Cysteine depletion induces pancreatic tumor ferroptosis in mice.

Ferroptosis is a form of cell death that results from the catastrophic accumulation of lipid reactive oxygen species (ROS). Oncogenic signaling elevates lipid ROS production in many tumor types and is counteracted by metabolites that are derived from the amino acid cysteine. In this work, we show that the import of oxidized cysteine (cystine) via system xC - is a critical dependency of pancreatic ductal adenocarcinoma (PDAC), which is a leading cause of cancer mortality. PDAC cells used cysteine to synthesize glutathione and coenzyme A, which, together, down-regulated ferroptosis. Studying genetically engineered mice, we found that the deletion of a system xC - subunit, Slc7a11, induced tumor-selective ferroptosis and inhibited PDAC growth. This was replicated through the administration of cyst(e)inase, a drug that depletes cysteine and cystine, demonstrating a translatable means to induce ferroptosis in PDAC.

An Immunosuppressive Dendritic Cell Subset Accumulates at Secondary Sites and Promotes Metastasis in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) after complete surgical resection is often followed by distant metastatic relapse for reasons that remain unclear. In this study, we investigated how the immune response at secondary sites affects tumor spread in murine models of metastatic PDAC. Early metastases were associated with dense networks of CD11b+CD11c+MHC-II+CD24+CD64lowF4/80low dendritic cells (DC), which developed from monocytes in response to tumor-released GM-CSF. These cells uniquely expressed MGL2 and PD-L2 in the metastatic microenvironment and preferentially induced the expansion of T regulatory cells (Treg) in vitro and in vivo Targeted depletion of this DC population in Mgl2DTR hosts activated cytotoxic lymphocytes, reduced Tregs, and inhibited metastasis development. Moreover, blocking PD-L2 selectively activated CD8 T cells at secondary sites and suppressed metastasis, suggesting that the DCs use this particular pathway to inhibit CD8 T-cell-mediated tumor immunity. Phenotypically similar DCs accumulated at primary and secondary sites in other models and in human PDAC. These studies suggest that a discrete DC subset both expands Tregs and suppresses CD8 T cells to establish an immunosuppressive microenvironment conducive to metastasis formation. Therapeutic strategies to block the accumulation and immunosuppressive activity of such cells may help prevent PDAC progression and metastatic relapse after surgical resection. Cancer Res; 77(15); 4158-70. ©2017 AACR.

Control of inflammation by stromal Hedgehog pathway activation restrains colitis.

Inflammation disrupts tissue architecture and function, thereby contributing to the pathogenesis of diverse diseases; the signals that promote or restrict tissue inflammation thus represent potential targets for therapeutic intervention. Here, we report that genetic or pharmacologic Hedgehog pathway inhibition intensifies colon inflammation (colitis) in mice. Conversely, genetic augmentation of Hedgehog response and systemic small-molecule Hedgehog pathway activation potently ameliorate colitis and restrain initiation and progression of colitis-induced adenocarcinoma. Within the colon, the Hedgehog protein signal does not act directly on the epithelium itself, but on underlying stromal cells to induce expression of IL-10, an immune-modulatory cytokine long known to suppress inflammatory intestinal damage. IL-10 function is required for the full protective effect of small-molecule Hedgehog pathway activation in colitis; this pharmacologic augmentation of Hedgehog pathway activity and stromal IL-10 expression are associated with increased presence of CD4+Foxp3+ regulatory T cells. We thus identify stromal cells as cellular coordinators of colon inflammation and suggest their pharmacologic manipulation as a potential means to treat colitis.

Restoring Retinoic Acid Attenuates Intestinal Inflammation and Tumorigenesis in APCMin/+ Mice.

Chronic intestinal inflammation accompanies familial adenomatous polyposis (FAP) and is a major risk factor for colorectal cancer in patients with this disease, but the cause of such inflammation is unknown. Because retinoic acid (RA) plays a critical role in maintaining immune homeostasis in the intestine, we hypothesized that altered RA metabolism contributes to inflammation and tumorigenesis in FAP. To assess this hypothesis, we analyzed RA metabolism in the intestines of patients with FAP as well as APCMin/+ mice, a model that recapitulates FAP in most respects. We also investigated the impact of intestinal RA repletion and depletion on tumorigenesis and inflammation in APCMin/+ mice. Tumors from both FAP patients and APCMin/+ mice displayed striking alterations in RA metabolism that resulted in reduced intestinal RA. APCMin/+ mice placed on a vitamin A-deficient diet exhibited further reductions in intestinal RA with concomitant increases in inflammation and tumor burden. Conversely, restoration of RA by pharmacologic blockade of the RA-catabolizing enzyme CYP26A1 attenuated inflammation and diminished tumor burden. To investigate the effect of RA deficiency on the gut immune system, we studied lamina propria dendritic cells (LPDC) because these cells play a central role in promoting tolerance. APCMin/+ LPDCs preferentially induced Th17 cells, but reverted to inducing Tregs following restoration of intestinal RA in vivo or direct treatment of LPDCs with RA in vitro These findings demonstrate the importance of intestinal RA deficiency in tumorigenesis and suggest that pharmacologic repletion of RA could reduce tumorigenesis in FAP patients. Cancer Immunol Res; 4(11); 917-26. ©2016 AACR.

Brg1 promotes both tumor-suppressive and oncogenic activities at distinct stages of pancreatic cancer formation.

Pancreatic ductal adenocarcinoma (PDA) develops predominantly through pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) precursor lesions. Pancreatic acinar cells are reprogrammed to a "ductal-like" state during PanIN-PDA formation. Here, we demonstrate a parallel mechanism operative in mature duct cells during which functional cells undergo "ductal retrogression" to form IPMN-PDA. We further identify critical antagonistic roles for Brahma-related gene 1 (Brg1), a catalytic subunit of the SWI/SNF complexes, during IPMN-PDA development. In mature duct cells, Brg1 inhibits the dedifferentiation that precedes neoplastic transformation, thus attenuating tumor initiation. In contrast, Brg1 promotes tumorigenesis in full-blown PDA by supporting a mesenchymal-like transcriptional landscape. We further show that JQ1, a drug that is currently being tested in clinical trials for hematological malignancies, impairs PDA tumorigenesis by both mimicking some and inhibiting other Brg1-mediated functions. In summary, our study demonstrates the context-dependent roles of Brg1 and points to potential therapeutic treatment options based on epigenetic regulation in PDA.

Vascular endothelial growth factor receptor type 2-targeted contrast-enhanced US of pancreatic cancer neovasculature in a genetically engineered mouse model: potential for earlier detection.

PURPOSE: To test ultrasonographic (US) imaging with vascular endothelial growth factor receptor type 2 (VEGFR2)-targeted microbubble contrast material for the detection of pancreatic ductal adenocarcinoma (PDAC) in a transgenic mouse model of pancreatic cancer development. MATERIALS AND METHODS: Experiments involving animals were approved by the Institutional Administrative Panel on Laboratory Animal Care at Stanford University. Transgenic mice (n = 44; Pdx1-Cre, KRas(G12D), Ink4a(-/-)) that spontaneously develop PDAC starting at 4 weeks of age were imaged by using a dedicated small-animal US system after intravenous injection of 5 × 10(7) clinical-grade VEGFR2-targeted microbubble contrast material. The pancreata in wild-type (WT) mice (n = 64) were scanned as controls. Pancreatic tissue was analyzed ex vivo by means of histologic examination (with hematoxylin-eosin staining) and immunostaining of vascular endothelial cell marker CD31 and VEGFR2. The Wilcoxon rank sum test and linear mixed-effects model were used for statistical analysis. RESULTS: VEGFR2-targeted US of PDAC showed significantly higher signal intensities (26.8-fold higher; mean intensity ± standard deviation, 6.7 linear arbitrary units [lau] ± 8.5; P < .001) in transgenic mice compared with normal, control pancreata of WT mice (mean intensity, 0.25 lau ± 0.25). The highest VEGFR2-targeted US signal intensities were observed in smaller tumors, less than 3 mm in diameter (30.8-fold higher than control tissue with mean intensity of 7.7 lau ± 9.3 [P < .001]; and 1.7-fold higher than lesions larger than 3 mm in diameter with mean intensity of 4.6 lau ± 5.8 [P < .024]). Ex vivo quantitative VEGFR2 immunofluorescence demonstrated that VEGFR2 expression was significantly higher in pancreatic tumors (P < .001; mean fluorescent intensity, 499.4 arbitrary units [au] ± 179.1) compared with normal pancreas (mean fluorescent intensity, 232.9 au ± 83.7). CONCLUSION: US with clinical-grade VEGFR2-targeted microbubbles allows detection of small foci of PDAC in transgenic mice.

USP33 regulates centrosome biogenesis via deubiquitination of the centriolar protein CP110.

Centrosome duplication is critical for cell division, and genome instability can result if duplication is not restricted to a single round per cell cycle. Centrosome duplication is controlled in part by CP110, a centriolar protein that positively regulates centriole duplication while restricting centriole elongation and ciliogenesis. Maintenance of normal CP110 levels is essential, as excessive CP110 drives centrosome over-duplication and suppresses ciliogenesis, whereas its depletion inhibits centriole amplification and leads to highly elongated centrioles and aberrant assembly of cilia in growing cells. CP110 levels are tightly controlled, partly through ubiquitination by the ubiquitin ligase complex SCF(cyclin F) during G2 and M phases of the cell cycle. Here, using human cells, we report a new mechanism for the regulation of centrosome duplication that requires USP33, a deubiquitinating enzyme that is able to regulate CP110 levels. USP33 interacts with CP110 and localizes to centrioles primarily in S and G2/M phases, the periods during which centrioles duplicate and elongate. USP33 potently and specifically deubiquitinates CP110, but not other cyclin-F substrates. USP33 activity antagonizes SCF(cyclin F)-mediated ubiquitination and promotes the generation of supernumerary centriolar foci, whereas ablation of USP33 destabilizes CP110 and thereby inhibits centrosome amplification and mitotic defects. To our knowledge, we have identified the first centriolar deubiquitinating enzyme whose expression regulates centrosome homeostasis by countering cyclin-F-mediated destruction of a key substrate. Our results point towards potential therapeutic strategies for inhibiting tumorigenesis associated with centrosome amplification.

Invasive mucinous cystic neoplasms of the pancreas.

Mucinous cystic neoplasms (MCN) and intraductal papillary mucinous neoplasms (IPMN) of the pancreas both appear to have been included and intermixed in some early reports of pancreatic cystic neoplasms. Recognition of their distinguishing features evolved during the last decade of the twentieth century. One legacy of the early period is the statement that mucinous cystic neoplasms sometimes progress to invasive colloid carcinoma. It is now recognized that colloid carcinomas characteristically arise from IPMN. We set out to see if we could find MCN that invaded as colloid carcinomas and found no examples in MCN collected in two academic medical centers. We then sought to expand the number of MCN by evaluating series from additional centers. This yielded no examples of colloid carcinomas associated with 291 MCN, however one MCN exhibited a minor component with colloid (non-cystic mucinous) growth pattern within the fibrous wall of the neoplasm. The expression of CDX2, a marker of intestinal differentiation that is found in colloid carcinomas was examined by immunostaining in the original MCN series and in the MCN with the intratumoral colloid growth pattern. Focal expression of CDX2 was found in 22 of 43 MCN including the MCN that exhibited the intratumoral colloid growth pattern. Overall, the data suggest that MCN rarely, if ever, invade as colloid carcinoma but the expression of CDX2 by some MCN and the observation of intratumoral colloid growth pattern in one MCN seems to leave open the possibility that MCN might rarely invade as colloid carcinoma. The majority of malignant MCN invade with a tubular (ductal) pattern, and rarely the invasive component was anaplastic.

Primary cilium depletion typifies cutaneous melanoma in situ and malignant melanoma.

Cutaneous melanoma is a lethal malignancy that arises spontaneously or via in situ precursor neoplasms. While melanoma in situ and locally invasive malignant melanoma can be cured surgically, these lesions can sometimes be difficult to distinguish from melanocytic nevi. Thus, the identification of histolopathologic or molecular features that distinguish these biologically distinct lesions would represent an important advance. To this end, we determined the abundance of melanocytic primary cilia in a series of 62 cases composed of typical cutaneous melanocytic nevi, melanoma in situ, invasive melanoma, and metastatic melanoma. Primary cilia are sensory organelles that modulate developmental and adaptive signaling and notably, are substantially depleted from the neoplastic epithelium of pancreatic carcinoma at a stage equivalent to melanoma in situ. In this series, we find that while nearly all melanocytes in 22 melanocytic nevi possessed a primary cilium, a near-complete loss of this organelle was observed in 16 cases of melanoma in situ, in 16 unequivocal primary invasive melanomas, and in 8 metastatic tumors, each associated with a cutaneous primary lesion. These findings suggest that the primary cilium may be used to segregate cutaneous invasive melanoma and melanoma in situ from melanocytic nevi. Moreover, they place the loss of an organelle known to regulate oncogenic signaling at an early stage of melanoma development.

The perennial organelle: assembly and disassembly of the primary cilium.

Primary cilia contain signaling receptors of diverse classes, and ciliary dysfunction results in a variety of developmental defects. Thus, primary cilia are thought to have an important role in sensing and transducing cellular signals. Although there is clear evidence demonstrating that these organelles are assembled and disassembled dynamically as cells progress through the cell cycle, the mechanisms by which the cell cycle controls the assembly and disassembly of the primary cilium remain poorly understood. In this Commentary, we review the basic cellular mechanisms that underlie the early stages of cilium assembly and discuss how the cell cycle communicates with the ciliation program. A commonly held view is that ciliation occurs exclusively in cells that have exited the cell cycle and entered quiescence or differentiation. However, this concept is at odds with the finding that, during development, many actively proliferating cells require cilia-mediated signaling pathways to instruct their developmental fate. Here, we reassess the quiescence-centric view of ciliation by reviewing historic and current literature. We discuss ample evidence that cilia are in fact present on many proliferating cells, and that a transient peak of ciliation before the G1-S transition might be tightly coupled to entry into the DNA replication phase. Finally, we touch on the relationship between the ciliation and cell-division cycles and the tissue distribution of primary cilia in order to highlight potential roles for the primary cilium in restraining cells from the hyperproliferative state that contributes to cancer.

Trafficking to the ciliary membrane: how to get across the periciliary diffusion barrier?

The primary cilium organizes numerous signal transduction cascades, and an understanding of signaling receptor trafficking to cilia is now emerging. A defining feature of cilia is the periciliary diffusion barrier that separates the ciliary and plasma membranes. Although lateral transport through this barrier may take place, polarized exocytosis to the base of the cilium has been the prevailing model for delivering membrane proteins to cilia. Key players for this polarized exocytosis model include the GTPases Rab8 and Rab11, the exocyst, and possibly the intraflagellar tranport machinery. In turn, the sorting of membrane proteins to cilia critically relies on the recognition of ciliary targeting signals by sorting machines such as the BBSome coat complex or the GTPase Arf4. Finally, some proteins need to exit from cilia, and ubiquitination may regulate this step. The stage is now set to dissect the interplay between signaling and regulated trafficking to and from cilia.

Pancreatic cancer and precursor pancreatic intraepithelial neoplasia lesions are devoid of primary cilia.

Primary cilia have been proposed to participate in the modulation of growth factor signaling pathways. In this study, we determined that ciliogenesis is suppressed in both pancreatic cancer cells and pancreatic intraepithelial neoplasia (PanIN) lesions in human pancreatic ductal adenocarcinoma (PDAC). Primary cilia were absent in these cells even when not actively proliferating. Cilia were also absent from mouse PanIN cells in three different mouse models of PDAC driven by an endogenous oncogenic Kras allele. Inhibition of Kras effector pathways restored ciliogenesis in a mouse pancreatic cancer cell line, raising the possibility that ciliogenesis may be actively repressed by oncogenic Kras. By contrast, normal duct, islet, and centroacinar cells retained primary cilia in both human and mouse pancreata. Thus, arrested ciliogenesis is a cardinal feature of PDAC and its precursor PanIN lesions, does not require ongoing proliferation, and could potentially be targeted pharmacologically.

Constructing and deconstructing roles for the primary cilium in tissue architecture and cancer.

Primary cilia are exquisitely designed sensory machines that have evolved at least three distinct sensory modalities to monitor the extracellular environment. The presence and activation of growth factor, morphogen, and hormone receptors within the confines of the ciliary membrane, the intrinsic physical relationship between the ciliary axoneme and the centriole, and the preferential assembly of primary cilia on the apical surfaces of tissue epithelia highlight the importance of this organelle in the establishment and maintenance of tissue architecture and homeostasis. Accordingly, recent studies begin to suggest roles for these organelles in oncogenesis and tumor suppression. Here, we review the sensory properties of primary cilia, assess the "history" of the primary cilium in cancer, and draw upon recent findings in a discussion of how the primary cilium may influence tissue architecture and neoplasia.

Genomic analysis of homotypic vacuole fusion.

Yeast vacuoles undergo fission and homotypic fusion, yielding one to three vacuoles per cell at steady state. Defects in vacuole fusion result in vacuole fragmentation. We have screened 4828 yeast strains, each with a deletion of a nonessential gene, for vacuole morphology defects. Fragmented vacuoles were found in strains deleted for genes encoding known fusion catalysts as well as 19 enzymes of lipid metabolism, 4 SNAREs, 12 GTPases and GTPase effectors, 9 additional known vacuole protein-sorting genes, 16 protein kinases, 2 phosphatases, 11 cytoskeletal proteins, and 28 genes of unknown function. Vacuole fusion and vacuole protein sorting are catalyzed by distinct, but overlapping, sets of proteins. Novel pathways of vacuole priming and docking emerged from this deletion screen. These include ergosterol biosynthesis, phosphatidylinositol (4,5)-bisphosphate turnover, and signaling from Rho GTPases to actin remodeling. These pathways are supported by the sensitivity of the late stages of vacuole fusion to inhibitors of phospholipase C, calcium channels, and actin remodeling. Using databases of yeast protein interactions, we found that many nonessential genes identified in our deletion screen interact with essential genes that are directly involved in vacuole fusion. Our screen reveals regulatory pathways of vacuole docking and provides a genomic basis for studies of this reaction.

Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle.

Three membrane microdomains can be identified on docked vacuoles: "outside" membrane, not in contact with other vacuoles, "boundary" membrane that contacts adjacent vacuoles, and "vertices," where boundary and outside membrane meet. In living cells and in vitro, vacuole fusion occurs at vertices rather than from a central pore expanding radially. Vertex fusion leaves boundary membrane within the fused organelle and is an unexpected pathway for the formation of intralumenal membranes. Proteins that regulate docking and fusion (Vac8p, the GTPase Ypt7p, its HOPS/Vps-C effector complex, the t-SNARE Vam3p, and protein phosphatase 1) accumulate at these vertices during docking. Their vertex enrichment requires cis-SNARE complex disassembly and is thus part of the normal fusion pathway.