The Functional Characterization Of Novel Lung Cancer-associated Genes CT120A/B

Lung cancer is by far the leading cause of global cancer-related death, with an incidence estimating 1,240,000 in 2001 all over the world. While median survival has improved from 7 months in 1960 to 16.8 months in 2001 for all patients diagnosed with this malignancy, much of the progress relates to the improved surgical techniques, combined modality therapy for locally advanced disease. However, many problems remain to be solved, as only 15% to 17% of patients survive over 5 years. With the development of multiple small molecules and monoclonal antibodies targeting important growth factor receptors, oncogenes and tumor-suppressor genes known to be aberrantly mutated, there leaves a vast space for further incremental improvements in the treatment of this fatal disease.CT120A, a novel human plasma membrane-associated gene, was isolated from chromosome 17pl3.3 by using positional cloning and RACE (rapid amplification of cDNA ends) by our laboratory (GeneBank accession no. AF477201). CT120A gene consists of 2145 bp and encodes a protein with 257 amino acids. CT120A, with seven transmembrane domains, has an extracellular NH2 and an intracellular COOH-terminus, in addition to three extracellularly and three intracellularly connecting loops. Subcellular localization analysis showed that CT120A was a novel membrane-associated protein. In our previous report, transcript of CT120A was not detectable in normal lung tissues, but was abundant in the human lung adenocarcinoma SPC-A-1 cell line. Ectopic expression of CT120A by cDNA transfection could promote the malignant transformation of NIH3T3 cells and overexpression of CT120A in the A549 (human lung adenocarcinoma) cells enhanced rumorigenicity in nude mice. All these data indicated that CT120A might be a novel candidate gene closely related to pulmonary carcinogenesis or cancer progression.To validate our obtained outcomes and clarify the relationship between CT120A and oncogenesis of lung cancers, here we employed RNA interference (RNAi) to silence the endogenous CT120A expression in the SPC-A-1 cells. The knockdown ofCT120A expression by small hairpin RNA (shRNA) reduced the cell growth rate, suppressed the cell clonogenicity in soft agarose and inhibited tumorigenicity in a xenograft model. Furthermore, it sensitized the cancer cells to ultraviolet (UV)-induced apoptosis. Atlas cDNA expression array revealed that the expressions of several apoptosis- and growth-associated genes were altered underlying the molecular mechanisms of these cell biological behaviors, including up-regulated procaspase 8, 9, 10, Tob (transducer of ErbB2), DDIT3 (DNA-damage-inducible transcript 3) genes and down-regulated ErbB2 gene. By Western blotting analysis, CT120A was overexpressed at protein levels in 15 cases of the 16 primary lung cancer specimens so far examined. All these results confirmed that CT120A was involved in lung cancer development or progression, and the RNAi of CT120A might hold promise for development of a new strategy for treating lung cancers.A BLAST search in human genome database of NCBI revealed that there existed the other splice variant designated as CT120B (GenBank accession no. BC026023). CT120B, with the absence of the fourth ex on when compared with CT120A, deleted 96 nucleotides and led to an in-frame loss of 32 amino acids between the codon 136 and 167. The search with TMHMM programs predicted that CT120B had six transmembrane domains, with intracellular NH2 and COOH-terminus, two intracellularly and three extracellularly connecting loops. To determine the effects of CT120B on lung cancer cells, we transfected CT120B ORF into the SPC-A-1 cells and established the stable transfectants. The growth inhibitory activities of overexpressed CT120B in SPC-A-1 cells represented to inhibit cell growth and maintain cancer cells at a less malignant state by its suppressive effects on the clonogenicity in soft agarose and the tumorigenicity in nude mice.We tried to use cDNA microarray technology to address the molecular mechanisms of CT120B growth inhibitory activities. Among the differentially expressed genes there were down-regulated cyclin El, cdk 2, c-kit, CXCR4 and up-regulated procaspase 8. Cyclin E/CDK2 complexes are the key to the Gl-to-S phase transition, thus we investigated the effects of CT120B overexpression on cell cycle progression. The cells overexpressing CT120B showed dramatically delayed Gl/S phase transition as compared with control cells.Our previous data indicated that CT120A acted as positive regulation on cell growth. It appeared that the functions of CT120A and CT120B were antagonistic. Thedistinct molecular conformation of CT120A and CT120B was anticipated to lead to remarkably different functions between them. Furthermore, the expression pattern and the relative ratio of the two isoforms might be relevant to the regulatory complex systems in cell proliferation and cell survival.