Ilona Linnoila

Rapid Akt activation by nicotine and a tobacco carcinogen modulates the phenotype of normal human airway epithelial cells

Tobacco-related diseases such as lung cancer cause over 4.2 million deaths annually, with approximately 400,000 deaths per year occurring in the US. Genotoxic effects of tobacco components have been described, but effects on signaling pathways in normal cells have not been described. Here, we show activation of the serine/threonine kinase Akt in nonimmortalized human airway epithelial cells in vitro by two components of cigarette smoke, nicotine and the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Activation of Akt by nicotine or NNK occurred within minutes at concentrations achievable by smokers and depended upon alpha(3)-/alpha(4)-containing or alpha(7)-containing nicotinic acetylcholine receptors, respectively. Activated Akt increased phosphorylation of downstream substrates such as GSK-3, p70(S6K), 4EBP-1, and FKHR. Treatment with nicotine or NNK attenuated apoptosis caused by etoposide, ultraviolet irradiation, or hydrogen peroxide and partially induced a transformed phenotype manifest as loss of contact inhibition and loss of dependence on exogenous growth factors or adherence to ECM. In vivo, active Akt was detected in airway epithelial cells and lung tumors from NNK-treated A/J mice, and in human lung cancers derived from smokers. Redundant Akt activation by nicotine and NNK could contribute to tobacco-related carcinogenesis by regulating two processes critical for tumorigenesis, cell growth and apoptosis.

Tobacco Carcinogen-Induced Cellular Transformation Increases Activation of the Phosphatidylinositol 3′-Kinase/Akt Pathway in Vitro and in Vivo

The role of the phosphatidylinositol 3′-kinase (PI3K)/Akt pathway during tobacco carcinogen-induced transformation is unknown. To address this question, we evaluated this pathway in isogenic immortalized or tumorigenic human bronchial epithelial cells in vitro, as well as in progressive murine lung lesions induced by a tobacco-specific carcinogen, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Compared with immortalized cells, tumorigenic cells had greater activation of the PI3K/Akt pathway, enhanced survival, and increased apoptosis in response to inhibition of the pathway. In vivo, increased activation of Akt and mammalian target of rapamycin was observed with increased phenotypic progression. Collectively, these results support the hypothesis that maintenance of Akt activity is necessary for survival of preneoplastic as well as transformed lung epithelial cells and suggest that inhibition of the PI3K/Akt pathway might be a useful approach to arrest lung tumorigenesis.

Identification of an integrated SV40 T/t-antigen cancer signature in aggressive human breast, prostate, and lung carcinomas with poor prognosis

Understanding the genetic architecture of cancer pathways that distinguishes subsets of human cancer is critical to developing new therapies that better target tumors based on their molecular expression profiles. In this study, we identify an integrated gene signature from multiple transgenic models of epithelial cancers intrinsic to the functions of the Simian virus 40 T/t-antigens that is associated with the biological behavior and prognosis for several human epithelial tumors. This genetic signature, composed primarily of genes regulating cell replication, proliferation, DNA repair, and apoptosis, is not a general cancer signature. Rather, it is uniquely activated primarily in tumors with aberrant p53, Rb, or BRCA1 expression but not in tumors initiated through the overexpression of myc, ras, her2/neu, or polyoma middle T oncogenes. Importantly, human breast, lung, and prostate tumors expressing this set of genes represent subsets of tumors with the most aggressive phenotype and with poor prognosis. The T/t-antigen signature is highly predictive of human breast cancer prognosis. Because this class of epithelial tumors is generally intractable to currently existing standard therapies, this genetic signature identifies potential targets for novel therapies directed against these lethal forms of cancer. Because these genetic targets have been discovered using mammary, prostate, and lung T/t-antigen mouse cancer models, these models are rationale candidates for use in preclinical testing of therapies focused on these biologically important targets.

Immunohistochemical demonstration of VIP, [Met5]- and [Leu5]-enkephalin immunoreactive nerve fibres in the human prostate and seminal vesicles

SummaryThe distribution of nerves containing immunoreactivity for the VIP and enkephalins has been demonstrated in the human prostate and seminal vesicles using the immunoperoxidase bridge. VIP-containing nerves were detected in both organs studied mainly in association with the epithelium, while nerves containing ELI seemed to be related to smooth muscle. Compared with the distribution of adrenergic and cholinergic nerves in the prostate marked differences in the density of the innervation were detected. The possible nature of these peptide-containing nerves is discussed.

Neuroendocrine-specific protein (NSP)-reticulons as independent markers for non-small cell lung cancer with neuroendocrine differentiation. An in vitro histochemical study

Abstract Neuroendocrine-specific protein (NSP)-reticulons have recently been discovered and were shown to exhibit a restricted, neuroendocrine/neural-specific expression pattern. These protein aggregates are anchored to the membranes of the endoplasmic reticulum and occur in small cell lung cancer (SCLC), but not in typical non-SCLC. In the current study we have examined the occurrence of NSP-reticulons in non-SCLC cell lines known to express neuroendocrine features (non-SCLC-NE). NSP-reticulon expression was observed in all three non-SCLC-NE cell lines studied, albeit with variable intensity and in varying numbers of cells. Western blot analysis confirmed the presence of NSP-reticulon expression in these non-SCLC-NE cell lines, and showed that they were predominantly of the NSP-A type. When compared to conventional neuroendocrine markers, NSP-reticulons revealed a distinct staining profile, showing only partial overlap with the other markers. The non-SCLC-NE cell lines combined these neuroendocrine characteristics with some features of non-SCLC. We conclude that NSP-reticulon expression is restricted to lung carcinoma cells with a neuroendocrine phenotype and predict that these constituents may become clinically relevant markers for the detection of neuroendocrine differentiation in solid tumours.

Monoclonal antibodies for the in vitro detection of small cell lung cancer metastases in human bone marrow

Three rat monoclonal antibodies were selected for the immunodetection of small cell lung cancer metastases in bone marrow and other hematologic samples. By membrane immunofluorescence, they define three distinct surface antigens here termed lung cancer-associated antigens or LCAs. The latter are widely expressed on small cell lung cancer and non-small cell lung cancer cells/cell lines, but not detectable on a variety of normal and transformed bone marrow, blood and lymphoid cells. Anti-LCA1 (IgM) is similar to the many anti-lacto-N-fucopentaose III IgM antibodies rasied against human tumors. In contrast, anti-LCA2 (IgG2b) and anti-LCA3 (IgG2a) define surface proteins of 29, 32, 41 and 98 kilodaltons, respectively, that have not been reported earlier. These three reagents have immunodiagnostic potential, since in combination they label all 49 lung cancer cell lines tested. Their ability to detect lung cancer metastases in patients bone marrow samples is documented in an accompanying paper.

Coupling of amines to and cross-linking of endogenous cytosol or membrane proteins by hepatic transglutaminase

Abstract Endogenous protein acceptors for rabbit transglutaminase were demonstrated in the cytosol fraction of liver homogenates by the coupling of labeled compounds containing primary amino groups, such as putrescine, and by the apparent cross-linking of proteins, as indicated by a low mobility band observed in gel electrophoresis in 0.1% sodium dodecyl sulfate. When Ca 2+ was omitted or when iodoacetamide was added to either of these reactions, no coupling or cross-linking occurred. Cross-linking of proteins of isolated rabbit liver plasma membranes could be demonstrated in the presence of purified rabbit liver transglutaminase, Ca 2+ , and a reducing agent. A marked diminution of at least two peaks corresponding to proteins of high molecular weight (> 150,000 daltons) was observed in densitometric scans of gels of proteins from membranes or cytosol cross-linked by the enzyme. It is proposed that these proteins participated in the crosslinking reaction. Since cross-linking was inhibited only weakly by relatively high concentrations of various amines, it is probable that a physiological role of hepatic transglutaminase is the catalysis of ϵ(γ-glutamyl) lysine bonds between cellular proteins, a process that might be controlled by the amount of intracellular Ca 2+ available to the enzyme.

Abstract 344: Subpopulations of lung neuroendocrine cells mirror neuronal development.

Pulmonary neuroendocrine cells (PNECs) which comprise less than 1% of airway epithelium were recently shown to be the cell of origin for small cell lung cancer (SCLC). SCLC is the most aggressive form of human lung cancers and characterized by early dissemination or metastases and neuroendocrine features. We used immunohistochemistry (IHC) and animal models in order to delineate the cellular hierarchy of PNEC potentially leading to cancer. PNECs were first identified in the early lung development (E12.5) as positive for the proneural basic helix-loop-helix transcription factor Achaete-scute homolog-1 (Ascl1) which is pivotal for PNEC differentiation and highly expressed in SCLC. Later in the development (E16.5) and in adulthood, PNECs coexpressed Ascl1 and calcitonin gene-related peptide (CGRP), a primary neuropeptide in mouse lung. Interestingly, we showed that doublecortin (DCX), a marker of immature migrating neurons, is transiently expressed in PNECs during the early development. Later in the development (E16.5), we observed a subpopulation expressing the neural-specific β3-tubulin (TUBB3), a neuronal marker. We also observed that neural cell adhesion molecule (NCAM), a marker of SCLC, is transiently expressed in a low number of PNECs in the late embryonic period. In parallel, using the transgenic CC10-hASH1 mouse model, where Ascl1 is overexpressed in non-neuroendocrine airway epithelial cells, we determined that it is not sufficient to induce neuroendocrine differentiation or expression of DCX, TUBB3 and NCAM. We conclude that Ascl1, DCX and TUBB3 are consecutively expressed in PNECs during development, similarly to the pattern observed during neuronal development. However, Ascl1 expression seems to be maintained even in the mature PNECs unlike in the mature neurons where it is turned off. On the other hand, NCAM expression in PNECs is transient and maybe reactivated during the instance of SCLC. To establish the relationships between the different PNEC populations and Ascl1 expression, we are currently evaluating Cre-dependent conditional Ascl1 deletion in mouse lung. Further work will be necessary to address the functional potential of the PNEC subpopulations in tumorigenesis. Citation Format: Florent Suau, Ilona Linnoila, Xu Naizhen. Subpopulations of lung neuroendocrine cells mirror neuronal development. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 344. doi:10.1158/1538-7445.AM2013-344

Abstract IA10: Pathology of lung neuroendocrine tumors: In search of evidence for classification

Neuroendocrine (NE) tumors comprise approximately 25% of all tumors in human lung. As a group they continue to provide challenges at biological, molecular and pathological level for uniform classification. The prototype of NE tumors is the highly aggressive small cell lung cancer (SCLC) that accounts 15% of human lung cancers. For years lung cancers were clinically classified mainly into SCLCs and non-SCLCs (NSCLCs) while it has become evident that lung NE tumors actually form a complex spectrum from SCLC to carcinoid with shifting boundaries. The current WHO classification recognizes SCLC, its variant form combined SCLC, typical and atypical carcinoids as well as large cell NE carcinoma (LCNEC) and its variant form combined large cell carcinoma, the last two cancers under the main NSCLC category of large cell carcinoma. With the exception of the LCNEC, it is expected that the diagnoses will be rendered by routine hematoxylin-eosin stain according the established criteria. Benign entities include diffuse idiopathic pulmonary NE cell hyperplasia which is described in the chapter of preinvasive lesions. Over time there have been several efforts to place lung NE tumors into two to four-tier categories according to their malignant potential (high versus low grade) or degree of differentiation often leaving behind confusing terminology that is later misused. Unlike surgically resected NSCLCs, most NE tumors are frequently diagnosed from biopsies which remains a seldom recognized fact that may create its own biases. The diagnosis that experts most often agree with is SCLC, while distinctions between atypical carcinoid and LCNEC that are in the middle of the spectrum are often disputed. Many molecular studies have also “correctly” classified the endpoints of the NE spectrum while grouping in the middle of it has produced varied results. Moreover, several studies have suggested that NSCLCs with NE features (NSCLC-NEs) is a distinct carcinoma type with worse prognosis than ordinary non-NE NSCLC, while others disagree. Biologically this category is very interesting as NSCLCs and SCLCs are ultimately thought to be derived from the same cell. The regulation of NE differentiation is poorly understood. The proneural achaete-scute homolog 1 (ASCL1), a lineage-dependent oncogene may be a key factor for NE differentiation. On the other hand, it was recently shown that the deletion of the tumor suppressor genes Rb1 and p53 specifically in NE cells, a subpopulation of cells in normal airways, or in alveolar non-NE type2 pneumocytes of mice resulted in SCLCs closely mimicking human disease. These results provide experimental evidence for the close relationship of NE and non-NE epithelial cells in carcinogenesis.

Abstract 1308: Achaete-scute homolog 1 (Ascl1) lineage in the lung gives rise to multiple cell types

Lung contains many highly specialized cells. Type I pneumocytes are responsible for gas exchange, while type II pneumocytes secrete surfactant which prevents alveoli from collapsing. Airways are lined by non-ciliated secretory (Clara) and ciliated cells that eliminate excess mucus and harmful particles, and rare pulmonary neuroendocrine cells (PNECs) which regulate breathing and contribute to carcinogenesis. PNECs have unknown histogenesis but differentiate prior to the other cells. The neural transcription factor achaete-scute homolog 1 (Ascl1) is critical for PNEC development and is associated with stem cell marker expression in normal and neoplastic lung. Hence, we used in vivo lineage and fate tracing strategy to investigate Ascl1-defined cells (ASDCs) during lung development and injury repair. R26R-stop-lacZ (Rosa) reporter mice were crossed with Ascl1-Cre and Ascl1-CreERTM mice, in which the Ascl1 promoter drives the expression of Cre or inducible Cre recombinase, respectively (Battiste et al., Development (2007) 134:285-93). Following recombination, ASDCs and their descendants will be permanently labeled and were further characterized by immunohistochemistry using cellular differentiation markers. Labeled cells in Ascl1-Cre/Rosa mice gave rise not only to PNECs but also to neural, Clara, ciliated, alveolar type I and type II cells. Interestingly, tamoxifen (TM) administration to Ascl1-CreERTM/Rosa dams at embryonic day E9.5 labeled both airway and alveolar cells, while TM injected at E11.5 only labeled airway cells. Moreover, adult Ascl1-CreERTM/Rosa mice were administered TM followed by naphthalene (Nap), which kills Clara cells. It is notable that in addition to PNECs which are resistant to Nap, regenerating Clara cells were now labeled during the injury repair. We conclude that ASDCs are not limited to giving rise to neural cells or PNECs during pulmonary development, but they also contribute to a range of non-neuroendocrine cells such as Clara and ciliated cells as well as alveolar pneumocytes. Further, the lineage participates in injury repair of the airway lining in mature lung. The current Ascl1 lineage study provides a basis for recent studies on mouse small cell lung cancer (Sutherland et al., Cancer Cell (2011) 19:754-64) which suggested that both PNECs and non-neuroendocrine cells such as alveolar type II pneumocytes may serve as progenitors for this very virulent lung cancer type. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1308. doi:1538-7445.AM2012-1308