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DOCK8 is a Novel Regulator of Lysosome Mediated Pancreatic Cancer Invasion

ABSTRACT: Amplified lysosome activity is a hallmark of pancreatic ductal adenocarcinoma (PDAC) orchestrated by oncogenic KRAS that mediates tumor progression and metastasis, though the mechanisms underlying this phenomenon remain unclear. Using comparative proteomics, we found that oncogenic KRAS significantly enriches levels of the guanine nucleotide exchange factor (GEF) DOCK8 on lysosomes. Surprisingly, we found that DOCK8 is aberrantly expressed in a subset of PDAC where it promotes cell invasion and proliferation. Further, DOCK8 associates with lysosomes and regulates lysosomal morphology and motility. DOCK8 promotes actin polymerization at the surface of lysosomes, while increasing the levels and proteolytic activity of the lysosomal protease cathepsin B. Depletion of DOCK8 significantly reduces extracellular matrix degradation and impairs the invasive capacity of PDAC cells. Further, CRISPR-based knockout of DOCK8 dramatically reduces tumor size and metastasis in vivo. Together, these findings implicate ectopic expression of DOCK8 as a key driver of KRAS-driven lysosomal regulation, tumor progression, and metastasis.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Mus Musculus (ncbitaxon:10090)

SUBMITTER: Gina L Razidlo

PROVIDER: MSV000091036 | MassIVE | Tue Jan 10 09:25:00 GMT 2023

SECONDARY ACCESSION(S): PXD039351

REPOSITORIES: MassIVE

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Project description:The maintenance of advanced malignancies relies on continued activity of driver oncogenes, although their rate-limiting role is highly context-dependent with respect to tumor types and associated genetic alterations. Oncogenic Kras mutation is the signature event in human pancreatic ductal adenocarcinoma (PDAC), serving a critical role in tumor initiation. Here, an inducible KrasG12D-driven p53 mutant PDAC mouse model establishes that advanced PDAC remains strictly dependent on continued KrasG12D expression and that KrasG12D serves a vital role in the control of tumor metabolism, through stimulation of glucose uptake and channeling of glucose intermediates through the hexosamine biosynthesis pathway (HBP) and the pentose phosphate pathway (PPP). Notably, these studies reveal that oncogenic Kras regulates ribose biogenesis. Unlike canonical models of PPP-mediated ribose biogenesis, we demonstrate that oncogenic Kras drives intermediates from enhanced glycolytic flux into the non-oxidative arm of the PPP, thereby decoupling ribose biogenesis from NADPNADPH-mediated redox control. Together, this work provides in vivo mechanistic insights into how oncogenic Kras promotes metabolic reprogramming in native tumors and illuminates potential metabolic targets that can be exploited for therapeutic benefit in Kras-driven PDAC. Primary pancreatic tumor lines were established from p48Cre tetO_LKrasG12D ROSA_rtTAL+ p53L+ mice. Five independent tumor lines (iKras1-5) were used for pancreatic injection into nude mice to generate orthotopic tumors. The mice were kept on doxycycline for 2 weeks until obvious tumor formation. Half of the animals were pulled off doxycycline for 24 hours. Tumors with over 75% cellularity were collected for total RNA prepartion. For in vitro expression profiles, the same five tumor lines were cultured in the presence or absence of doxycycline for 24 hours and total cellular RNA was prepared. For control samples, two independent tumor lines from LSL-KrasG12D p53L+ tumors were cultured in the presence or absence of doxycycline for 24 hours and total cellular RNA was prepared.

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DOCK8 is a Novel Regulator of Lysosome Mediated Pancreatic Cancer Invasion

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