TGF-Beta Signaling and Radiation Response in Lung Carcinoma

Gene expression studies revealed that there exist more than one pathways regulating growth inhibition and apoptosis processes. The few important pathways include: (1) pathway mediated through p53 gene for cell cycle arrest and apoptosis; (2) pathway mediated through TGF-~ signaling for negative growth regulation; and (3) pathways downstream to p53 and TGF-~ acting as cross-point regulators that can induce, enhance, delay or inhibit apoptosis. Lung carcinomas are known to have accelerated tumor progression and this phenomenon can be attributed to loss of TGF-~ signaling due to loss of function of RII and Smad4 genes. This observation underscores the fact that intact TGF-~ signaling is pivotal in inhibiting accelerated tumor progression. Our recent preliminary experiments in lung tumors showed that there exists aberrant expression of genes (TGF-f3type II (RII) receptor and Smad4) downstream to TGF- f3pathway. In particular, our data indicated in non-small cell lung cancer cells, that defective TGF~ signaling due to a lack of RII expression contributed to the radiation resistant phenotype ofNCI-H460 cells. In addition, clinical samples from lung cancer exhibited aberrant expression of RII or no expression of Smad4 when compared to normal lung tissue. Based on our preliminary findings, we hypothesize that radiation-induced clonogenic inhibition and apoptosis require intact TGF- 6 signaling. Dysregulation of TGF- Bsignaling caused by lack of RII or Smad4 expression will render enhanced resistance to radiation-induced clonogenic inhibition. To test this hypothesis, we propose the following specific aims: 1. To determine the induction of TGF-~ signaling in established cultures of lung cancer cell lines (SCLC and NSLC) exposed to radiation and characterize the radiosensitivity profile of these cell lines with respect to clonogenic inhibition, apoptosis and cell cycle changes. Assessment ofTGF-~ signaling will be performed by (a) analyzing the radiation-induced kinetics of TGF-~ expression by determining the effect of radiation on TGF-~ promoter activity, the induction ofTGF-~l mRNA using RNAse protection assays and by determining the protein in the medium of irradiated cultures using ELISA; (b) analyzing the TGF-~ responsive promoter activity by p3TP-Iuciferase assay in the untreated and irradiated cultures; and (c) evaluating the expression of RI, RII and RII and Smad4 mRNA using Northern blot analysis, RTPCR and RNAse protection assays. Induction of TGF-~ signaling by radiation will be correlated with p53 status. 2. To determine the functional role of TGF-~ signaling in the regulation of radiation-inducible clonogenic inhibition in lung cancer cells. This will be performed in cell lines treated with neutralizing antibody to TGF-~ or in the parent cells where RII/Smad4 expression or function is blocked by transfecting cells with vectors expressing an RII-dominant negative mutant or a Smad4 antisense. Similarly, the effect of restoration of TGF -~ signaling will be determined in transfectant cell lines overexpressing the cDNA of RII or infecting the cells with AdlSmad4. 3. To elucidate the pathway of TGF-~ signaling mediated up-regulation of the downstream effector genes, such as CDK inhibitor p21wafI/ciplandpro-apoptotic gene Bax (a presumed effector gene of TGF-~ signaling based on the presence of Smad binding element), so that the mechanis~ of.radiation-inducible clonogenic inhibition and apoptosis in lung cancer cells can be unqerstood. ThIS wIll be performed by studying the radiation-induced kinetics of effector gene's (p21wafI/cIPlandBax)promoter activity (using CAT and gel-shift reporter assay), mRNA and protein levels in cell lines treated with neutralizing antibody to TGF-~ or transfectant cell line expressing RII-dominant negative mutant or cells. treated with Smad4-antisense oligomers or in transfectant cell lines overexpressing the cDNA of RII or mfectmg thecells with AdlSmad4. 4. To determine the clinical relevance ofTGF-~ receptors (RI, RII and RIll) and Smad4 expression and p53 status in primary tumor specimens of lung carcinoma. This will be performed by documenting the incidence of alterations in TGF-~ signaling machinery gene in tumor samples of untreated lung carcinoma at University of Kentucky using PCR-SSCP, immunohistochemical and RT-PCR analyses.