Mechanistic Study of TTF-1 Modulation of Cellular Sensitivity to Cisplatin.

The lung lineage master regulator gene, Thyroid Transcription Factor-1 (TTF-1, also known as NKX2-1), is used as a marker by pathologists to identify lung adenocarcinomas since TTF-1 is expressed in 60 ~ 70% of lung ADs. Much research has been conducted to investigate roles of TTF-1 in lung cancer biology. But, how it modulates cellular chemosensitivity remains poorly characterized. Our study shows that TTF-1 sensitizes the KRAS-mutated A549 and NCI-H460 lung cancer cells to cisplatin, a common chemotherapy used to treat lung cancer. This chemosensitization activity does not appear to be mediated by a TTF-1-imposed alteration on nucleotide excision repair. Mechanistically, TTF-1 induced a reduction in p-AKT (S473), which in turn activated glycogen synthase kinase 3 (GSK3) and reduced ß-catenin. Intriguingly, in the EGFR-mutated NCI-H1975 and HCC827 cells, TTF-1 desensitized these cells to cisplatin; concomitantly, TTF-1 conferred an increase in p-AKT. Finally, the conditioned media of TTF-1-transefected cells sensitized TTF-1- cells to cisplatin, implicating that the TTF-1-driven chemosensitization activity may be dually pronged in both intracellular and extracellular compartments. In short, this study highlights the enigmatic activities of TTF-1 in lung cancer, and calls for future research to optimally manage chemotherapy of patients with TTF-1+ lung ADs.

Thyroid transcription factor 1 enhances cellular statin sensitivity via perturbing cholesterol metabolism.

We have discovered an unexpected connection between a critical lung development and cancer gene termed thyroid transcription factor 1 (TTF-1 also known as NKX2-1) and cholesterol metabolism. Our published work implicates that TTF-1 positively regulates miR-33a which is known to repress ATP-binding cassette transporter 1 (ABCA1) and thus its cholesterol efflux activity. We set out to demonstrate that a higher TTF-1 expression would presumably inhibit cholesterol efflux and consequently raise intracellular cholesterol level. Surprisingly, raising TTF-1 expression actually lowers intracellular cholesterol level, which, we believe, is attributed to a direct transactivation of ABCA1 by TTF-1. Subsequently, we show that lung cancer cells primed with a TTF-1-driven decrease of cholesterol were more vulnerable to simvastatin, a frequently prescribed cholesterol biosynthesis inhibitor. In view of the fact that pathologists routinely interrogate human lung cancers for TTF-1 immunopositivity to guide diagnosis and the prevalent use of statins, TTF-1 should be further investigated as a putative biomarker of lung cancer vulnerability to statins.

Roles of Thyroid Transcription Factor 1 in Lung Cancer Biology.

Thyroid transcription factor 1 (TTF-1 or NKX2-1) is a transcription factor of fundamental importance in driving lung maturation and morphogenesis. In the last decade, scientists began to appreciate the functional roles of TTF-1 in lung tumorigenesis. This movement was triggered by the discoveries of genetic alterations of TTF-1 in the form of gene amplification in lung cancer. Many downstream target genes of TTF-1 relevant to the lung cancer biology of TTF-1 have been documented. One of the most surprising findings was that TTF-1 may exhibit either pro- or antitumorigenic activities, an outcome with the complexity exceeding the original anticipation purely based on the fact that TTF-1 undergoes gene amplification in lung cancer. In the coming decade, we believe, we will witness additional surprises as the research exploring the cancer roles of TTF-1 progresses.

Thyroid Transcription Factor 1 Reprograms Angiogenic Activities of Secretome.

Through both gain- and loss-of-TTF-1 expression strategies, we show that TTF-1 positively regulates vascular endothelial growth factor (VEGF) and that the VEGF promoter element contains multiple TTF-1-responsive sequences. The major signaling receptor for VEGF, i.e VEGFR2, also appears to be under a direct and positive regulation of TTF-1. The TTF-1-dependent upregulation of VEGF was moderately sensitive to rapamycin, implicating a partial involvement of mammalian target of rapamycin (mTOR). However, hypoxia did not further increase the secreted VEGF level of the TTF-1(+) lung cancer cells. The TTF-1-induced VEGF upregulation occurs in both compartments (exosomes and exosome-depleted media (EDM)) of the conditioned media. Surprisingly, the EDM of TTF-1(+) lung cancer cells (designated EDM-TTF-1(+)) displayed an anti-angiogenic activity in the endothelial cell tube formation assay. Mechanistic studies suggest that the increased granulocyte-macrophage colony-stimulating factor (GM-CSF) level in the EDM-TTF-1(+) conferred the antiangiogenic activities. In human lung cancer, the expression of TTF-1 and GM-CSF exhibits a statistically significant and positive correlation. In summary, this study provides evidence that TTF-1 may reprogram lung cancer secreted proteome into an antiangiogenic state, offering a novel basis to account for the long-standing observation of favorable prognosis associated with TTF-1(+) lung adenocarcinomas.

Peptide Inhibitor of Complement C1 (PIC1) Rapidly Inhibits Complement Activation after Intravascular Injection in Rats.

The complement system has been increasingly recognized to play a pivotal role in a variety of inflammatory and autoimmune diseases. Consequently, therapeutic modulators of the classical, lectin and alternative pathways of the complement system are currently in pre-clinical and clinical development. Our laboratory has identified a peptide that specifically inhibits the classical and lectin pathways of complement and is referred to as Peptide Inhibitor of Complement C1 (PIC1). In this study, we determined that the lead PIC1 variant demonstrates a salt-dependent binding to C1q, the initiator molecule of the classical pathway. Additionally, this peptide bound to the lectin pathway initiator molecule MBL as well as the ficolins H, M and L, suggesting a common mechanism of PIC1 inhibitory activity occurs via binding to the collagen-like tails of these collectin molecules. We further analyzed the effect of arginine and glutamic acid residue substitution on the complement inhibitory activity of our lead derivative in a hemolytic assay and found that the original sequence demonstrated superior inhibitory activity. To improve upon the solubility of the lead derivative, a pegylated, water soluble variant was developed, structurally characterized and demonstrated to inhibit complement activation in mouse plasma, as well as rat, non-human primate and human serum in vitro. After intravenous injection in rats, the pegylated derivative inhibited complement activation in the blood by 90% after 30 seconds, demonstrating extremely rapid function. Additionally, no adverse toxicological effects were observed in limited testing. Together these results show that PIC1 rapidly inhibits classical complement activation in vitro and in vivo and is functional for a variety of animal species, suggesting its utility in animal models of classical complement-mediated diseases.