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A7106 - IGF2 as a Potential Novel Therapeutic Target for Rapamycin-Insensitive LAM Patients
Author Block: K. Obraztsova1, S. Hur2, R. Rue1, J. F. Evans1, M. Bartolomei2, V. P. Krymskaya1; 1Pulmonary, Allergy, Critical care, University of Pennsylvania, Philadelphia, PA, United States, 2Cell and Developmental Biology, University of Pennsylvania, Philadelphia, PA, United States.
Rationale: Pulmonary Lymphangioleiomyomatosis (LAM) is a rare progressive lung disease that mostly affects women of childbearing age. It is characterized by a metastasizing growth of abnormal smooth muscle-like cells originating from an unknown source. Over time, LAM cells destroy the lung parenchyma causing lung collapse and pulmonary failure. LAM is attributed to loss of function in the tumor suppressor gene Tuberous sclerosis complex 2 (TSC2) which upregulates mTOR signaling, one of the main metabolic pathways in the cell. The only existing therapy is treatment with the mTOR inhibitor sirolimus (rapamycin) which stabilizes lung function, treats chylothorax, and improves overall quality of life, but due to its cytostatic effect does not reverse the disease. Moreover, this treatment is effective for only approximately 50% of the patients. Previously we demonstrated the overexpression of the pro-oncogenic gene IGF2 in LAM cells by RNA-seq. IGF2 gene dosage is known to be crucial for the normal cell proliferation and is achieved through paternal imprinting. Upregulation of IGF2 is linked to a variety of malignant tumors. We have shown that IGF2 overexpression contributes to LAM cell growth and survival. Methods: To investigate the mechanism of IGF2 over-expression we examined the imprinting status of this gene in LAM cells by pyro-sequencing of the related to IGF2 gene imprinting control region (IC1). In addition, we observed that IGF2 over-expression was partially STAT3-dependent. We explored a potential transcriptional activation mechanism by STAT3 by Chromatin Immunoprecipitation(ChIP)-qPCR analysis of the IGF2 promoter region. To address mTOR-dependence of IGF2 over-expression we quantified IGF2 protein expression in LAM cells treated and untreated with rapamycin by immunoblot. Results: Imprinting status analysis showed normal IGF2 imprinting patterns for all analyzed LAM cells, concluding that the observed increased levels of IGF2 were independent of chromosomal gene dosage. ChIP-qPCR analysis revealed predominant binding of STAT3 within the predicted STAT3 binding regions of IGF2 promoter in both mouse and human LAM cells, suggesting that STAT3 acts as a direct transcriptional activator of IGF2 gene expression. Rapamycin treatment showed that both gene and protein expression levels of IGF2 were mTOR-independent. Conclusions: Increased IGF2 expression in LAM cells is independent of its imprinting status and is most likely partially induced by activation of the transcription factor STAT3. Most importantly, we demonstrated that both gene and protein expression levels of IGF2 are mTOR-independent. The latter finding makes inhibition of IGF2 a good potential target in novel therapies for rapamycin-insensitive LAM patients.