Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Biology (Cell Biology, Microbiology, Molecular Biology)

Major Professor

Jie Wu

Keywords

mouse model, NSCLC, SHP2, Transgenic

Abstract

The gene PTPN11 was identified in the early 1990s, and encodes the non-transmembrane protein tyrosine phosphatase SHP2. SHP2 is expressed ubiquitously in cells, and plays an important role in cancer. Unlike most phosphatases, SHP2 positively regulates several signaling pathways including the Ras/MAPK and Src signaling pathways and acts as a proto-oncogene. SHP2 is also a cancer essential gene in certain types of carcinomas, and promotes growth, survival, and epithelial to mesenchymal transformation. Gain of function (GOF) SHP2 mutations are known leukemic oncogenes, and have been identified to a smaller extent in solid tumors as well. Currently, the roles of SHP2 in lung carcinoma are not fully understood. While GOF SHP2 mutations have been detected in lung cancer, their contributions to cellular transformation had not been established. In addition, SHP2 is known to promote EGF growth factor receptor (EGFR) signaling. Since GOF EGFR mutations induce transformation of lung epithelial cells, it is possible that SHP2 plays a role in promoting GOF EGFR mutant driven tumorigenesis. The objective of this dissertation is to determine whether SHP2 can act as an oncogene in lung epithelial cells and whether SHP2 inhibition can affect GOF EGFR mutant induced lung cancer. To achieve these aims, we generated two novel doxycycline (Dox) inducible transgenic mouse models which express either the GOF SHP2E76K leukemic oncogene or the dominant negative SHP2CSDA mutant under the control of the Clara cell secretory protein (CCSP) promoter to regulate transgene expression to type II pneumocytes.

To determine whether SHP2 plays a role in promoting GOF EGFR mutant signaling, we started by disrupting SHP2 function in vitro. Two non small lung cancer cell lines were used for this project: HCC827 carries the LREA deletion in exon 19, and H1975 co-expresses the EGFRL858R point mutation and the EGFRT790M gatekeeper mutation. After SHP2 PTP inhibition or knock-down by shRNA and siRNA, both cell lines exhibited decreased cell proliferation and reduced levels of pErk1/2 and c-Myc. Based on these results, we acquired a transgenic mouse line which expresses the EGFRL858R mutant under the control of the tet-O promoter and generated bitransgenic CCSP-rtTA/tetO-EGFRL858R and tritransgenic CCSP-rtTA/tetO-EGFRL858R/tetO-SHP2CSDA mice to study the effects of the dominant negative SHP2CDSA mutant on EGFRL858R mediated carcinogenesis in vivo. After 4, 6, and 8 weeks of Dox induction, pErk1/2 and pSrc levels were increased in the lungs of bitransgenic mice compared to wild type controls. Both kinases were suppressed by SHP2CSDA expression in tritransgenic mice. In addition, SHP2CSDA expression delayed tumor onset and prevented progression to a more aggressive phenotype. Tritransgenic mice also developed a smaller tumor burden compared to bitransgenic animals. These results suggest that SHP2 is critical for GOF EGFR mutant mediated lung tumorigenesis and describe a new role of SHP2 as a potential therapeutic target for the development of novel NSCLC drugs.

Once we generated our CCSP-rtTA/tetO-SHP2E76K transgenic mouse model, we administered Dox for one month and found that SHP2E76K expression upregulates pErk1/2, pSrc, pGab1, c-Myc and Mdm2 levels in the lungs of bitransgenic mice compared to controls. After six to nine months of Dox induction, SHP2E76K expression caused formation of lung adenomas and adenocarcinoma. We then took advantage of the reversible feature of our mouse model to test whether lung tumors are dependent on sustained SHP2E76K expression for survival. MRI analysis of lung adenocarcinomas showed full regression of the lung tumors after Dox withdrawal. Histological evaluation of lung tissues revealed residual hyperplastic lesions as well as evidence of necrosis, while biochemical analysis showed that pGab1, pErk1/2, pSrc and c-Myc returned to basal levels. These results demonstrate that sustained SHP2E76K expression is required for lung tumor maintenance. Moreover, this data describe a novel function of SHP2E76K as an oncogene in lung carcinoma.

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