Abstract A021: Divergent roles for BNIP3 and NIX in tumor progression and metastasis
Authors: Berardi Damian , Macleod Kay 
Published: January 2023

Abstract
BNIP3 and NIX (BNIP3L) are mitochondrial cargo receptors (MCRs) induced by physiological stresses, including hypoxia and nutrient deprivation, to promote turnover of mitochondria at the autolysosome in a process known as mitophagy. We and others have shown that BNIP3 and NIX function similarly in mitophagy by integrating into the outer mitochondrial membrane (OMM) via their conserved C-terminal transmembrane domain while interacting via conserved N-terminal LIR motifs with processed LC3 at nascent phagophores. To address functional roles of BNIP3 and NIX in tumorigenesis and metastasis in vivo, we examined the effect of loss of BNIP3 and/or NIX in two different mouse models: (1) the 4T1 orthotopic model of mammary tumorigenesis, and (2) the KRasG12D driven autochthonous model of pancreatic ductal adenocarcinoma (PDAC). Using CRISPR/Cas9 gene editing to delete BNIP3 (DBNIP3), NIX (DNIX) or both BNIP3 and NIX (DKO) in 4T1 cells, we showed that while loss of BNIP3 increased tumor growth, promoted invasion/migration and progression to metastasis in vivo, loss of NIX decreased primary tumor growth, inhibited invasion/migration and markedly attenuated metastasis to the lungs. Intriguingly, the effect of NIX loss was dominant over the effect of BNIP3 loss, such that DKO 4T1 cells phenocopied DNIX 4T1 cells in exhibiting decreased growth, invasion/migration properties and inability to metastasize from the mammary gland. Similar findings were observed in Pdx1-Cre;LSL-KRasG12D (KC) mice lacking BNIP3 and/or NIX with KC-DBNIP3 mice showing accelerated progression from PanIN to PDAC, increased organoid growth in vitro and increased metastasis to the liver compared to control KC mice, while KC-DNIX and KC-DKO mice showed increased tumor latency and absence of metastases. Thus, despite 55% homology in primary amino acid sequence and a shared role in mitophagy, our work indicates that BNIP3 and NIX do not function analogously in vivo in tumorigenesis and metastasis. In summary, our work shows that BNIP3 suppresses tumorigenesis in mouse models of breast, pancreas (and liver) cancer and that loss of BNIP3 correlates with worse prognosis in several human cancers. Conversely, NIX is required for progression to malignancy in models of both breast and pancreatic ductal adenocarcinoma (PDAC), and elevated NIX levels correlated with worse overall patient survival. These opposing roles for BNIP3 and NIX in progression to malignancy were unexpected and work presented will discuss mechanistic insight to their differential roles in tumorigenesis and metastasis, including a novel role for BNIP3 in suppressing RAS pathway signaling and a new role for NIX in promoting EMT. These novel functions are in addition to their canonical roles in mitophagy.
Citation Format: Damian Berardi, Kay Macleod. Divergent roles for BNIP3 and NIX in tumor progression and metastasis [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr A021.