Role of ER-phagy and proteostatic defects in pancreatic cancer

Abstract

Despite being the most frequently mutated oncogene in pancreatic ductal adenocarcinoma (PDAC), Kras mutation in pancreatic acinar cells in mouse models does not lead to immediate morphological changes. Indeed, the molecular events driven by Kras that precede and facilitate the earliest transformation towards malignancy, such as acinar-ductal metaplasia (ADM) and PanIN formation, are unclear.

The precursors of PDAC, pancreatic acinar cells, are highly-specialised secretory cells and thus, vulnerable to changes in ER homeostasis. In fact, it has been shown that the recently characterised ER-phagy cargo receptor CCPG1, plays a role in maintaining ER proteostasis in these cells. However, it is unclear whether this is due to its role in ER-phagy, a selective form of degradation of the endoplasmic reticulum (ER) that involves autophagy-mediated delivery of ER fragments – which may contain pathologic aggregates of proteins – to the lysosomes. Here, initial experiments in PDAC cells reveal no significant role for ER-phagy in PDAC cell physiology. I thus developed an in vivo model to study CCPG1 function in acinar cell physiology – as these primary cells cannot be cultured in vitro – and to interrogate its potential contribution to tumourigenesis. Preliminary data using a novel ER-phagy reporter (ss-SRAI-KDEL) showed that indeed, deletion of Ccpg1 led to reduction in ER-phagy flux in pancreatic acinar cells. Not only that, but in a Kras mutant mouse model of PDAC initiation (KC model), heterogeneous degrees of suppression of ER-phagy across the acinar cell compartment of the KC mouse pancreas were observed, and the areas of greatest downregulation were tightly spatially correlated with ADM formation.

Therefore, to interrogate whether such loss of ER-phagy would drive ADM and PanINs, a genetically engineered mouse model with a conditional deletion of the key ER-phagy gene, Ccpg1, was generated. This led to homogenous ER proteostatic defects across the acinar cell compartment and widespread acceleration of inflammation and ADM in the KC model. Proteomic analysis of purified acini revealed a small group of highly-aggregation prone ER luminal proteins that accumulated and aggregated upon ER-phagy loss. Crucially, Kras mutation alone is sufficient to drive these protein aggregates in acinar cells, given sufficient time, and this phenotype is tightly spatially associated with the formerly observed suppression of ER-phagy and incipient ADM. It is then conceivable to assume that Kras-driven proteostatic defects are molecular events in acinar cells that facilitate their transdifferentiation to ADM.

Thus, to understand how impaired proteostasis predisposes acinar cells towards malignant transformation, initial transcriptional analyses were performed in pancreata of CCPG1-deficient mice. Gene Set Enrichment Analysis (GSEA) showed that gene sets associated with pancreatic injury were enriched in these mice, suggesting ER-phagy deficiency causes an injury-like phenotype in acinar cells. Finally, spatial transcriptomics was used in KC murine pancreata to identify the transcriptional changes in the subpopulation of acinar cells presenting sporadic proteostatic defects. In these cells, the injury-specific genes were also enriched, confirming that Kras mutation alone drives the same molecular changes as ER-phagy failure.

Taken together, the results presented in this thesis suggest that dysregulation of ER proteostasis and aggregation of proteins predispose acinar cells to undergo ADM by inducing a phenotype similar to inflammation and injury and thus, are mechanistic hallmarks of early steps in Kras-driven tumourigenesis.