Role of Novel Cytokine-like Molecule Secretoglobin (SCGB) 3A2 in Lung

Division Of Basic Sciences - Nci

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Abstract

(1) SCGB3A2 peptides as a therapeutic. Long-term goal of this project is to develop SCGB3A2 as a therapeutic to treat various lung diseases. To this end, producing a large amount of pure recombinant SCGB3A2 protein with LPS below FDA approved level is absolutely necessary. E. Coli expression system is most frequently used to produce a large quantity of recombinant protein because of its rather inexpensive system. As mentioned in the Goals and Objectives above, SCGB3A2 has a high affinity towards LPS. This makes the purification of recombinant SCGB3A2 protein with low levels of LPS contamination extremely difficult. We experienced that LPS removal step from the crude SCGB3A2 preparation ended up with the loss of almost all SCGB3A2 protein. In order to circumvent this problem, we started working on producing serially overlapping peptides of ~20 amino acids that cover the entire SCGB3A2 amino acid sequence. These peptides were subjected to various assays to determine their biological activities; in vitro assay for cell growth and anti-apoptotic activities, ex vivo assay for fetal lung branching morphogenesis, and in vivo anti-inflammatory activity in allergic airway inflammation model mice. The results demonstrated that most of peptides derived from the C-terminal half of the SCGB3A2 amino acid sequence exhibited various aforementioned activities. These SCGB3A2 peptides when intratracheally administered to mice, successfully suppressed allergic airway inflammation in vivo, suggesting that the bioactivity resides towards the C-terminal region of SCGB3A2 sequence, and the peptides covering this region could be used as a therapeutic in treating lung inflammation. (2) Thyroid carcinogenesis. SCGB3A2 is normally expressed at high levels in airway club cells. Using CC10Tag transgenic mice that express SV40 large T antigen under the mouse Scgb1a1 (CC10) gene promoter, we previously found that SCGB3A2 expression was negative in Type II cell hyperplasias and adenomas developed in these mouse lungs, while it was expressed in lung alveolar Type II cell carcinomas and club cell adenocarcinomas. This suggested that SCGB3A2 can be used as a marker for adenocarcinomas. Further, SCGB3A2 expression was observed in the portion of the tumor where NKX2-1 expression was reduced or almost abolished, suggesting that SCGB3A2 and NKX2-1 may function in an opposite direction. Although SCGB3A2 expression in thyroid is extremely low, we were interested in examining the role of SCGB3A2 in thyroid carcinogenesis if there is any, as described in the Project 1 (Thyroid regeneration and carcinogenesis). In the KrasG12D-induced thyroid carcinogenesis model (in the Project 1, Section 2-2), the loss of Scgb3a2 gene resulted in the increased incidence of adenoma and thus, the function of Scgb3a2 gene appeared to be opposite to that of Nkx2-1 as we previously observed, that is that NKX2-1 serves as a tumor suppressor while SCGB3A2 as a tumor promoter. Single cell RNA sequencing was carried out and we are currently analyzing what genes/pathways are affected by the loss of Nkx2-1 vs Scgb3a2, or both genes in KrasG12D-induced carcinogenesis, and how they interact each other.