High expression of SERPINA1 gene encoding acute phase protein, alpha1-antitrypsin (AAT), is associated with various tumors. We sought to examine the significance of SERPINA1 and AAT protein in non-small-cell lung cancer (NSCLC) patients and NSCLC cell lines. Tumor and adjacent non-tumor lung tissues and serum samples from 351 NSCLC patients were analyzed for SERPINA1 expression and AAT protein levels. We also studied the impact of SERPINA1 expression and AAT protein on H1975 and H661 cell behavior, in vitro. Lower SERPINA1 expression in tumor but higher in adjacent non-tumor lung tissues (n = 351, p = 0.016) as well as higher serum levels of AAT protein (n = 170, p = 0.033) were associated with worse survival rates. Specifically, in NSCLC stage III patients, higher blood AAT levels (>2.66 mg/mL) correlated with a poor survival (p = 0.002). Intriguingly, levels of serum AAT do not correlate with levels of C-reactive protein, neutrophils-to-leukocyte ratio, and do not correlate with SERPINA1 expression or AAT staining in the tumor tissue. Additional experiments in vitro revealed that external AAT and/or overexpressed SERPINA1 gene significantly improve cancer cell migration, colony formation and resistance to apoptosis. SERPINA1 gene and AAT protein play an active role in the pathogenesis of lung cancer and not just reflect inflammatory reaction related to cancer development.

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Elevated levels of plasma alpha1-antitrypsin (AAT) correlate with a poor prognosis of various cancers. Herein, we investigated effects of exogenous AAT on non-small lung cancer cell lines with high (H1975) and very low (H661) baseline expression of SERPINA1 gene encoding AAT protein. Comparison of cells grown for 3 weeks in a regular medium versus medium supplemented with 2 mg/ml of AAT revealed that in the presence of AAT cells acquire better proliferative properties, resistance to staurosporine (STS)-induced apoptosis, and show higher expression of CLU, a pro-tumorigenic gene coding clusterin protein. Similarly, the co-administration of STS with AAT or addition of AAT to the cells pre-treated with STS abrogated effects of STS in both cell lines. Following experiments with H1975 cells have shown that AAT blocks critical steps in STS-induced cell death: inhibition of AKT/MAPK pathways, and activation of caspase 3 and autophagy. AAT does not inhibit apoptosis-triggered by chloroquine (inhibitor of autophagy) or streptonigrin (inducer of p53 pathway). The anti-apoptotic effects of AAT were unaffected by lipopolysaccharide (LPS). However, AAT induced TLR4 levels and enhanced LPS effects on the production of IL-6, a tumor-promoting cytokine. Our data provide further evidence that AAT plays a significant role in the tumorigenesis. In recent years, an importance of the inflammation in cancer progression became more recognised 1. In both early and advanced stages of cancer, the development and maintenance of a systemic inflammation confer poorer outcome 2. Repeated studies show that neutrophil, lymphocyte and platelet counts, and acute phase proteins, such as C-reactive protein and albumin and their combinations, have a prognostic value 3-5. Besides, the major hallmark of human cancers is the resistance to apoptosis and treatments, since most current anticancer therapies, including chemotherapy, radio-and immunotherapy, primarily act by activating cell death pathways 6. Different research groups reported that acute phase proteins interfere with apoptosis induction. For example, α1-acid glycoprotein expresses a direct anti-apoptotic mode on tumor necrosis factor (TNF)-α-induced apoptosis 7. The C-reactive protein enhances secretion of interleukin 6 (IL-6) and, synergized with IL-6, protects cancer cells from chemotherapy-induced apoptosis 8,9. There are observations that certain members of serine protease inhibitor (serpin) superfamily, such as plasminogen activator inhibitor-1, are also associated with poor outcome in several types of cancer 10. Alpha1-antitrypsin (AAT) is another acute phase protein and an archetype member of serpin superfamily, well recognized for its role as a serine protease inhibitor, but also known as an inhibitor of caspase 3, anti-apoptotic and immunomodulatory protein in vitro and in vivo 11. Various studies provide evidence that higher AAT levels are associated with cancer and a poor prognosis 12,13. AAT seems to be directly involved in the distant metastasis of various cancer types, including...

33Motile cilia serve vital functions in development, homeostasis and regeneration. We recently 34 demonstrated that TAp73 is an essential transcriptional regulator of respiratory multiciliogenesis. 35Here, we show that TAp73 is expressed in multiciliated cells (MCCs) of diverse tissues. Analysis 36 of TAp73 mutant animals revealed that TAp73 regulates Foxj1, Rfx2, Rfx3, axonemal dyneins 37 Dnali1 and Dnai1, plays a pivotal role in the generation of MCCs in male and female reproductive 38 ducts, and contributes to fertility. However, the function of MCCs in the brain appears to be 39 preserved despite the loss of TAp73, and robust activity of cilia-related networks is maintained in 40 the absence of TAp73. Notably, TAp73 loss leads to distinct changes in ciliogenic microRNAs: 41 miR34bc expression is reduced, whereas the miR449 cluster is induced in diverse multiciliated 42 epithelia. Among different MCCs, choroid plexus (CP) epithelial cells in the brain display prominent 43 miR449 expression, whereas brain ventricles exhibit significant increase in miR449 levels along 44 with an increase in the activity of ciliogenic E2F4/MCIDAS circuit in TAp73 mutant animals. 45Conversely, E2F4 induces robust transcriptional response from miR449 genomic regions. To 46 address whether increased miR449 levels in the brain maintain the multiciliogenesis program in 47 the absence of TAp73, we deleted both TAp73 and miR449 in mice. Although loss of miR449 48 alone led to a mild ciliary defect in the CP, more pronounced ciliary defects and hydrocephalus 49 were observed in the brain lacking both TAp73 and miR449. In contrast, miR449 loss in other 50MCCs failed to enhance ciliary defects associated with TAp73 loss. Together, our study shows 51 that, in addition to the airways, TAp73 is essential for generation of MCCs in male and female 52 reproductive ducts, whereas miR449 and TAp73 complement each other to support 53 multiciliogenesis and CP development in the brain. 54

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