Faculty Bibliography

Oncogenic activation of RAS is associated with the acquisition of a unique set of metabolic dependencies that contribute to tumour cell fitness. Cells that express oncogenic RAS are able to internalize and degrade extracellular protein via a fluid-phase uptake mechanism termed macropinocytosis¹. There is increasing recognition of the role of this RAS-dependent process in the generation of free amino acids that can be used to support tumour cell growth under nutrient-limiting conditions². However, little is known about the molecular steps that mediate the induction of macropinocytosis by oncogenic RAS. Here we identify vacuolar ATPase (V-ATPase) as an essential regulator of RAS-induced macropinocytosis. Oncogenic RAS promotes the translocation of V-ATPase from intracellular membranes to the plasma membrane via a pathway that requires the activation of protein kinase A by a bicarbonate-dependent soluble adenylate cyclase. Accumulation of V-ATPase at the plasma membrane is necessary for the cholesterol-dependent plasma-membrane association of RAC1, a prerequisite for the stimulation of membrane ruffling and macropinocytosis. These observations establish a link between V-ATPase trafficking and nutrient supply by macropinocytosis that could be exploited to curtail the metabolic adaptation capacity of RAS-mutant tumour cells.

Pancreatic adenocarcinoma (PDA) is an aggressive disease driven by oncogenic KRAS and characterized by late diagnosis and therapeutic resistance. Here we show that deletion of the ataxia-telangiectasia group D-complementing (Atdc) gene, whose human homolog is up-regulated in the majority of pancreatic adenocarcinoma, completely prevents PDA development in the context of oncogenic KRAS. ATDC is required for KRAS-driven acinar-ductal metaplasia (ADM) and its progression to pancreatic intraepithelial neoplasia (PanIN). As a result, mice lacking ATDC are protected from developing PDA. Mechanistically, we show ATDC promotes ADM progression to PanIN through activation of β-catenin signaling and subsequent SOX9 up-regulation. These results provide new insight into PDA initiation and reveal ATDC as a potential target for preventing early tumor-initiating events.

The immune microenvironment of pancreatic ductal adenocarcinoma (PDA) is comprised of a heterogeneous population of cells that are critical for disease evolution. Prominent among these are the specialized CD1d^hiCD5⁺ regulatory B (B_reg) cells that exert a pro-tumorigenic role by promoting tumor cell proliferation. Dissecting the molecular pathways regulating this immune sub-population can thus be valuable for uncovering potential therapeutic targets. Here, we investigate Bruton's tyrosine kinase (BTK), a key B-cell kinase, as a potential regulator of CD1d^hiCD5⁺ B_reg differentiation in the pancreatic tumor microenvironment. Treatment of cytokine-induced B cells in vitro with the high specificity BTK inhibitor Tirabrutinib inhibited CD1d^hiCD5⁺ B_reg differentiation and production of IL-10 and IL-35, essential mediators of B_reg immunosuppressive functions. The BTK signaling pathway was also found to be active in vivo in PanIN-associated regulatory B cells. Tirabrutinib treatment of mice bearing orthotopic Kras^G12D-pancreatic lesions severely compromised stromal accumulation of the CD1d^hiCD5⁺ B_reg population. This was accompanied by an increase in stromal CD8⁺IFNγ⁺ cytotoxic T cells and significant attenuation of tumor cell proliferation and PanIN growth. Our results uncover a novel role for BTK in regulating CD1d^hiCD5⁺ B_reg differentiation and emphasize its potential as a therapeutic target for pancreatic cancer.

The BRCA1-BRCA2-RAD51 axis is essential for homologous recombination repair (HRR) and is frequently disrupted in breast cancers. PARP inhibitors (PARPis) are used clinically to treat BRCA-mutated breast tumors. Using a genetic screen, we identified EMI1 as a modulator of PARPi sensitivity in triple-negative breast cancer (TNBC) cells. This function requires the F-box domain of EMI1, through which EMI1 assembles a canonical SCF ubiquitin ligase complex that constitutively targets RAD51 for degradation. In response to genotoxic stress, CHK1-mediated phosphorylation of RAD51 counteracts EMI1-dependent degradation by enhancing RAD51's affinity for BRCA2, leading to RAD51 accumulation. Inhibition of RAD51 degradation restores HRR in BRCA1-depleted cells. Human breast cancer samples display an inverse correlation between EMI1 and RAD51 protein levels. A subset of BRCA1-deficient TNBC cells develop resistance to PARPi by downregulating EMI1 and restoring RAD51-dependent HRR. Notably, reconstitution of EMI1 expression reestablishes PARPi sensitivity both in cellular systems and in an orthotopic mouse model.

With the recent breakthroughs in immunotherapy as curative treatments in certain tumor types, there has been renewed interest in the relationship between immunity and tumor growth. Although we are gaining a greater understanding of the complex interplay of immune modulating components in the tumor microenvironment, the specific role that tumor cells play in shaping the immune milieu is still not well characterized. In this review, we focus on how mutant Kras tumor cells contribute to tumor immunity, with a specific focus on processes induced directly or indirectly by the oncogene.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease and a major health problem in the United States. While the cytokine TGF-β has been implicated in PDAC development, it can exert both pro-tumorigenic and anti-tumorigenic effects that are highly context dependent and incompletely understood. Using three-dimensional (3D) cultures of Kras ^G12D -expressing mouse pancreatic epithelial cells we demonstrated that while exposure to exogenous TGF-β induced growth arrest of the Kras ^G12D cells, its subsequent removal allowed the cells to enter a hyper-proliferative, partially mesenchymal (PM), and progenitor-like state. This state was highly stable and was maintained by autocrine TGF-β signaling. While untreated Kras ^G12D cells formed cystic lesions in vivo, PM cells formed ductal structures resembling human PanINs, suggesting that they had attained increased oncogenic potential. Supporting this hypothesis, we determined that the PM cells share salient molecular and phenotypic features with the quasi-mesenchymal/squamous subtype of human PDAC, which has the worst prognosis of any of the recently identified subtypes. Transient pulses of TGF-β have been observed during pancreatitis, a major risk factor for PDAC. Our data suggest that transient TGF-β exposure is sufficient to induce the acquisition of stable PDAC-associated phenotypes in pre-neoplastic Kras ^G12D cells, providing novel molecular insight into the complex role of TGF-β in tumorigenesis.

Epithelial-mesenchymal transition (EMT) is strongly implicated in tumor cell invasion and metastasis. EMT is thought to be regulated primarily at the transcriptional level through the repressive activity of EMT transcription factors. However, these classical mechanisms have been parsed out almost exclusively in vitro, leaving questions about the programs driving EMT in physiological contexts. Here, using a lineage-labeled mouse model of pancreatic ductal adenocarcinoma to study EMT in vivo, we found that most tumors lose their epithelial phenotype through an alternative program involving protein internalization rather than transcriptional repression, resulting in a "partial EMT" phenotype. Carcinoma cells utilizing this program migrate as clusters, contrasting with the single-cell migration pattern associated with traditionally defined EMT mechanisms. Moreover, many breast and colorectal cancer cell lines utilize this alternative program to undergo EMT. Collectively, these results suggest that carcinoma cells have different ways of losing their epithelial program, resulting in distinct modes of invasion and dissemination.

The product of the KRAS oncogene, KRAS4B, promotes tumor growth when associated with the plasma membrane (PM). PM association is mediated, in part, by farnesylation of KRAS4B, but trafficking of nascent KRAS4B to the PM is incompletely understood. We performed a genome-wide screen to identify genes required for KRAS4B membrane association and identified a G protein-coupled receptor, GPR31. GPR31 associated with KRAS4B on cellular membranes in a farnesylation-dependent fashion, and retention of GPR31 on the endoplasmic reticulum inhibited delivery of KRAS4B to the PM. Silencing of GPR31 expression partially mislocalized KRAS4B, slowed the growth of KRAS-dependent tumor cells, and blocked KRAS-stimulated macropinocytosis. Our data suggest that GPR31 acts as a secretory pathway chaperone for KRAS4B.