Abstract
Progress in the understanding of fundamental aspects of carcinogenesis as well as the discovery and validation of new approaches to cancer prevention and therapy have been facilitated and enhanced by the development and use of animal models for human cancer. Mouse models, which emulate processes triggering the development and progression of human lung cancer, are especially desired because this cancer continues to be the leading cause of cancer death and new insights are urgently needed to improve its diagnosis, prevention and treatment. Lung cancer is caused mainly by tobacco smoking, which causes both genetic and epigenetic changes. Genetic changes including mutations in K‐Ras and p53 (mainly among smokers) and epidermal growth factor receptor (mainly among nonsmokers) have been implicated in the development of non‐small cell lung cancers (NSCLC). In addition, epigenetic silencing of many tumor suppressor genes has been found in the majority of NSCLC. The several decades long latency period characteristic of most lung cancers indicates that many incremental changes due to chronic processes augment the insults caused by tobacco smoke carcinogenic substances. One such process is chronic pulmonary inflammation, which has been implicated in the development of lung cancer in humans (both smokers and non‐smokers) and in mouse models. We discovered a retinoic acid‐inducible gene encoding a seven transmembrane glycoprotein named GPRC5A, which is presumed to be an orphan G‐protein‐coupled receptor. GPRC5A is expressed preferentially in normal human and murine lung epithelial cells. Further characterization of GPRC5A revealed that it behaves like a class II tumor suppressor in that it is inactivated in lung tumor cells by epigenetic suppression of its expression rather than by mutations. GPRC5A expression was found to decrease at both the mRNA and protein level in over 60% of non‐small cell lung cancer cell lines and tumor tissue specimens compared to normal lung tissues. Furthermore, restoration of GPRC5A expression in certain cancer cells by transfection decreased their anchorage‐independent colony forming ability. The strongest support for the idea that GPRC5A functions as a tumor suppressor has come from studies of mice in which its orthologous murine gene ‐ Gprc5a ‐ had been knocked out. Such mice (Gprc5a−/−) were found to develop spontaneous lung tumors (primarily adenoma and a few adenocarcinomas). The carcinogenesis process in this mouse model takes 1 to 2 years, which is reminiscent of the long latency period required for human lung cancer development. This observation suggested that the loss of Gprc5a gene is not sufficient for transforming the lung epithelial cells and that additional factors may be required to induce subsequent changes during the latency period. Examination under the microscope of specimens from normal lung tissue and tumors from 51 Gprc5a+/+ (9.8% adenoma incidence) and 38 Gprc5a−/− mice (63% adenoma and 21% adenocarcinoma incidence) revealed enhanced infiltration of macrophages into lungs of 45% of the Gprc5a−/− mice and 8% of Gprc5a+/+ mice and the direct association of macrophages with 45% of adenomas and 88% of adenocarcinomas in the knockout mice. Macrophage infiltration is the hallmark of chronic inflammation. It has been reported that activated macrophages secrete many proinflammatory cytokines, which promote lung tumor development and progression. Therefore, our observation raised the possibility that macrophage‐mediated inflammation plays a role in promoting lung carcinogenesis, especially in the progression of “premalignant” adenomas to malignant adenocarcinomas, in the Gprc5a knockout model. Indeed, we found that Gprc5a−/− mouse lungs contained higher constitutive levels of a variety of proinflammatory cytokines and chemokines than those of Gprc5a+/+ mice and were much more sensitive to stimulation by the inflammation inducer lipopolysaccharide (LPS) in vivo. Because many effects of chronic inflammation are mediated by nuclear factor kappa B (NF‐kB), which controls the expression of genes involved in inflammation, immune responses, cell cycle, apoptosis, and angiogenesis, we analyzed the status of this transcription factor in wild type and knockout mice before and after administration of LPS and found a greater activation of NF‐kB in the lungs of Gprc5a−/− mice as evidenced by increased nuclear translocation of the p65 subunit of NF‐kB in lung epithelial cells in vivo and higher DNA‐binding activity measured by EMSA in nuclear extracts of lung tissue. More in depth studies comparing epithelial cells that had been cultured from lungs of Gprc5a knockout (Gprc5a−/− cells) and wildtype mice (Gprc5a+/+ cells) revealed that the loss of Gprc5a in lung epithelial cells confers on them a premalignant phenotype including immortality, enhanced resistance to apoptosis, and expression of various genes and pathways associated with cancer. They also exhibit a high level of constitutively activated NF‐kB revealed by electrophoretic mobility shift assay. This level is comparable to the one achieved in WT cells only after treatment with tumor necrosis factor alpha (TNFa). Interestingly, these changes were reversed partially in Gprc5a−/− adenocarcinoma cells by re‐expression of Gprc5a. The high activation of NF‐kB in the lung epithelial cells from the knockout mice may cause both autocrine and paracrine effects that could lead to their high proliferation rate and resistance to apoptosis and can also explain, at least in part, the higher macrophage infiltration in the knockout mouse lungs. In support of this contention, we found that the cultured Gprc5a−/− mouse lung epithelial cells produced higher levels of chemokines and cytokines that can attract macrophages and, indeed, their conditioned medium induced more extensive macrophage migration through a polyporous membrane than the medium from the corresponding Gprc5a+/+ cells. Silencing of Gprc5a and NF‐kB in Gprc5a+/+ and Gprc5a−/− cells, respectively reversed these effects. Therefore, we suggest that the activation of NF‐kB in Gprc5a−/− mice is a major driving force in the carcinogenesis process via two major mechanisms for oncogenesis: 1) by enhancing proliferation potential, survival, and immortalization of target lung airway epithelial cells; and 2) by increasing production of chemokines and cytokines from epithelial cells leading to increased recruitment of inflammatory cells for creation of proinflammatory microenvironment that promotes tumorigenesis. This model should be useful for assessing the chemopreventive potential of anti‐inflammatory agents. Preliminary studies with the corticosteroid Budesonide showed promising results.
Citation Information: Cancer Prev Res 2010;3(1 Suppl):PL02-03.
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