Autotaxin-lysolipid signaling suppresses a CCL11-eosinophil axis to promote pancreatic cancer progression.

Lipids and their modifying enzymes regulate diverse features of the tumor microenvironment and cancer progression. The secreted enzyme autotaxin (ATX) hydrolyzes extracellular lysophosphatidylcholine to generate the multifunctional lipid mediator lysophosphatidic acid (LPA) and supports the growth of several tumor types, including pancreatic ductal adenocarcinoma (PDAC). Here we show that ATX suppresses the accumulation of eosinophils in the PDAC microenvironment. Genetic or pharmacologic ATX inhibition increased the number of intratumor eosinophils, which promote tumor cell apoptosis locally and suppress tumor progression. Mechanistically, ATX suppresses eosinophil accumulation via an autocrine feedback loop, wherein ATX-LPA signaling negatively regulates the activity of the AP-1 transcription factor c-Jun, in turn suppressing the expression of the potent eosinophil chemoattractant CCL11 (eotaxin-1). Eosinophils were identified in human PDAC specimens, and rare individuals with high intratumor eosinophil abundance had the longest overall survival. Together with recent findings, this study reveals the context-dependent, immune-modulatory potential of ATX-LPA signaling in cancer.

A Cancer Cell-Intrinsic GOT2-PPARδ Axis Suppresses Antitumor Immunity.

Despite significant recent advances in precision medicine, pancreatic ductal adenocarcinoma (PDAC) remains near uniformly lethal. Although immune-modulatory therapies hold promise to meaningfully improve outcomes for patients with PDAC, the development of such therapies requires an improved understanding of the immune evasion mechanisms that characterize the PDAC microenvironment. Here, we show that cancer cell-intrinsic glutamic-oxaloacetic transaminase 2 (GOT2) shapes the immune microenvironment to suppress antitumor immunity. Mechanistically, we find that GOT2 functions beyond its established role in the malate-aspartate shuttle and promotes the transcriptional activity of nuclear receptor peroxisome proliferator-activated receptor delta (PPARδ), facilitated by direct fatty acid binding. Although GOT2 is dispensable for cancer cell proliferation in vivo, the GOT2-PPARδ axis promotes spatial restriction of both CD4+ and CD8+ T cells from the tumor microenvironment. Our results demonstrate a noncanonical function for an established mitochondrial enzyme in transcriptional regulation of immune evasion, which may be exploitable to promote a productive antitumor immune response. SIGNIFICANCE: Prior studies demonstrate the important moonlighting functions of metabolic enzymes in cancer. We find that the mitochondrial transaminase GOT2 binds directly to fatty acid ligands that regulate the nuclear receptor PPARδ, and this functional interaction critically regulates the immune microenvironment of pancreatic cancer to promote tumor progression. See related commentary by Nwosu and di Magliano, p. 2237.. This article is highlighted in the In This Issue feature, p. 2221.

Mesenchymal Lineage Heterogeneity Underlies Nonredundant Functions of Pancreatic Cancer-Associated Fibroblasts.

Cancer-associated fibroblast (CAF) heterogeneity is increasingly appreciated, but the origins and functions of distinct CAF subtypes remain poorly understood. The abundant and transcriptionally diverse CAF population in pancreatic ductal adenocarcinoma (PDAC) is thought to arise from a common cell of origin, pancreatic stellate cells (PSC), with diversification resulting from cytokine and growth factor gradients within the tumor microenvironment. Here we analyzed the differentiation and function of PSCs during tumor progression in vivo. Contrary to expectations, we found that PSCs give rise to a numerically minor subset of PDAC CAFs. Targeted ablation of PSC-derived CAFs within their host tissue revealed nonredundant functions for this defined CAF population in shaping the PDAC microenvironment, including production of specific extracellular matrix components and tissue stiffness regulation. Together, these findings link stromal evolution from distinct cells of origin to transcriptional heterogeneity among PDAC CAFs and demonstrate unique functions for CAFs of a defined cellular origin. SIGNIFICANCE: By tracking and ablating a specific CAF population, we find that a numerically minor CAF subtype from a defined cell of origin plays unique roles in establishing the pancreatic tumor microenvironment. Together with prior studies, this work suggests that mesenchymal lineage heterogeneity and signaling gradients diversify PDAC CAFs.See related commentary by Cukierman, p. 296.This article is highlighted in the In This Issue feature, p. 275.

Acidic fibroblast growth factor underlies microenvironmental regulation of MYC in pancreatic cancer.

Despite a critical role for MYC as an effector of oncogenic RAS, strategies to target MYC activity in RAS-driven cancers are lacking. In genetically engineered mouse models of lung and pancreatic cancer, oncogenic KRAS is insufficient to drive tumorigenesis, while addition of modest MYC overexpression drives robust tumor formation, suggesting that mechanisms beyond the RAS pathway play key roles in MYC regulation and RAS-driven tumorigenesis. Here we show that acidic fibroblast growth factor (FGF1) derived from cancer-associated fibroblasts (CAFs) cooperates with cancer cell-autonomous signals to increase MYC level, promoter occupancy, and activity. FGF1 is necessary and sufficient for paracrine regulation of MYC protein stability, signaling through AKT and GSK-3ß to increase MYC half-life. Patient specimens reveal a strong correlation between stromal CAF content and MYC protein level in the neoplastic compartment, and identify CAFs as the specific source of FGF1 in the tumor microenvironment. Together, our findings demonstrate that MYC is coordinately regulated by cell-autonomous and microenvironmental signals, and establish CAF-derived FGF1 as a novel paracrine regulator of oncogenic transcription.

EHD1 and RUSC2 Control Basal Epidermal Growth Factor Receptor Cell Surface Expression and Recycling.

Epidermal growth factor receptor (EGFR) is a prototype receptor tyrosine kinase and an oncoprotein in many solid tumors. Cell surface display of EGFR is essential for cellular responses to its ligands. While postactivation endocytic trafficking of EGFR has been well elucidated, little is known about mechanisms of basal/preactivation surface display of EGFR. Here, we identify a novel role of the endocytic regulator EHD1 and a potential EHD1 partner, RUSC2, in cell surface display of EGFR. EHD1 and RUSC2 colocalize with EGFR in vesicular/tubular structures and at the Golgi compartment. Inducible EHD1 knockdown reduced the cell surface EGFR expression with accumulation at the Golgi compartment, a phenotype rescued by exogenous EHD1. RUSC2 knockdown phenocopied the EHD1 depletion effects. EHD1 or RUSC2 depletion impaired the EGF-induced cell proliferation, demonstrating that the novel, EHD1- and RUSC2-dependent transport of unstimulated EGFR from the Golgi compartment to the cell surface that we describe is functionally important, with implications for physiologic and oncogenic roles of EGFR and targeted cancer therapies.

A Stromal Lysolipid-Autotaxin Signaling Axis Promotes Pancreatic Tumor Progression.

Pancreatic ductal adenocarcinoma (PDAC) develops a pronounced stromal response reflecting an aberrant wound-healing process. This stromal reaction features transdifferentiation of tissue-resident pancreatic stellate cells (PSC) into activated cancer-associated fibroblasts, a process induced by PDAC cells but of unclear significance for PDAC progression. Here, we show that PSCs undergo a dramatic lipid metabolic shift during differentiation in the context of pancreatic tumorigenesis, including remodeling of the intracellular lipidome and secretion of abundant lipids in the activated, fibroblastic state. Specifically, stroma-derived lysophosphatidylcholines support PDAC cell synthesis of phosphatidylcholines, key components of cell membranes, and also facilitate production of the potent wound-healing mediator lysophosphatidic acid (LPA) by the extracellular enzyme autotaxin, which is overexpressed in PDAC. The autotaxin-LPA axis promotes PDAC cell proliferation, migration, and AKT activation, and genetic or pharmacologic autotaxin inhibition suppresses PDAC growth in vivo. Our work demonstrates how PDAC cells exploit the local production of wound-healing mediators to stimulate their own growth and migration. SIGNIFICANCE: Our work highlights an unanticipated role for PSCs in producing the oncogenic LPA signaling lipid and demonstrates how PDAC tumor cells co-opt the release of wound-healing mediators by neighboring PSCs to promote their own proliferation and migration.See related commentary by Biffi and Tuveson, p. 578.This article is highlighted in the In This Issue feature, p. 565.

Cdc42 activation couples fluid shear stress to apical endocytosis in proximal tubule cells.

Cells lining the kidney proximal tubule (PT) respond to acute changes in glomerular filtration rate and the accompanying fluid shear stress (FSS) to regulate reabsorption of ions, glucose, and other filtered molecules and maintain glomerulotubular balance. Recently, we discovered that exposure of PT cells to FSS also stimulates an increase in apical endocytic capacity (Raghavan et al. PNAS, 111:8506-8511, 2014). We found that FSS triggered an increase in intracellular Ca2+ concentration ([Ca2+]i) that required release of extracellular ATP and the presence of primary cilia. In this study, we elucidate steps downstream of the increase in [Ca2+]i that link FSS-induced calcium increase to increased apical endocytic capacity. Using an intramolecular FRET probe, we show that activation of Cdc42 is a necessary step in the FSS-stimulated apical endocytosis cascade. Cdc42 activation requires the primary cilia and the FSS-mediated increase in [Ca2+]i Moreover, Cdc42 activity and FSS-stimulated endocytosis are coordinately modulated by activators and inhibitors of calmodulin. Together, these data suggest a mechanism by which PT cell exposure to FSS is translated into enhanced endocytic uptake of filtered molecules.

The endocytic recycling regulatory protein EHD1 Is required for ocular lens development.

The C-terminal Eps15 homology domain-containing (EHD) proteins play a key role in endocytic recycling, a fundamental cellular process that ensures the return of endocytosed membrane components and receptors back to the cell surface. To define the in vivo biological functions of EHD1, we have generated Ehd1 knockout mice and previously reported a requirement of EHD1 for spermatogenesis. Here, we show that approximately 56% of the Ehd1-null mice displayed gross ocular abnormalities, including anophthalmia, aphakia, microphthalmia and congenital cataracts. Histological characterization of ocular abnormalities showed pleiotropic defects that include a smaller or absent lens, persistence of lens stalk and hyaloid vasculature, and deformed optic cups. To test whether these profound ocular defects resulted from the loss of EHD1 in the lens or in non-lenticular tissues, we deleted the Ehd1 gene selectively in the presumptive lens ectoderm using Le-Cre. Conditional Ehd1 deletion in the lens resulted in developmental defects that included thin epithelial layers, small lenses and absence of corneal endothelium. Ehd1 deletion in the lens also resulted in reduced lens epithelial proliferation, survival and expression of junctional proteins E-cadherin and ZO-1. Finally, Le-Cre-mediated deletion of Ehd1 in the lens led to defects in corneal endothelial differentiation. Taken together, these data reveal a unique role for EHD1 in early lens development and suggest a previously unknown link between the endocytic recycling pathway and regulation of key developmental processes including proliferation, differentiation and morphogenesis.

Eps 15 Homology Domain (EHD)-1 Remodels Transverse Tubules in Skeletal Muscle.

We previously showed that Eps15 homology domain-containing 1 (EHD1) interacts with ferlin proteins to regulate endocytic recycling. Myoblasts from Ehd1-null mice were found to have defective recycling, myoblast fusion, and consequently smaller muscles. When expressed in C2C12 cells, an ATPase dead-EHD1 was found to interfere with BIN1/amphiphysin 2. We now extended those findings by examining Ehd1-heterozygous mice since these mice survive to maturity in normal Mendelian numbers and provide a ready source of mature muscle. We found that heterozygosity of EHD1 was sufficient to produce ectopic and excessive T-tubules, including large intracellular aggregates that contained BIN1. The disorganized T-tubule structures in Ehd1-heterozygous muscle were accompanied by marked elevation of the T-tubule-associated protein DHPR and reduction of the triad linker protein junctophilin 2, reflecting defective triads. Consistent with this, Ehd1-heterozygous muscle had reduced force production. Introduction of ATPase dead-EHD1 into mature muscle fibers was sufficient to induce ectopic T-tubule formation, seen as large BIN1 positive structures throughout the muscle. Ehd1-heterozygous mice were found to have strikingly elevated serum creatine kinase and smaller myofibers, but did not display findings of muscular dystrophy. These data indicate that EHD1 regulates the maintenance of T-tubules through its interaction with BIN1 and links T-tubules defects with elevated creatine kinase and myopathy.

ESCRT proteins: Double-edged regulators of cellular signaling.

ESCRT pathway proteins play a key role in sorting ubiquitinated membrane receptors towards lysosomes providing an important mechanism for attenuating cell surface receptor signaling. However, recent studies point to a positive role of ESCRT proteins in signal transduction in multiple species studied under physiological and pathological conditions. ESCRT components such as Tsg101 and Hrs are overexpressed in human cancers and Tsg101 depletion is detrimental for cell proliferation, survival and transformed phenotype of tumor cells. However, the mechanisms underlying the positive contributions of ESCRT pathway to surface receptor signaling have remained unclear. In a recent study, we showed that Tsg101 and Vps4 are essential for translocation of active Src from endosomes to focal adhesion and invadopodia, thereby revealing a role of ESCRT pathway in promoting Src-mediated migration and invasion. We discuss the implications of these and other recent studies which together suggest a role for the ESCRT pathway in recycling of endocytic cargo proteins, aside from its role in lysosomal targeting, potentially explaining the positive roles of ESCRT proteins in signal transduction.