Mutational And Expressional Analyses Of The Transforming Growth Factor Beta Type Ⅱ Receptor Gene In Primary Non-small Cell Lung Cancer

Transforming growth factor beta (TGF-β) is a multifunctional cytokine that plays a central role in the signaling networks that regulate cell growth, differentiation, adhesion, apoptosis, migration and modification of the microenvironment in which the cells reside. The TGF-βsignal is transduced through a pair of transmembrane serine-threonine kinase receptors named receptor type II and type I and Smad proteins. Many malignant tumor cells including non-small lung carcinoma (NSCLC) cells are frequently resistant to TGF-β-mediated signal transduction, suggesting that alterations of the TGF-βsignaling pathway maybe involved in tumorigenesis. TGF-βbinds directly to TGF-βreceptor type II (TGFBR2), a constitutively active transmembrane serine/threonine kinase, and is then recognized by TGF-βreceptor type I, which is phosphorylated and activated by TGFBR2. Therefore, inactivation of TGFBR2 can result in TGF-βresistance, and TGFBR2 has been shown to function as a tumor suppressor in many solid tumors. Variants of the TGFBR2 gene are rarely seen in primary NSCLC cells and tissues, even though reduction of TGFBR2 expression has been found frequently. Whether the changes of the protein in NSCLC is genetic changes or epigenetic changes, however, remains unclear. The importance of TGF-βsignaling pathway in suppressing tumorigenesis, together with the high frequency of TGFBR2 mutations in many other types of cancers, directed us to screen for TGFBR2 mutations in NSCLC and to explore further the effect of the identified mutations on TGF-βsignal transduction and their role in tumorigenesis. The correlation between the expression and the mutation status of TGFBR2 were also analyzed.Part 1 Mutational analyses of the transforming growth factor beta typeâ…¡receptor gene in primary non-small cell lung cancerObjectives: 1. To detect the mutation status of TGFBR2 in 17 human lung carcinoma cell lines, two normal human fetal lung fibroblast cell lines and 115 pairs of NSCLC tissues. 2. To investigate the effect of the mutated gene on TGFBR2 mRNA and protein expressions in giant cell carcinoma (GCC) cell lines.Methods: 1. Mutation of TGFBR2 was detected by polymerase chain reaction-denatured gradient gel electrophoresis (PCR-DGGE) and direct sequencing in 17 human lung carcinoma cell lines, 2 normal human fetal lung fibroblast cell lines and 115 pairs of NSCLC tissues. (1)Genomic DNA was extracted from the cell lines, tumor and corresponding normal tissue samples by standard proteinase K digestion and phenol-chloroform extraction. (2)All seven exons of the TGFBR2 gene were assayed by PCR-DGGE. (3)DNA sequencing analysis: The PCR products were separated on agarose gels. Bands of the expected size were excised and purified and then sequenced directly on an automatic DNA sequencer. 2. The expression of mutated TGFBR2 on the mRNA level in GCC cell lines was investigated by RT-PCR. Total RNA from the cells was prepared by use of TRIzol reagent and was reverse transcribed with the SUPERSCRIPTTM II RNase HˉReverse Transcriptase Kit. Two sets of primers were designed so that the amplified fragments spanned exons 1 to 4 and 4 to 7 with several nucleotides overlapping allowing to check the integrity of the mRNA expression. Glyceralaldehyde 3-phosphate dehydrogenase (GAPDH) cDNA was also amplified as an internal control. The RT-PCR products were separated on 8% PAGE. 3. Expression of TGFBR2 in GCC cell lines was detected using immunoprecipitation and Western blotting and confirmed by immunocytochemical analysis. Immunoprecipitation was performed using the antibody targeted to the extracellular domain. Western blot analysis was performed using monoclonal antibody against the extracellular domain of TGFBR2 and horseradish peroxidase-conjugated secondary antibody. For immunocytochemical analysis, the cells were incubated with monoclonal antibody and stained with FITC-conjugated goat antimouse antibody.Results: 1. A novel homozygous microdeletion (c.492₅07del) was identified in two cell lines(95D and PG) derived from the same GCC and was confirmed in the corresponding tumor tissues. Furthermore, a heterozygous c.492₅07del was found in the germ-line of one patient, as well as in the other GCC cases and some large cell carcinomas (LCC). The total frequency of the mutation was 2/2 GCC and 7/13 LCC. No mutation was detected in any of the other 100 NSCLC samples, including the subtypes of AdC, SqC and Ad-SqC, and no mutation was detected in any of the corresponding normal lung tissues and other cell lines. 2. HFL1 cells had amplified the two products of 717 bp and 805 bp; whereas the expression of both fragments of 701 bp and 805 bp were detected from the TGFBR2 mutated GCC cells. 3. The truncated protein was identified in GCC cells and the full length TGFBR2 was detected in normal HFL1 cells. The mutant protein could be seen on the membrane of the GCC cells.Conclusions: 1. A Novel Microdeletion(c.492₅07del) was found in two cell lines derived from the same giant cell carcinoma (GCC), as well as in the GCC cases and some large cell carcinomas (LCC) but not in other subtypes of NSCLC. The mutated TGFBR2 seems to play an important role in the abrogation of TGF-βsignal transduction and tumorigenesis of GCC and LCC. 2. The16 bp microdeletion in TGFBR2 has not affected the integrity of transcription and splicing of the mRNA. 3. The c.492₅07del mutation causes a premature stop of translation which results in a truncated protein. The alteration in transmembrane domain had not made the protein to be released from the cell surface.Part 2 Effect of the mutated TGFBR2 on TGF-βsignal transduction in the GCC CellsObjective: To confirm the mutated TGFBR2 plays an important role in the abrogation of TGF-βsignal transduction and tumorigenesis of GCC.Methods: 1. For assessing the sensitivity to exogenous TGF-βof GCC cells carrying microdeletion in TGFBR2: (1)Growth inhibition assay was used to assess the antiproliferation effect of TGF-β1 on GCC cells carrying microdeletion in TGFBR2. The cell numbers were determined in response to increasing concentrations of exogenous TGF-β1 (0, 0.2, 1 or 5 ng/ml) by MTS assay. Cells that showed over 40% reduction of cell proliferation by TGF-β1 (5 ng/ml) were determined to be TGF-β1 responsive. (2)To study the effect of exogenous TGF-β1 on fibronectin (FN) expression of GCC cells carrying microdeletion in TGFBR2, the relative quantification of fibronectin mRNA levels from cells treated with or without 5 ng/ml of TGF-β1 for 48 h was performed using real-time, fluorescence quantitative PCR and the△△C t method. The FN gene expression was presented relative to the housekeeping gene GAPDH. 2. To investigate whether the abrogated TGF-βsignal transduction is solely due to the inactivation of TGFBR2, we sought to restore TGF-β1 responsiveness in GCC cells by introducing wild-type TGFBR2. A TGF-β1 responsive luciferase reporter construct, p3TP-Lux, was used to cotransfect the GCC cells with the vector pcDNA3-TGFBR2 which expressed wild-type TGFBR2 or empty vector pcDNA3. Cells were treated with or without 5 ng/ml of TGF-β1 in medium. Renilla luciferase reporter gene pRL-SV40 was cotransfected to normalize the transfection efficiency. Luciferase activity in cell lysate was determined by the Dual-luciferase Reporter Assay System.Results: 1. The TGFBR2-defective GCC cells showed loss of sensitivity to TGF-β1 induction both in growth inhibition and stimulation of extracellular matrix proteins. 2. After the transfection of wild-type TGFBR2 into the mutant cells, the sensitivity to TGF-β1 was restored.Conclusions: The mutant TGFBR2 plays an important role in the abrogation of the TGF-βpathway in GCC cells.Part 3 Expression of TGFBR2 in NSCLC and its possible implicationsObjectives: 1. To examine the expression level of TGFBR2 both in LCC (including GCC) and non-LCC tumors, which include adenocarcinoma (AdC), squamous cell carcinoma (SqC) and adenosquamous carcinoma (Ad-SqC). Analyses were also conducted to determine the associations between TGFBR2 expression and clinical features, such as pathological types, age, gender, tumor size, nodal involvement, metastasis and stage. 2. The correlation between the expression and the mutation status of TGFBR2 in LCC samples were also analyzed.Methods: 115 pairs of NSCLC samples (tumor tissues and their corresponding normal tissues) and one case of human normal bronchial epithelium were accumulated and made into three tissue microarrays which containing 90 dots, 90 dots and 53 dots respectively. Expression of TGFBR2 was detected by immunohistochemistry assay using the monoclonal antibody against the extracellular domain of TGFBR2 via the two-step immunohistochemical staining with the MaxVision system. After the paraffin sections being deparaffinized and hydrated, sections were placed into a 3% hydrogen peroxide solution for 10 min to block endogenous peroxide activity. For antigen retrieval, the sections were treated with boiling 0.01 mol/L citrate buffer for 20 min and were then incubated with 10% goat serum for 20 min at room temperature. After the blocking serum was removed, the sections were incubated with the primary antibody (1:50) at room temperature for 1 h. The sections were then incubated with the working solution of horseradish peroxidase-labeled goat anti-mouse immunoglobulin for 30 min. Finally, the peroxidase activity was developed with 3,3'-diaminobenzidine tetrahydrochloride and hydrogen peroxide. Human normal bronchial epithelium was used as a positive control. Negative control for each specimen has been done by treating the sections with PBS instead of the primary antibody. All sections were examined by using standard light microscopy and scored semi-quantitatively on the basis of the percentage of cells that displayed immunoreactivity and on the intensity of the staining reaction. The expression of TGFBR2 was predominantly on the membrane and in the cytoplasm of the epithelial cells. All the normal tissues stained strongly in over 75% of cells and their staining scores were 9.The samples were first grouped into four grades according to the staining intensity: 0 (negative staining, the same with that of negative control), 1 (weak staining), 2 (medium staining) and 3 (strong staining). The percentages of positive staining cells were assigned as 0 for 0²5%, 1 for 26⁵0%, 2 for 51⁷5% and 3 for 76¹00%, respectively. The final score was determined as the product of the proportion and intensity scores and will be 0⁹. The sample was assigned as negative staining if the final score was 0 or positive staining when the final score was 1⁹. Moreover, a cancer sample was assigned as a preserved (Pr) or a reduced type (Re) of TGFBR2 expression if its final score was the same with or less than that of its corresponding normal lung tissue.Results: 1. All the normal tissues stained strongly in over 75% of cells and their staining scores were 9. According to the criteria to classify the tumor samples, 2 cases showed negative TGFBR2 expression (1.7%) and other 113 cases showed positive expression. Furthermore, 47.8% (55/115) of tumor tissues showed reduced or loss of TGFBR2 expression and 52.2% (60/115) of tumors had preserved TGFBR2 expression. In case of adenocarcinoma, squamous cell carcinoma and adenosquamous cell carcinoma, the frequency of reduced type were 44%(22/50), 43.2%(19/44) and 33.3%(2/6) respectively, while in case of large cell carcinoma, the frequency of reduced type was extremely higher(80.0%, 12/15), indicating that most of the LCC cells had reduced TGFBR2 expression. No significant association was found between TGFBR2 expression and pathological types, age, gender, tumor size and local invasion, nodal involvement, metastasis or stage. 2. No correlation was found between TGFBR2 expression and c.492 507del mutation in LCC tissue sections.Conclusions: 1. The differences in the expression level of TGFBR2 between NSCLC samples and corresponding normal lung tissues suggest that the defective TGFBR2 expression might contribute to the carcinogenesis and/or development of NSCLC. 2. There is significant difference on the TGFBR2 expression level between the LCC and non-LCC subtypes. 3. No correlation was found between TGFBR2 expression and c.492 507del mutation in LCC tissue sections. 4. No significant association was found between TGFBR2 expression and age, gender, tumor size and local invasion, nodal involvement, metastasis or stage. Summary1. A novel homozygous TGFBR2 microdeletion (c.492₅07del) was identified in two cell lines derived from the same giant cell carcinoma (GCC) and was confirmed in the corresponding tumor tissues. Furthermore, a heterozygous c.492₅07del was found in the germ-line of one patient, as well as in the other GCC cases and some large cell carcinomas (LCC) but not in other subtypes of NSCLC.2. The 16 bp-microdeletion introduced a premature stop codon at positions 590–592 of the cDNA, resulting in a truncated TGFBR2 protein with a mutated transmembrane domain and loss of kinase domain. The GCC cells were characterized as being unresponsive to TGF-βinduction both in growth inhibition and stimulation of extracellular matrix protein. Moreover, after the reconstitution of wild-type TGFBR2 expression, the sensitivity to TGF-βwas restored. Therefore, mutated TGFBR2 seems to play an important role in the abrogation of TGF-βsignal transduction in GCC cells.3. The differences in the expression level of TGFBR2 between NSCLC samples and corresponding normal lung tissues suggest that the defective TGFBR2 expression might contribute to the carcinogenesis and/or development of NSCLC. There is significant difference on the TGFBR2 expression level between the LCC and non-LCC subtypes, but no correlation was found between TGFBR2 expression and c.492 507del mutation in LCC tissue sections. No significant association was found between TGFBR2 expression and age, gender, tumor size and local invasion, nodal involvement, metastasis or stage.4. The reduced expression of TGFBR2 and the existence of c.492₅07del mutation inactivate the TGF-βsignal transduction, leading to the loss of growth inhibition of the corresponding cells and acceleration of tumor formation. Our findings in LCC provided genotypic and expression supports in the classification of LCC and non-LCC subtypes which was previously classified solely by phenotypes. LCC is distinct from AdC and SqC not only in differentiation, but also in the pattern of TGFBR2 expression and TGFBR2 mutation.