Characterizing PIAS1 Function During Embryogenesis and Lung Cancer Progression
Constanzo, Jerfiz D.
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The main focus of this work is to provide a better understanding about the function of small ubiquitin like modifiers (SUMO) conjugating enzymes in eukaryotes during embryonic organogenesis, tissue differentiation and human tumor progression; with a focus on non-small cell lung cancers (NSCLC). The SUMO E3 ligase protein inhibitor of activated STAT-1 (PIAS1) is one the few known SUMO conjugating enzymes known to date. PIAS1 was first discovered as a negative regulator of signal transducer and activator of transcription 1 (STAT-1), from which its name is derived. PIAS1 facilitates STAT-1 conjugation with SUMO, which subsequently blocks STAT-1 ability to interact with DNA and promote gene transcription. PIAS1 is also known for its involvement in the coordinated response to DNA damage repair and immune repression. During DNA damage PIAS1 SUMOylation of target proteins, such as, breast cancer susceptibility gene 1 (BRCA1) promotes its recruitment to sites of DNA damage and promotes efficient DNA repair. In contrast, PIAS1 repression of necrosis factor kappa B (NFκB), blocks interaction with downstream target genes and suppresses the secretion of inflammatory cytokines by immune cells. Thus, the ability to interact with widely diverse targets, including transcription factors and genome integrity proteins places PIAS1 at the epicenter of SUMOylation biology making it a valuable protein to study in this field. We used mice (Mus musculus) as our model system for studying the function of Pias1 by global inactivation of this gene in mammals to discover tissues and biological processes affected by the loss of Pias1. We found that, in agreement with previous reports, inactivation of Pias1 results in embryonic lethality in 90% of mouse embryos, while the remaining 10% are born about 40% smaller and anemic. Upon close examination of mouse embryos prior to their death, we discovered severe growth retardation, anemia and defective angiogenesis. We determined that hematopoietic cells in the embryonic yolk sac (YS) were depleted early during development as a result of defective differentiation and reduced cell survival. Although blood differentiation and angiogenic defects were detected first, the lack of blood circulation and a deficient endothelial cell layer in the vasculature of Pias1 null embryos also results in loss of myocardium muscle mass and defective heart formation. However, with in vivo analysis we could not rule out heart tissue intrinsic defects. In contrast, in vitro studies show that PIAS1 may cooperate with the cardiac transcription factors Myocardin and GATA-4 in promoting cardiac differentiation. Because Pias1 loss primarily affects erythrogenesis and vascular development of the YS, we also tested how conditional Pias1 deletion in endothelial cells of the YS would affect erythrogenesis and vascular development. Pias1 loss in endothelial cells recapitulates the erythroid and angiogenic defects in the YS of Pias1 null embryos, but not the growth retardation phenotype. In this study we also examined PIAS1 involvement in tumorigenesis with the hypothesis that PIAS1 functions during tissue growth and survival can be exploited by malignant neoplasms during tumor progression. Previous studies have found that PIAS1 can either repress or enhance tumorigenesis depending on tumor type. In prostate cancer, PIAS1 has been found to promote tumor proliferation by repression of the p21 tumor suppressor. In addition, PIAS1 protein loss correlates with colon cancer development. Previous reports suggested that amplification of the PIAS1 gene locus is observed in human NSCLC. Using single nucleotide polymorphism (SNPs) data we identified concurrent copy number alterations for PIAS1 and the focal adhesion kinase (FAK) in a subset of NSCLC cell lines. PIAS1 and FAK were previously found to interact by a yeast two-hybrid screen. Moreover, FAK SUMOylation by PIAS1 was reported to increase its active phosphorylation at Tyrosine 397 (Y397). We decided to characterize FAK-PIAS1 interaction further and discovered that upon mitogenic stimulation, endogenous FAK and PIAS1 proteins interact in endosomes in the cytoplasm of NSCLC cells. Moreover PIAS1 promotes a modest increase in FAK SUMOylation and C-terminal cleavage by the calcium dependent protease Calpain. Ectopic expression of PIAS1 induces FAK nuclear accumulation and affects gene transcription favoring DNA damage repair and metabolism. Importantly, we found that pharmacologic inhibition of FAK protein or its deletion by CRISPR/CAS9 technology results in susceptibility to DNA damage in vitro and in vivo, providing a valuable strategy for combination therapy using FAK inhibitors (FAKi) and ionizing radiation (IR) in the treatment of human NSCLC. Our results provide new knowledge about PIAS1 and FAK requirement for basic biology and in human disease.