Lung developmental process requires the precise activation of several signaling cascades, namely, FGF, TGF?-BMP, WNT, HIPPO, RA, NOTCH, and SHH. Mainly, Shh play a critical role during this developmental process and appear in correlation with other developmental signaling pathway, in which the retinoic acid (RA) induce the SHH endoderm expression, then, SHH signals back to the mesoderm in order to activate GLI2/3 transcription factors that, consequently, stimulate the expression of WNT2/2b ligands and BMP4 that then induce nkx2.1 expression which is a homeodomain tissue-specific gene that determine the respiratory lineage and, consequently, lung specification programs, lung bud formation and branching morphogenesis will be induced. However, dysfunctions of these pathways is associated with different types of lung developmental disorders and cancers and are evident in multiple tumor types and malignancies.
When the SHH pathway is active, the SHH ligand binds to PTCH1 receptor, and eliminate the SMO inhibition that will then move to the primary cilia and induce GLI activation through SUFU detachment. Thus, the activator form of GLIs will move to the nucleus and induce the transcription of specific target genes. Thus, in order to study the crosstalk between SHH pathway and the other signalling pathways implicated in lung cancer, an experiments can be designed by: Inactivation of the SHH signaling, by blocking of the Shh ligands from binding to PTCH1 receptor by Shh antibodies, activation of SMO in the cell membrane, modulating intracytoplasmic regulators of Hh signaling like protein kinase A and SuFu, or altering the intranuclear functioning of Gli. The expression of FGF, BMP, WNT, NOTCH and other signalling pathways can be examine in Shh inducible knockout mouse model, where a histological analysis and in situ hybridization can be perform, and, consequently observe any alterations in their expression in Shh mutant mouse. In addition, the expression of the activator form of GLIs transcription factors in SHH pathway such as GLI1 can be analyze in mouse models for lung cancer, in order to investigate the correlation of GLI expression with transcription factors of other signalling pathways implicated in lung cancer such as Wnt2, Fgf10, Sox2 and others.
GLI1 mRNA of mouse models for lung cancer will be collected and converted to cDNA. The cDNA will be labeled and a specific primers will be designed. Then cDNA will be amplified using real-time PCR in order to assess the expression of GLI1 and analyze the correlation between GLI1 and selected transcription factors of other pathways in lung cancer mouse. For example, if the result shows that overexpression of GLI1 in lung cancer tissues is associated with other transcription factor such as SOX2, this will indicate that there is a correlation between GLI1 and SOX2, suggesting that SHH signaling pathway is in crosstalk with BMP pathway within lung cancer tissues.
