Lung cancer: Premalignant biology and medical prevention.

Lung cancer (both adenocarcinoma and squamous cell) progress through a series of pre-malignant histologic changes before the development of invasive disease. Each of these carcinogenic cascades is defined by genetic and epigenetic alterations in pulmonary epithelial cells. Additionally, alterations in the immune response, progenitor cell function, mutational burden, and microenvironmental mediated survival of mutated clones contribute to the risk of pre-malignant lesions progressing to cancer. Medical preventions studies have been completed and current and future trials are informed by the improved understanding of pre-malignancy. This will lead to precision chemoprevention trials based on lesional biology and histologic characteristics.

Prostacyclin reverses the cigarette smoke-induced decrease in pulmonary Frizzled 9 expression through miR-31.

Half of lung cancers are diagnosed in former smokers, leading to a significant treatment burden in this population. Chemoprevention in former smokers using the prostacyclin analogue iloprost reduces endobronchial dysplasia, a premalignant lung lesion. Iloprost requires the presence of the WNT receptor Frizzled 9 (Fzd9) for inhibition of transformed growth in vitro. To investigate the relationship between iloprost, cigarette smoke, and Fzd9 expression, we used human samples, mouse models, and in vitro studies. Fzd9 expression was low in human lung tumors and in progressive dysplasias. In mouse models and in vitro studies, tobacco smoke carcinogens reduced expression of Fzd9 while prostacyclin maintained or increased expression. Expression of miR-31 repressed Fzd9 expression, which was abrogated by prostacyclin. We propose a model where cigarette smoke exposure increases miR-31 expression, which leads to decreased Fzd9 expression and prevents response to iloprost. When smoke is removed miR-31 is reduced, prostacyclin can increase Fzd9 expression, and progression of dysplasia is inhibited. Fzd9 and miR-31 are candidate biomarkers for precision application of iloprost and monitoring of treatment progress. As we continue to investigate the mechanisms of prostacyclin chemoprevention and identify biomarkers for its use, we will facilitate clinical trials and speed implementation of this valuable prevention approach.

Mer or Axl receptor tyrosine kinase inhibition promotes apoptosis, blocks growth and enhances chemosensitivity of human non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is a prevalent and devastating disease that claims more lives than breast, prostate, colon and pancreatic cancers combined. Current research suggests that standard chemotherapy regimens have been optimized to maximal efficiency. Promising new treatment strategies involve novel agents targeting molecular aberrations present in subsets of NSCLC. We evaluated 88 human NSCLC tumors of diverse histology and identified Mer and Axl as receptor tyrosine kinases (RTKs) overexpressed in 69% and 93%, respectively, of tumors relative to surrounding normal lung tissue. Mer and Axl were also frequently overexpressed and activated in NSCLC cell lines. Ligand-dependent Mer or Axl activation stimulated MAPK, AKT and FAK signaling pathways indicating roles for these RTKs in multiple oncogenic processes. In addition, we identified a novel pro-survival pathway-involving AKT, CREB, Bcl-xL, survivin, and Bcl-2-downstream of Mer, which is differentially modulated by Axl signaling. We demonstrated that short hairpin RNA (shRNA) knockdown of Mer or Axl significantly reduced NSCLC colony formation and growth of subcutaneous xenografts in nude mice. Mer or Axl knockdown also improved in vitro NSCLC sensitivity to chemotherapeutic agents by promoting apoptosis. When comparing the effects of Mer and Axl knockdown, Mer inhibition exhibited more complete blockade of tumor growth while Axl knockdown more robustly improved chemosensitivity. These results indicate that Mer and Axl have complementary and overlapping roles in NSCLC and suggest that treatment strategies targeting both RTKs may be more effective than singly-targeted agents. Our findings validate Mer and Axl as potential therapeutic targets in NSCLC and provide justification for development of novel therapeutic compounds that selectively inhibit Mer and/or Axl.

Role of biomarkers for early detection of lung cancer and chemoprevention.

Lung cancer is the leading cause of cancer deaths in developed countries. The poor prognosis associated with this disease is closely related to the fact that most lung cancer patients are not identified until their malignancy has reached an advanced stage. Recent advances have added to the understanding of the morphological and molecular characteristics of preinvasive bronchial lesions and early lung cancers. Such information is being used to provide new tests for the detection of lung cancer at early or preinvasive stages, and for identifying targets for therapeutic intervention that can prevent progression to advanced disease. Laser induced fluorescence endoscope bronchoscopy has improved the sensitivity with which preinvasive dysplastic bronchial lesions and early invasive malignancies can be detected. Morphological features of such lesions have been described and can be monitored by follow-up bronchoscopies in order to validate potential chemoprevention treatments. Distinct morphological characteristics such as angiogenic squamous dysplasia also suggest that processes like angiogenesis are present early in the development of lung cancer. Furthermore, tissue obtained from these early lesions has been used to describe alterations in the expression of a number of factors that distinguish these early lesions from normal bronchial epithelium. This could provide molecular markers and targets for the detection and treatment of early lung cancer. Studies to detect these alterations by polymerase chain reaction and/or immunhistochemical analyses of easily obtained specimens such as sputa are helping identifing molecular markers that could be utilized in effective screening programmes. The current article reviews new findings regarding the molecular biology of preinvasive bronchial lesions and early lung cancers, and describes new developments regarding their application in the early detection and chemoprevention of lung cancer.

Congenital duplication of the tricuspid valve presenting as a circumscribed obstructive mass in the right ventricular outflow tract.

Congenital duplication of the tricuspid valve is a rare condition. We present a case of a 32-year-old man with an increase in intensity of a long-standing heart murmur and intermittent palpitations. The murmur was evaluated by transthoracic and transesophageal echocardiography that identified a mass in the right ventricle and right ventricular outflow tract resulting in partial obstruction of the right ventricular outflow tract. This mass was determined to be congenital duplication of the tricuspid valve by histologic examination after surgical excision.

Re-expression of interleukin 1 in human papillomavirus 18 immortalized keratinocytes inhibits their tumorigenicity in nude mice.

The ability to form tumors in nude mice developed spontaneously in the human papillomavirus (HPV)-18 immortalized keratinocyte cell line, 18-11, and is shown here to be accompanied by a loss of interleukin 1 (IL-1) alpha and beta expression at both the RNA and protein level. In addition, a separate tumorigenic 18-11 derivative and two cervical carcinoma-derived cell lines, HeLa and Caski, were found to have significantly decreased or lost IL-1 alpha and IL-1 beta expression. Using retroviral expression vectors, we re-established IL-1 expression in tumorigenic 18-11 cells (18-11S3) in an effort to evaluate whether loss of IL-1 expression represented an important phenotypic change in the development of tumorigenicity in these cells. IL-1-expressing 18-11S3 cells showed a range of tumorigenic potential, depending on the type and combination of IL-1 alpha and IL-1 beta expressed. Although 18-11S3 expressing the precursor forms of both IL-1 alpha and IL-1 beta normally found in keratinocytes showed moderate inhibition of tumorigenicity, other IL-1-expressing lines showed complete inhibition of tumor formation. Co-injection of nontumorigenic, IL-1-expressing 18-11S3 with parental 18-11S3 also inhibited tumor formation. These results suggest that maintenance of IL-1 expression may play an important role in preventing progression to tumorigenicity in cervical carcinoma and other epithelial cancers.

Human papillomavirus-immortalized keratinocytes are resistant to the effects of retinoic acid on terminal differentiation.

In order to study how human papillomaviruses (HPVs) can alter normal epithelial cell differentiation, we looked at the response to retinoic acid (RA) of HPV-immortalized keratinocytes grown on organotypic cultures. Ten- to 30-fold higher concentrations of RA were required to block terminal differentiation in these cultures when compared to organotypic cultures of control cells. This resistance to RA was associated with maintained expression of differentiation-specific markers and, for keratin K1, Northern analysis showed that K1 mRNA was also detectable at 30-fold higher concentrations of RA in HPV organotypic cultures when compared to controls. These differences were reproducible and characteristic of all HPV cell lines studied, including very early passage HPV16-containing cell lines, suggesting that expression of HPV genes leads to this phenotype. Expression of epithelia-specific components of the RA response pathway was also studied by Northern analysis. At all RA concentrations, there were no detectable differences in overall levels of retinoic acid receptor gamma or cytosolic RA-binding protein II mRNA found. Retinoid X receptor alpha expression was also evaluated, and, in two of three HPV-immortalized cell lines, it was found to be 2 to 3 times as abundant as in controls. Although this difference in retinoid X receptor alpha expression could contribute to RA resistance, the mechanism involved in producing this resistance could not be fully elucidated in these studies. However, resistance to the effects of RA on epithelial differentiation is demonstrated in organotypic cultures of HPV-containing cells, and it is shown that this is associated with maintenance of RNA and protein expression of differentiation-associated genes at abnormally high concentrations of RA.

Altered expression of proliferation and differentiation markers in human papillomavirus 16 and 18 immortalized epithelial cells grown in organotypic culture.

The patterns of expression of keratins K1 and K8, filaggrin, and the proliferation-associated protein, proliferating cell nuclear antigen (PCNA), were studied in normal and human papillomavirus (HPV) 16 or 18 immortalized keratinocyte cell lines grown on organotypic raft cultures. Normal keratinocytes produced an epithelial sheet that closely resembled epidermis in vivo, characterized by lack of K8 expression, PCNA expression restricted to the basal layer, and K1 and filaggrin expression in the suprabasal layers. Although morphologically abnormal in many respects, some HPV-immortalized cell lines produced cornified epithelial layers and approximated the normals in their patterns of expression of keratins and filaggrin. Other HPV-immortalized cell lines produced poorly differentiated epithelial layers that were characterized by loss of filaggrin expression, and the single tumorigenic cell line, 18-11, was distinguished by abundant K8 expression. All of the HPV-immortalized cell lines were distinguished from normal keratinocytes by a common pattern of full-thickness PCNA expression in the epithelial layers they produced, suggesting that maintenance of the proliferative state may be an important contribution made by HPV 16 or 18 sequences toward the production of a malignant phenotype.

Epithelial cells immortalized by human papillomaviruses have premalignant characteristics in organotypic culture.

Three HPV-16--and four HPV-18--immortalized human foreskin keratinocyte cell lines were analyzed on organotypic epidermal raft cultures at various passage levels. This culture system allowed normal cultured keratinocytes to stratify and differentiate in a manner similar to normal epidermis. All seven HPV-immortalized cell lines displayed epidermal morphologies on organotypic cultures, which were clearly abnormal and resembled premalignant lesions in vivo. Features of premalignant lesions that were shared by all of the HPV-immortalized cell lines included disorganized tissue architecture, mitotic cells present throughout the living layers of the epidermal sheet, abnormal mitoses, enlarged nuclei, and variable cell size and shape. Most HPV-immortalized cell lines were stable in terms of epidermal morphology with long-term passage in culture. Two of the HPV-18--immortalized cell lines, however, lost all morphologically apparent terminal squamous differentiation potential after long-term passage in monolayer culture. These results strongly support the idea that immortalization of squamous epithelial cells in culture by HPV-transforming genes generates a morphologically premalignant cell.

Lethal effects and cardiovascular effects of purified alpha- and theta-toxins from Clostridium perfringens.

Shock, a common and frequently fatal manifestation of gas gangrene caused by Clostridium perfringens, is probably mediated by extracellular toxins. Previous studies implicating alpha-toxin as the major lethal factor were frequently done with preparations contaminated with a second lethal factor, theta-toxin. We purified alpha- and theta-toxins from C. perfringens and demonstrated that both were lethal to mice. We investigated the effects of these purified toxins on cardiovascular function in intact rabbits; both toxins caused profound hypotension and bradycardia within 40 min. Reduced cardiac output preceded the development of hypotension and bradycardia. Purified alpha-toxin produced a dose-dependent reduction in myocardial function in isolated rabbit atrial preparations. Purified theta-toxin did not directly inhibit myocardial function. Shock induced by alpha-toxin may be partly mediated by direct depression of myocardial function. theta-Toxin reduced cardiac output in intact animals but had no direct effects on isolated heart preparations at concentrations that induced shock in intact animals. These data suggest that theta-toxin-induced shock could be mediated by an endogenous myocardial depressant factor.