Distinct roles of autophagy-dependent and -independent functions of FIP200 revealed by generation and analysis of a mutant knock-in mouse model

Abstract

Autophagy is an evolutionarily conserved cellular process controlled through a set of essential autophagy genes (Atgs). However, there is increasing evidence that most, if not all, Atgs also possess functions independent of their requirement in canonical autophagy, making it difficult to distinguish the contributions of autophagy-dependent or -independent functions of a particular Atg to various biological processes. To distinguish these functions for FIP200 (FAK family-interacting protein of 200 kDa), an Atg in autophagy induction, we examined FIP200 interaction with its autophagy partner, Atg13. We found that residues 582-585 (LQFL) in FIP200 are required for interaction with Atg13, and mutation of these residues to AAAA (designated the FIP200-4A mutant) abolished its canonical autophagy function in vitro. Furthermore, we created a FIP200-4A mutant knock-in mouse model and found that specifically blocking FIP200 interaction with Atg13 abolishes autophagy in vivo, providing direct support for the essential role of the ULK1/Atg13/FIP200/Atg101 complex in the process beyond previous studies relying on the complete knockout of individual components. Analysis of the new mouse model showed that nonautophagic functions of FIP200 are sufficient to fully support embryogenesis by maintaining a protective role in TNFα-induced apoptosis. However, FIP200-mediated canonical autophagy is required to support neonatal survival and tumor cell growth. These studies provide the first genetic evidence linking an Atg's autophagy and nonautophagic functions to different biological processes in vivo.

Targeting autophagy in cancer

“…Genetically engineered mouse models of lung cancer, pancreatic cancer, and melanoma, driven by either mutant rat sarcoma ( RAS ) or B‐Raf proto‐oncogene, serine/threonine kinase ( BRAF ) in which autophagy genes were deleted, have demonstrated that autophagy suppresses the growth of benign tumors but accelerates the growth of advanced cancers . This also was observed in a mouse model of breast cancer …”

“…[30][31][32][33][34] This also was observed in a mouse model of breast cancer. 35,36 Mouse models of tumorigenesis should not be used to understand the utility of inhibiting autophagy in the therapeutic context, because autophagy genes are usually deleted in utero at the same time that oncogenes and tumor-suppressor genes are altered. In patients, autophagy inhibitors will be deployed after the cancer is already formed in the adult, and likely in combination with other agents.…”

Autophagy in cancer metastasis

“…Numerous factors affecting FIP200 levels and post-translational modification likely play into which functions of FIP200 are dominant or indeed exclusive in any given cell type or cellular stress condition. Recent studies have begun to separate out the functions of FIP200 in autophagy from those in other cellular processes (109). By mutating amino acids 582–585 to alanine (FIP200-4A) and preventing the interaction of FIP200 with Atg13 (Autophagy related gene 13), Chen et al were able to block the function of FIP200 in autophagy (109).…”

“…Recent studies have begun to separate out the functions of FIP200 in autophagy from those in other cellular processes (109). By mutating amino acids 582–585 to alanine (FIP200-4A) and preventing the interaction of FIP200 with Atg13 (Autophagy related gene 13), Chen et al were able to block the function of FIP200 in autophagy (109). Interestingly, knock-in of the FIP200-4A mutant allele into mice extended the lifespan of Fip200−/− mice from E16.5 (110) to birth when other Atg gene knockout mice are known to die (111).…”

Therapeutic Targeting of Autophagy

“…A requirement for autophagy to maintain tumors in vivo has also been demonstrated for cancers driven by PTEN loss (Santanam et al, 2016). Moreover, additional autophagy regulators such as FIP200, have been shown to maintain tumor growth due to autophagy and not other functions of the protein (Chen et al, 2016). One caveat for many of the mouse studies is that deletion of the Atg gene occurs simultaneously with activation of the oncogenic driver and occurs only in the tumor cells.…”