Xu Naizhen

Achaete-scute homologue-1 (ASH1) stimulates migration of lung cancer cells through Cdk5/p35 pathway

Cyclin-dependent kinase 5 (Cdk5) activity is important for the migration and invasion of cancer cells. Our results indicate that in the lung one of the mechanisms that hASH1 regulates—migration—takes place through induction of Cdk5 activity. Our data suggest that Cdk5 and its activator p35 promote lung cancer cell migration through hASH1-mediated signaling.

Achaete-scute homolog-1 linked to remodeling and preneoplasia of pulmonary epithelium.

The basic helix–loop–helix protein achaete-scute homolog-1 (ASH1) is involved in lung neuroendocrine (NE) differentiation and tumor promotion in SV40 transgenic mice. Constitutive expression of human ASH-1 (hASH1) in mouse lung results in hyperplasia and remodeling that mimics bronchiolization of alveoli (BOA), a potentially premalignant lesion of human lung carcinomas. We now show that this is due to sustained cellular proliferation in terminal bronchioles and resistance to apoptosis. Throughout the airway epithelium the expression of anti-apoptotic Bcl-2 and c-Myb was increased and Akt/mTOR pathway activated. Moreover, the expression of matrix metalloproteases (MMPs) including MMP7 was specifically enhanced at the bronchiolo-alveolar duct junction and BOA suggesting that MMPs play a key role in this microenvironment during remodeling. We also detected MMP7 in 70% of human BOA lesions. Knockdown of hASH1 gene in human lung cancer cells in vitro suppressed growth by increasing apoptosis. We also show that forced expression of hASH1 in immortalized human bronchial epithelial cells decreases apoptosis. We conclude that the impact of hASH1 is not limited to cells with NE phenotype. Rather, constitutive expression of hASH1 in lung epithelium promotes remodeling through multiple pathways that are commonly activated during lung carcinogenesis. The collective results suggest a novel model of BOA formation via hASH1-induced suppression of the apoptotic pathway. Our study yields a promising new preclinical tool for chemoprevention of peripheral lung carcinomas.

Mouse lung neuroendocrine carcinomas: Distinct morphologies, same transcription factors

Constitutive expression of human achaete-scute homolog-1 (hASH-1) in combination with simian virus large T antigen under the Clara cell 10-kDa secretory protein (CC10) promoter results in adenocarcinomas with focal neuroendocrine (NE) differentiation. Mice carrying conditional alleles for both Rb-1 and p53 in lung epithelial cells develop aggressive lung tumors with similarities to human small cell lung cancers, including high level expression of ASH-1, NE markers, and extrapulmonary metastases. Tumors in both models originate from bronchiolar epithelium, reveal a range of premalignant changes, express thyroid transcription factor-1 (TTF-1), a marker of peripheral airway cell lineage, and display varying degrees of bidirectional epithelial/NE differentiation.

Secretoglobin 3A2/uteroglobin-related protein 1 is a novel marker for pulmonary carcinoma in mice and humans

Secretoglobin (SCGB) 3A2, also called uteroglobin-related protein (UGRP) 1, is a downstream target for a homeodomain transcription factor NKX2-1, which is critical for the development of lung, thyroid and ventral forebrain. Both SCGB3A2 and NKX2-1 are expressed in airway epithelial cells and the latter also in alveolar Type II cells. NKX2-1 has been used clinically for diagnosis of human pulmonary tumors. Recently, the expression of SCGB3A2 was reported in human carcinomas, suggesting the use of this protein as a tumor marker. In this study, 28 lung tumors from aging B6;129 mice and nine lung adenocarcinomas from CC10TAg transgenic mice that express SV40 large T antigen under the mouse Scgb1a1 (CC10) gene promoter, were subjected to histopathological and immunohistochemical analyses for the expression of NKX2-1 and SCGB3A2. NKX2-1 was expressed in all types of tumors albeit more focally in carcinomas. In contrast, SCGB3A2 normally expressed in Clara cells, was negative in Type II cell hyperplasias and adenomas. However, it was expressed in alveolar Type II cell carcinomas and Clara cell adenocarcinomas. In these carcinomas, SCGB3A2 expression was observed in the portion of the tumor where NKX2-1 expression was reduced or almost abolished. As a comparison, the expression of SCGB3A2 and NKX2-1 from 23 human non-small cell lung carcinoma specimens was also examined. The results demonstrate that SCGB3A2 is a useful marker for diagnosis of pulmonary tumors both in mice and humans.

Acute damage by naphthalene triggers expression of the neuroendocrine marker PGP9.5 in airway epithelial cells

Protein Gene Product 9.5 (PGP9.5) is highly expressed in nervous tissue. Recently PGP9.5 expression has been found to be upregulated in the pulmonary epithelium of smokers and in non-small cell lung cancer, suggesting that it also plays a role in carcinogen-inflicted lung epithelial injury and carcinogenesis. We investigated the expression of PGP9.5 in mice in response to two prominent carcinogens found in tobacco smoke: Naphthalene and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). By immunostaining, we found that PGP9.5 protein was highly expressed throughout the airway epithelium in the days immediately following a single injection of naphthalene. In contrast, PGP9.5 was exclusively confined to neurons and neuroendocrine cells in the control and NNK-exposed lungs. Furthermore, we investigated the expression of PGP9.5 mRNA in the lungs by quantitative RT-PCR (qPCR). PGP9.5 mRNA expression was highly upregulated in the days immediately following naphthalene injection and gradually returning to that of control mice 5 days after naphthalene injection. In contrast, exposure to NNK did not result in a significant increase in PGP9.5 mRNA 10 weeks after exposure. No increased expression of two other neuroendocrine markers was found in the non-neuroendocrine epithelial cells after naphthalene exposure. In contrast, immunostaining for the cell cycle regulator p27(Kip1), which has previously been associated with PGP9.5 in lung cancer cells, revealed transient downregulation of p27(Kip1) in naphthalene exposed airways compared to controls, indicating that the rise in PGP9.5 in the airway epithelium is related to downregulation of p27(Kip1). This study is the first to specifically identify the carcinogen naphthalene as an inducer of PGP9.5 expression in non-neuroendocrine epithelium after acute lung injury and further strengthens the accumulating evidence of PGP9.5 as a central player in lung epithelial damage and early carcinogenesis.

Co-expression of Achaete-Scute Homologue-1 and Calcitonin Gene-Related Peptide during NNK-Induced Pulmonary Neuroendocrine Hyperplasia and Carcinogenesis in Hamsters

Achaete-scute homologue-1 or ASCL1 (MASH1, hASH1) plays roles in neural development and pulmonary neuroendocrine (NE) differentiation, and it is expressed in certain lung cancers. This study was aimed to assess whether and/or how ASCL1 plays a role in 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced pulmonary NE hyperplasia and carcinogenesis in hamsters. Hamsters were injected 3 times weekly with either NNK or solvent alone (control) for treatment periods of 6 and 24 weeks, both without and with 6-week recovery. Immunohistochemical analysis was carried out to examine the expressions of ASCL1, CGRP (calcitonin gene-related peptide), secretoglobin SCGB1A1 (club [Clara] cell specific 10 kD protein, CC10, CCSP), synaptophysin (SYP), and PCNA (proliferating cell nuclear antigen). The number of ASCL1-expressing NE foci per airway increased from 0.8 in controls to 1.6 and 2.0 during NNK exposure for 6 and 24 weeks, respectively, and the number of cells per foci doubled after NNK exposure. Most ASCL1-expressing cells in NEBs (neuroepithelial bodies) were also CGRP immunoreactive; NNK enhanced this co-expression with CGRP, a NE marker with known proliferation-promoting properties. NNK also increased PCNA expression within NE foci. NNK-induced tumors showed no immunoreactivity for NE markers. This study confirms ASCL1 as an excellent marker for pulmonary NE cells and demonstrates CGRP co-expression in ASCL1-positive NEB cells participating in NNK-induced NE hyperplasia.

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