Abstract A04: Alpha-1-Antitrypsin is a secreted protein driven by mutant p53 and associated with EMT, migration and invasion in-vivo
Authors: Shakya Reshma , Turner Andrew G. , Cooper Wendy , Lokman Noor A. , Ricciardelli Carmela , Tarulli Gerard , Neilsen Paul M. , Callen David F. 
Published: May 2016

Half of all human tumors harbor mutations in TP53 gene. Such mutations not only lead to the expression of a protein unable to impart its normal tumor suppressor ability, but also endow the mutant p53 protein with newly acquired gain-of-function (GOF) properties that drive invasion and metastasis. Although it is widely established that mutant p53-expressing tumors have a high capacity to metastasize and are associated with poor outcomes, the key molecular mechanism utilized by mutant p53 are largely unknown. Since, p53 mutations are predominantly found in pre-invasive stages of lung adenocarcinomas, the identification of critical pathways that mediate mutant p53 gain of function (GOF) properties may uncover new targets for cancer therapy. In this study, we investigated the global impact of mutant p53 induced secretome on cancer cell proteome. A1AT is a secreted protein encoded by SERPINA1, a secreted serine protease inhibitor that neutralizes the effect of proteases, and A1AT involvement in mutant p53 driven tumorigenesis is unclear.

Methodology: We have developed a panel of H1299 lung adenocarcinoma derivatives that can inducibly re-express various mutant p53 proteins into a p53 null background. Expression levels were assessed by western blot analyses and real time PCR. Using iTRAQ (isobaric tag for relative and absolute quantification) and Liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses, the proteome of conditioned media from H1299 cells expressing either induced p53 mutants (R175H and R248) or their isogenic un-induced counterparts were identified and quantified. Mutant p53 and p63 bindings sites were analyzed by chromatin immune-precipitation (ChIP) assays. In vitro migration/invasion and in vivo Chicken Chorio-allantoic membrane (CAM) invasion were performed in A1AT knockdown lung adenocarcinoma cells harboring p53 mutation. Public microarray, Kaplan-Meir datasets of human non-small cell lung carcinoma (NSCLCs) and A1AT immunohistochemistry (IHC) of stage I-III lung adenocarcinoma patients tissue microarray (TMA) samples (n=107) with matching normal were analyzed to correlate A1AT with prognosis and clinical significance.

Results: Induction of mutant p53 in H1299 cells drove an invasive phenotype through the release of a pro-invasive secretome, providing a novel avenue through which mutant p53 may be driving invasion and metastasis. Through proteomic characterization of induced conditioned medium from H1299 versus un-induced counterparts, we identified A1AT as a novel secreted mediator and a putative metastasis marker of mutant p53 tumors. To determine the role for A1AT as a downstream mediator of mutant p53 oncogenic pathways, we developed a double inducible H1299 system whereby we can simultaneously (i) induce expression of EI-H1299 R248Q mutant p53 and (ii) induce knockdown of the expression of its target A1AT. Remarkably, silencing the expression of A1AT significantly attenuated mutant p53 dependent migration and invasion both in vitro and in vivo. Importantly, knockdown of A1AT did not alter the basal level of motility in the absence of induced mutant p53, suggesting that the role of A1AT is specific to the mutant p53 pathway. Knockdown of A1AT significantly altered epithelial-mesenchymal transition (EMT) markers expression and reduced the ability of p53 mutant cells to grow in an anchorage independent environment. Treatment of cells with conditioned medium containing secreted A1AT enhanced the cell invasion while A1AT knockdown elicited the opposite effect. A1AT-blocking antibody attenuated the mutant p53 driven migration and invasion, strongly suggesting that mutant p53 is using secretory gene targets to potentiate its gain-of-function. In addition, A1AT intracellular levels were directly regulated by mutant p53 through the involvement with p63, indicating that A1AT is a direct target of mutant p53 transcriptomic regulation.

In public microarray dataset, A1AT mRNA expression was higher in lung adenocarcinoma (AC) and associated with shorter overall survival. Consistent with these findings, immunohistochemistry of 107 lung adenocarcinoma TMA showed upregulation of A1AT expression compared with matching normal tissues. The A1AT signal was cytoplasmic in tumor but not in the core of the tumor. A1AT overexpression in tumor cells correlated with increased tumor size (P&lt;0.046), and strongly with increased tumor progression (advanced T stage; P&lt;0.0006) and shorter survival (P&lt;0.001). In addition, we detected a clear correlation between elevated p53 staining and high levels of A1AT (P&lt;0.018). This indicates A1AT expression is predominantly driven by mutant p53 gain-of-function.

Conclusion: We conclude A1AT is an essential mediator of mutant p53 driven gain-of-function properties and our results highlight crucial roles of A1AT in driving oncogenic transformations. We suggest that A1AT is a potential therapeutic target in lung adenocarcinoma patient tumors expressing mutant p53.

Citation Format: Reshma Shakya, Andrew G. Turner, Wendy Cooper, Noor A. Lokman, Carmela Ricciardelli, Gerard Tarulli, Paul M. Neilsen, David F. Callen. Alpha-1-Antitrypsin is a secreted protein driven by mutant p53 and associated with EMT, migration and invasion in-vivo. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr A04.