (pankeratins, keratin cocktail (CK), but specific keratin polypeptides (K) have not been examined for in schwannoma. Since we observed CK positivity in retroperitoneal schwannomas, we wanted to study a large group of retroperitoneal and peripheral schwannomas with GFAP, CK and Ks to explore the frequency and biologic background of this finding. We immunohistochemically evaluated a large number of retroperitoneal (n=115) and peripheral schwannomas (n=22) for GFAP, 16 individual K and AE1/AE3 keratin cocktail. The great majority (104/115, 90%) of retroperitoneal schwannomas were positive for GFAP, and 72/104 (69%) cases were positive for AE1/AE3, often extensively. Both markers highlighted the cellular Antoni A areas, particularly adjacent to the capsule, myxoid or degenerative areas, and perivascularly. Most cases 87/104 (84%) stained for both AE1/AE3 and GFAP at least focally. No tumors stained for keratins that were GFAP negative. None of the immunostains for individual K showed positivity comparable to that obtained with AE1/AE3 CK. However, 62% were focally positive for high molecular weight K1 and 8/61 (13%) for K7. None of the retroperitoneal schwannomas were positive for other keratins including K2, 4, 5, 8, 9, 10 and K14-20. Peripheral schwannomas showed GFAP-positivity in only three of 22 cases (14%), and all were negative for keratins, both cocktail and individual K. We conclude that crossreactivity of AE1/AE3 with other intermediate filament proteins, such as GFAP, as previously observed in brain and glioma tissue, probably accounts for the extensive keratin-positivity seen in some retroperitoneal schwannomas. However, focal expression of K1 and K7 cannot be ruled out. Keratin-positive schwannomas should not be confused with other keratin-positive tumors, such as sarcomatoid carcinoma, mesothelioma, and synovial sarcoma.