Hiroaki Shimoyamada

Down-Regulation of DUSP6 Expression in Lung Cancer —Its Mechanism and Potential Role in Carcinogenesis

Our preliminary studies revealed that oncogenic KRAS (KRAS/V12) dramatically suppressed the growth of immortalized airway epithelial cells (NHBE-T, with viral antigen-inactivated p53 and RB proteins). This process appeared to be a novel event, different from the so-called premature senescence that is induced by either p53 or RB, suggesting the existence of a novel tumor suppressor that functions downstream of oncogenic KRAS. After a comprehensive search for genes whose expression levels were modulated by KRAS/V12, we focused on DUSP6, a pivotal negative feedback regulator of the RAS-ERK pathway. A dominant-negative DUSP6 mutant, however, failed to rescue KRAS/V12-induced growth suppression, but conferred a stronger anchorage-independent growth activity to the surviving subpopulation of cells generated from KRAS/V12-transduced NHBE-T. DUSP6 expression levels were found to be weaker in most lung cancer cell lines than in NHBE-T, and DUSP6 restoration suppressed cellular growth. In primary lung cancers, DUSP6 expression levels decreased as both growth activity and histological grade of the tumor increased. Loss of heterozygosity of the DUSP6 locus was found in 17.7% of cases and was associated with reduced expression levels. These results suggest that DUSP6 is a growth suppressor whose inactivation could promote the progression of lung cancer. We have here identified an important factor involved in carcinogenesis through a comprehensive search for downstream targets of oncogenic KRAS.

Cancer stem cell: Implications in cancer biology and therapy with special reference to lung cancer

The cancer stem cell (CSC) theory is currently central to the field of cancer research, because it is not only a matter of academic interest but also crucial in cancer therapy. CSCs share a variety of biological properties with normal somatic stem cells in terms of self-renewal, the propagation of differentiated progeny, the expression of specific cell markers and stem cell genes, and the utilization of common signaling pathways and the stem cell niche. However, CSCs differ from normal stem cells in their tumorigenic activity. Thus, CSCs are also termed cancer initiating cells. In this paper, we briefly review hitherto described study results and refer to some excellent review articles to understand the basic properties of CSCs. In addition, we focus upon CSCs of lung cancers, since lung cancer is still increasing in incidence worldwide and remains the leading cause of cancer deaths. Understanding the properties of, and exploring cell markers and signaling pathways specific to, CSCs of lung cancers, will lead to progress in therapy, intervention, and improvement of the prognosis of patients with lung cancer. In the near future, the evaluation of CSCs may be a routine part of practical diagnostic pathology.

Early Growth Response-1 Induces and Enhances Vascular Endothelial Growth Factor-A Expression in Lung Cancer Cells

Vascular endothelial growth factor-A (VEGF-A) is crucial for angiogenesis, vascular permeability, and metastasis during tumor development. We demonstrate here that early growth response-1 (EGR-1), which is induced by the extracellular signal-regulated kinase (ERK) pathway activation, activates VEGF-A in lung cancer cells. Increased EGR-1 expression was found in adenocarcinoma cells carrying mutant K-RAS or EGFR genes. Hypoxic culture, siRNA experiment, luciferase assays, chromatin immunoprecipitation, electrophoretic mobility shift assays, and quantitative RT-PCR using EGR-1-inducible lung cancer cells demonstrated that EGR-1 binds to the proximal region of the VEGF-A promoter, activates VEGF-A expression, and enhances hypoxia inducible factor 1alpha (HIF-1alpha)-mediated VEGF-A expression. The EGR-1 modulator, NAB-2, was rapidly induced by increased levels of EGR-1. Pathology samples of human lung adenocarcinomas revealed correlations between EGR-1/HIF-1alpha and VEGF-A expressions and relative elevation of EGR-1 and VEGF-A expression in mutant K-RAS- or EGFR-carrying adenocarcinomas. Both EGR-1 and VEGF-A expression increased as tumors dedifferentiated, whereas HIF-1alpha expression did not. Although weak correlation was found between EGR-1 and NAB-2 expressions on the whole, NAB-2 expression decreased as tumors dedifferentiated, and inhibition of DNA methyltransferase/histone deacetylase increased NAB-2 expression in lung cancer cells despite no epigenetic alteration in the NAB-2 promoter. These findings suggest that EGR-1 plays important roles on VEGF-A expression in lung cancer cells, and epigenetic silencing of transactivator(s) associated with NAB-2 expression might also contribute to upregulate VEGF-A expression.

Small Cell Lung Cancer: Significance of RB Alterations and TTF-1 Expression in its Carcinogenesis, Phenotype, and Biology

Small cell lung cancer (SCLC) exhibits highly aggressive behavior and has a poor prognosis. While numerous investigations have been carried out, the exact mechanism of its carcinogenesis and aggressiveness is still unclear. SCLC is categorized as a neuroendocrine neoplasia and has a genetic profile characterized by universal alterations of the RB and TP53 genes. Epidemiological studies indicate the majority of SCLCs to be caused by smoking and the TP53 mutational pattern to be consistent with that evoked by smoke carcinogens; however, there is no direct evidence that such carcinogens induce alterations to RB in SCLC. While the importance of these alterations in the carcinogenesis of SCLC is strongly suggested, the exact molecular mechanism has been only little elucidated. SCLC cells almost always express mammalian achaete-scute homolog-1 (MASH1) and thyroid transcription factor-1 (TTF-1). MASH1 plays a critical role in neuroendocrine differentiation. TTF-1 is a characteristic marker of distal airway cells and pulmonary adenocarcinomas, but is also expressed in extrapulmonary neuroendocrine cancers. Thus, TTF-1 may well play a significant role in the development of neuroendocrine cancers. Recent studies indicate that the airway stem cell is committed to the neuroendocrine lineage through MASH1 and Notch signaling and that only RB-deleted neuroendocrine cells selectively proliferate in response to E2F3, eventually undergoing transformation to neuroendocrine cancer cells, probably in concert with TP53 gene aberrations. Thus, alterations of both the RB and TP53 genes are central to the carcinogenesis of SCLC, while many other factors including MASH1 and TTF-1 contribute to the development and biological behavior of SCLC.

POU domain transcription factor BRN2 is crucial for expression of ASCL1, ND1 and neuroendocrine marker molecules and cell growth in small cell lung cancer

BRN2 is a developmental neural cell‐specific POU domain transcription factor and is crucial for cell lineage determination. We investigated the importance of BRN2 in the expression of the lineage‐specific transcription factors (achaete‐scute homolog‐like 1 (ASCL1) and NeuroD1 (ND1)) and neural/neuroendocrine marker molecules (neural cell adhesion molecule 1 (NCAM1), synaptophysin (SYP) and chromogranin A (CHGA)) in small cell lung cancer (SCLC) using cultured lung cancer cells. All examined SCLC cell lines expressed BRN2, as well as ASCL1, ND1, NCAM1, SYP and CHGA. The expression levels of ASCL1, ND1, NCAM1, SYP and CHGA considerably decreased when BRN2 was knocked down in SCLC cells, and the addition of a BRN2 transgene into non‐SCLC (NSCLC) cells induced the expression of ASCL1, ND1, NCAM1, SYP and CHGA. However, the BRN2 gene was not activated by the forced expression of ASCL1 or ND1 in NSCLC cells. The knockdown of BRN2 caused significant growth retardation with decrease of S to G2 phase population and mitotic cell rates and unaltered Ki‐67‐labeled or apoptotic cell rates in SCLC cells, indicating increase of G1 phase population. These findings suggest that BRN2 is a higher level regulator than ASCL1 and ND1 and BRN2 might be involved in aggressiveness of SCLC.

Molecular and Genetic Pathogenesis of Lung Cancer: Differences Between Small-Cell and Non-Small-Cell Carcinomas

Lung cancer is classified into small-cell carcinoma (SCLC) and non-small-cell carcinoma (NSCLC). The pro- file of molecular and genetic alterations considerably differs between SCLC and NSCLC, as well as among the subtypes of NSCLC. Tp53 is inactivated in nearly 50% of NSCLC, while its mutations with functional inactivation are greatly prevalent in SCLC (70-100%). Rb gene alterations and protein loss are found in virtually all SCLC, but rarely in NSCLC. Instead, the Rb function is abrogated due to dysfunction of the upstream regulators of the Rb pathway. Tp53 alterations are later events in adenocarcinoma, while they occur early in squamous cell carcinoma carcinogenesis. Recent studies demonstrated activating mutations of the epidermal growth factor receptor (EGFR) gene play a significantly important role in adenocarcinoma carcinogenesis. Activation of PIK3 catalytic alpha gene was recently found in nearly half of squamous cell carcinoma cases. Smoking is associated strongly with alterations of Tp53, K-ras, and PIK3 catalytic alpha, but weakly with EGFR gene mutations. Taken together silencing alterations of both the Rb and Tp53 genes are most likely to be important and early events in the development of SCLC, whereas alterations of the EGFR signaling pathway play significant and important roles in NSCLC carcinogenesis. The biological behavior and phenotype of the respective types of lung cancer would be attributable to these molecular and genetic alterations, but also reflect the difference in the ability of their precursor cells. Identifying the airway stem cell(s) and elucidating the molecular mechanism of its mainte- nance and activation are required.

Morphometric profiling of lung cancers-its association with clinicopathologic, biologic, and molecular genetic features.

The traditional histologic classification of lung cancer is not satisfactory to describe the morphologic characteristics of individual tumors, because it does not fully cover cytologic features. This paper describes a novel typing system using morphometric profiling that covers a variety of morphologic features including histologic architecture, cell type, cytoplasmic color and internal structure, nuclear outline, chromatin pattern, nucleoli count and remarkableness, and average and deviation of nuclear size and circularity. In all, 201 cases of lung tumors (whose sizes are <20 mm) were examined. Results of a hierarchical clustering analysis were used to draw a dendrogram. We here tentatively focused on 8 morphometric clusters and analyzed their potential association with a variety of clinicopathologic and molecular genetic features. Significant differences in postoperative recurrent risk, growth activity, oncogenic mutation (EGFR or KRAS), impairments of tumor suppressors (p53 and p16), sex predisposition, and smoking status were found among the 8 clusters. The system has the potential to improve histopathologic diagnosis and our understanding of carcinogenesis in the lung.

Differences of molecular expression mechanisms among neural cell adhesion molecule 1, synaptophysin, and chromogranin A in lung cancer cells

Neural cell adhesion molecule 1 (NCAM1), synaptophysin (SYPT), and chromogranin A (CGA) are immunohistochemical markers for diagnosing lung neuroendocrine tumors (LNETs). However, the precise expression mechanisms have not been studied in enough detail. The purpose of the present study is to define the molecular mechanisms of NCAM1, SYPT, and CGA gene expressions, using cultivated lung cancer cells and focusing upon NeuroD1 (ND1), achaete‐scute homolog‐like 1 (ASCL1), and known transcription factors, repressor element 1 (RE1)‐silencing transcription factor (REST) and c‐AMP responsive element‐binding protein (CREB). Promoter assays, chromatin immunoprecipitation, and transfection experiments revealed that ND1 activated NCAM1, that ASCL1 weakly upregulated SYPT expression, and that CGA expression was not regulated by ND1 or ASCL1. REST expression was restricted in non‐small cell lung cancer (NSCLC) cells, and knockdown of REST could cause as much SYPT expression as in SCLC cells and weak CGA expression in NSCLC cells. However, CGA gene upregulation via CREB activation was not found in REST‐lacking NSCLC cells, indicating the requirement of some additional mechanism for sufficient expression. These results suggest that NCAM1, SYPT and CGA expressions are differently regulated by neuroendocrine phenotype‐specific transcription factors and provide a reason why NCAM1 and SYPT are frequently expressed in LNETs, irrespective of malignancy grade.

Neural lineage-specific homeoprotein BRN2 is directly involved in TTF1 expression in small-cell lung cancer.

Thyroid transcription factor 1 (TTF1) plays crucial roles in thyroid, lung, and developing brain morphogenesis. Because TTF1-expressing neoplasms are generated from organs and tissues that normally express TTF1, such as the thyroid follicular epithelium and peripheral lung airway epithelium, TTF1 is widely used as a cell lineage-specific and diagnostic marker for thyroid carcinomas and for lung adenocarcinomas with terminal respiratory unit (TRU) differentiation. However, among lung neuroendocrine tumors, small-cell carcinomas (small-cell lung cancers (SCLCs)), most of which are generated from the central airway, also frequently express TTF1 at high levels. To clarify how SCLCs express TTF1, we investigated the molecular mechanisms of its expression using cultivated lung cancer cells and focusing upon neural cell-specific transcription factors. Both SCLC cells and lung adenocarcinoma cells predominantly expressed isoform 2 of TTF1, and TTF1 promoter assays in SCLC cells revealed that the crucial region for activation of the promoter, which is adjacent to the transcription start site of TTF1 isoform 2, has potent FOX-, LHX-, and BRN2-binding sites. Transfection experiments using expression vectors for FOXA1, FOXA2, LHX2, LHX6, and BRN2 showed that BRN2 substantially upregulated TTF1 expression, whereas FOXA1/2 weakly upregulated TTF1 expression. BRN2 and FOXA1/2 binding to the TTF1 promoter was confirmed through chromatin immunoprecipitation experiments, and TTF1 expression in SCLC cells was considerably downregulated after BRN2 knockdown. Furthermore, the TTF1 promoter in SCLC cells was scarcely methylated, and immunohistochemical examinations using a series of primary lung tumors indicated that TTF1 and BRN2 were coexpressed only in SCLC cells. These findings suggest that TTF1 expression in SCLC is a cell lineage-specific phenomenon that involves the developing neural cell-specific homeoprotein BRN2.

Calcium oxalate crystal deposition in a patient with Aspergilloma due to Aspergillus niger

Discrimination between aspergilloma and chronic necrotizing pulmonary aspergillosis (CNPA) based on radiological findings can difficult. We describe a patient with aspergilloma and organizing pneumonia that was possibly caused by Aspergillus niger infection and radiologically mimicked CNPA. A postmortem histological analysis showed diffuse alveolar damage that had originated in peri-cavitary lung parenchyma. Calcium oxalate or Aspergillus niger was located inside, but not outside the cavity in the right upper lobe. Calcium oxalate or other unknown hyphal bioactive components might provoke severe lung inflammation not only adjacent to the cavity, but also on the contralateral side.