Xinchun Ding

Activation of mTOR pathway in myeloid-derived suppressor cells stimulates cancer cell proliferation and metastasis in lal(-/-) mice.

Inflammation critically contributes to cancer metastasis, in which myeloid-derived suppressor cells (MDSCs) are an important participant. Although MDSCs are known to suppress immune surveillance, their roles in directly stimulating cancer cell proliferation and metastasis currently remain unclear. Lysosomal acid lipase (LAL) deficiency causes systemic expansion and infiltration of MDSCs in multiple organs and subsequent inflammation. In the LAL-deficient (lal−/−) mouse model, melanoma metastasized massively in allogeneic lal−/− mice, which was suppressed in allogeneic lal+/+ mice owing to immune rejection. Here we report for the first time that MDSCs from lal−/− mice directly stimulated B16 melanoma cell in vitro proliferation and in vivo growth and metastasis. Cytokines, that is, interleukin-1β and tumor necrosis factor-α from MDSCs are required for B16 melanoma cell proliferation in vitro. Myeloid-specific expression of human LAL (hLAL) in lal−/− mice rescues these malignant phenotypes in vitro and in vivo. The tumor-promoting function of lal−/− MDSCs is mediated, at least in part, through overactivation of the mammalian target of rapamycin (mTOR) pathway. Knockdown of mTOR, Raptor or Rictor in lal−/− MDSCs suppressed their stimulation on proliferation of cancer cells, including B16 melanoma, Lewis lung carcinoma and transgenic mouse prostate cancer-C2 cancer cells. Our results indicate that LAL has a critical role in regulating MDSCs’ ability to directly stimulate cancer cell proliferation and overcome immune rejection of cancer metastasis in allogeneic mice through modulation of the mTOR pathway, which provides a mechanistic basis for targeting MDSCs to reduce the risk of cancer metastasis. Therefore MDSCs possess dual functions to facilitate cancer metastasis: suppress immune surveillance and stimulate cancer cell proliferation and growth.

Gene profile of myeloid-derived suppressive cells from the bone marrow of lysosomal acid lipase knock-out mice.

Background Lysosomal acid lipase (LAL) controls development and homeostasis of myeloid lineage cells. Loss of the lysosomal acid lipase (LAL) function leads to expansion of myeloid-derived suppressive cells (MDSCs) that cause myeloproliferative neoplasm. Methodology/Principal Findings Affymetrix GeneChip microarray analysis identified detailed intrinsic defects in Ly6G+ myeloid lineage cells of LAL knock-out (lal−/−) mice. Ingenuity Pathway Analysis revealed activation of the mammalian target of rapamycin (mTOR) signaling, which functions as a nutrient/energy/redox sensor, and controls cell growth, cell cycle entry, cell survival, and cell motility. Loss of the LAL function led to major alteration of large GTPase and small GTPase signal transduction pathways. lal−/− Ly6G+ myeloid cells in the bone marrow showed substantial increase of cell proliferation in association with up-regulation of cyclin and cyclin-dependent kinase (cdk) genes. The epigenetic microenvironment was significantly changed due to the increased expression of multiple histone cluster genes, centromere protein genes and chromosome modification genes. Gene expression of bioenergetic pathways, including glycolysis, aerobic glycolysis, mitochondrial oxidative phosphorylation, and respiratory chain proteins, was also increased, while the mitochondrial function was impaired in lal−/− Ly6G+ myeloid cells. The concentration of reactive oxygen species (ROS) was significantly increased accompanied by up-regulation of nitric oxide/ROS production genes in these cells. Conclusions/Significance This comprehensive gene profile study for the first time identifies and defines important gene pathways involved in the myeloid lineage cells towards MDSCs using lal−/− mouse model.

Critical Role of the mTOR Pathway in Development and Function of Myeloid-Derived Suppressor Cells in lal−/− Mice

Lysosomal acid lipase (LAL) is essential for the hydrolysis of cholesteryl esters and triglycerides to generate cholesterol and free fatty acids in cellular lysosomes. Ablation of the lal gene (lal(-/-)) systemically increased expansion of cluster of differentiation molecule 11b (CD11b), lymphocyte antigen 6G (Ly6G) myeloid-derived suppressor cells (MDSCs) that caused myeloproliferative neoplasms in mice. Study of lal(-/-) bone marrow Ly6G(+) MDSCs via transcriptional profiling showed increases in mammalian target of rapamycin (mTOR) signaling pathway transcripts. Injection of mTOR pharmacologic inhibitors into lal(-/-) mice significantly reduced bone marrow myelopoiesis and systemic CD11b(+)Ly6G(+) cell expansion. Rapamycin treatment of lal(-/-) mice stimulated a shift from immature CD11b(+)Ly6G(+) cells to CD11b(+) single-positive cells in marrow and tissues and partially reversed the increased cell proliferation, decreased apoptosis, increased ATP synthesis, and increased cell cycling of bone marrow CD11b(+)Ly6G(+) cells obtained from lal(-/-) mice. Pharmacologic and siRNA suppression of mTOR, regulatory-associated protein of mTOR, rapamycin-insensitive companion of mTOR, and Akt1 function corrected CD11b(+)Ly6G(+) cell in lal(-/-) mice development from Lin(-) progenitor cells and reversed the immune suppression on T-cell proliferation and function in association with decreased reactive oxygen species production, and recovery from impairment of mitochondrial membrane potential compared with control mutant cells. These results indicate a crucial role of LAL-regulated mTOR signaling in the production and function of CD11b(+)Ly6G(+) cells. The mTOR pathway may serve as a novel target to modulate the emergence of MDSCs in those pathophysiologic states in which these cells play an immunosuppressive role.

Stat3 Downstream Gene Product Chitinase 3-Like 1 Is a Potential Biomarker of Inflammation-induced Lung Cancer in Multiple Mouse Lung Tumor Models and Humans

Over-activation of the signal transducers and activators of the transcription 3 (Stat3) pathway in lung alveolar type II (AT II) epithelial cells induces chronic inflammation and adenocarcinoma in the lung of CCSP-rtTA/(tetO)7-CMV-Stat3C bitransgenic mice. One of Stat3 downstream genes products, chitinase 3-like 1 (CHI3L1) protein, showed increased concentration in both bronchioalveolar lavage fluid (BALF) and blood of doxycycline-treated CCSP-rtTA/(tetO)7-CMV-Stat3C bitransgenic mice. When tested in other inflammation-induced lung cancer mouse models, the CHI3L1 protein concentration was also highly increased in BALF and blood of these models with tumors. Immunohistochemical staining showed strong staining of CHI3L1 protein around tumor areas in these mouse models. Analysis of normal objects and lung cancer patients revealed a significant elevation of CHI3L1 protein concentration in human serum samples from all categories of lung cancers. Furthermore, recombinant CHI3L protein stimulated proliferation and growth of Lewis lung cancer cells. Therefore, secretory CHI3L1 plays an important role in inflammation-induced lung cancer formation and potentially serve as a biomarker for lung cancer prediction. Based on our previous publication and this work, this is the first animal study linking overexpression of CHI3L1 to various lung tumor mouse models. These models will facilitate identification of additional biomarkers to predict and verify lung cancer under various pathogenic conditions, which normally cannot be done in humans.

Hepatocyte-Specific Expression of Human Lysosome Acid Lipase Corrects Liver Inflammation and Tumor Metastasis in lal(-/-) Mice.

The liver is a major organ for lipid synthesis and metabolism. Deficiency of lysosomal acid lipase (LAL; official name Lipa, encoded by Lipa) in mice (lal(-/-)) results in enlarged liver size due to neutral lipid storage in hepatocytes and Kupffer cells. To test the functional role of LAL in hepatocyte, hepatocyte-specific expression of human LAL (hLAL) in lal(-/-) mice was established by cross-breeding of liver-activated promoter (LAP)-driven tTA transgene and (tetO)7-CMV-hLAL transgene with lal(-/-) knockout (KO) (LAP-Tg/KO) triple mice. Hepatocyte-specific expression of hLAL in LAP-Tg/KO triple mice reduced the liver size to the normal level by decreasing lipid storage in both hepatocytes and Kupffer cells. hLAL expression reduced tumor-promoting myeloid-derived suppressive cells in the liver of lal(-/-) mice. As a result, B16 melanoma metastasis to the liver was almost completely blocked. Expression and secretion of multiple tumor-promoting cytokines or chemokines in the liver were also significantly reduced. Because hLAL is a secretory protein, lal(-/-) phenotypes in other compartments (eg, blood, spleen, and lung) also ameliorated, including systemic reduction of myeloid-derived suppressive cells, an increase in CD4(+) and CD8(+) T and B lymphocytes, and reduced B16 melanoma metastasis in the lung. These results support a concept that LAL in hepatocytes is a critical metabolic enzyme in controlling neutral lipid metabolism, liver homeostasis, immune response, and tumor metastasis.

Establishment of lal-/- Myeloid Lineage Cell Line That Resembles Myeloid-Derived Suppressive Cells

Myeloid-derived suppressor cells (MDSCs) in mouse are inflammatory cells that play critical roles in promoting cancer growth and metastasis by directly stimulating cancer cell proliferation and suppressing immune surveillance. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an “MDSC-like” cell line. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lysosomal acid lipase (LAL) knock-out (lal-/-) mice, we have established a wild type (HD1A) and a lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas, dysfunction of mitochondria skewing toward fission structure, damaged membrane potential, and increased ROS production. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4+ T cell proliferation and function in vitro, and enhanced cancer cells proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an “MDSC-like” cell line that can be used as an alternative in vitro system to study how LAL controls various myeloid cell functions.

Myeloid-Derived Suppressor Cells Are Involved in Lysosomal Acid Lipase Deficiency–Induced Endothelial Cell Dysfunctions

The underlying mechanisms that lysosomal acid lipase (LAL) deficiency causes infiltration of myeloid-derived suppressor cells (MDSCs) in multiple organs and subsequent inflammation remain incompletely understood. Endothelial cells (ECs), lining the inner layer of blood vessels, constitute barriers regulating leukocytes transmigration to the site of inflammation. Therefore, we hypothesized that ECs are dysfunctional in LAL-deficient (lal−/−) mice. We found that Ly6G+ cells transmigrated more efficiently across lal−/− ECs than wild-type (lal+/+) ECs, which were associated with increased levels of PECAM-1 and MCP-1 in lal−/− ECs. In addition, lal−/− ECs showed enhanced migration and proliferation, decreased apoptosis, but impaired tube formation and angiogenesis. lal−/− ECs also suppressed T cell proliferation in vitro. Interestingly, lal−/− Ly6G+ cells promoted in vivo angiogenesis (including a tumor model), EC tube formation, and proliferation. Finally, the mammalian target of rapamycin (mTOR) pathway was activated in lal−/− ECs, and inhibition of mTOR reversed EC dysfunctions, including decreasing Ly6G+ cell transmigration, delaying migration, and relieving suppression of T cell proliferation, which was mediated by decreasing production of reactive oxygen species. Our results indicate that LAL regulates EC functions through interaction with MDSCs and modulation of the mTOR pathway, which may provide a mechanistic basis for targeting MDSCs or mTOR to rejuvenate EC functions in LAL deficiency–related diseases.

Endothelial Rab7 GTPase Mediates Tumor Growth and Metastasis in Lysosomal Acid Lipase Deficient Mice

Tumors depend on their microenvironment for sustained growth, invasion, and metastasis. In this environment, endothelial cells (ECs) are an important stromal cell type interacting with malignant cells to facilitate tumor angiogenesis and cancer cell extravasation. Of note, lysosomal acid lipase (LAL) deficiency facilitates melanoma growth and metastasis. ECs from LAL-deficient (lal−/−) mice possess enhanced proliferation, migration, and permeability of inflammatory cells by activating the mammalian target of rapamycin (mTOR) pathway. Here we report that lal−/− ECs facilitated in vivo tumor angiogenesis, growth, and metastasis, largely by stimulating tumor cell proliferation, migration, adhesion, and transendothelial migration via increased expression of IL-6 and monocyte chemoattractant protein 1 (MCP-1). This prompted us to look for lysosomal proteins that are involved in lal−/− EC dysfunctions. We found that lal−/− ECs displayed increased expression of Rab7, a late endosome/lysosome-associated small GTPase. Moreover, Rab7 and mTOR were co-increased and co-localized to lysosomes and physically interacted in lal−/− ECs. Rab7 inhibition reversed lal−/− EC dysfunctions, including decreasing their enhanced migration and permeability of tumor-stimulatory myeloid cells, and suppressed EC-mediated stimulation of in vitro tumor cell transmigration, proliferation, and migration and in vivo tumor growth and metastasis. Finally, Rab7 inhibition reduced overproduction of reactive oxygen species and increased IL-6 and MCP-1 secretion in lal−/− ECs. Our results indicate that metabolic reprogramming resulting from LAL deficiency enhances the ability of ECs to stimulate tumor cell proliferation and metastasis through stimulation of lysosome-anchored Rab7 activity.

Abstract A12: Establishment of myeloid lineage cell line that resembles myeloid-derived suppressive cells

Myeloid-derived suppressive cells (MDSCs) are inflammatory cells that play critical roles in promoting cancer growth and metastasis. In order to facilitate characterization of biochemical and cellular mechanisms of MDSCs, it is urgent to establish an “MDSC-like” cell line for pharmacological and immunotherapeutic applications. Lysosomal acid lipase (LAL) is a critical lipid enzyme in the metabolic signaling pathway that hydrolyzes cholesteryl esters (CE) and triglycerides (TG) in lysosomes. In mice, lack of LAL in genetically ablated knockout mice (lal-/-) shows systemic expansion of MDSCs, which directly stimulate cancer cell proliferation, and suppress T cell proliferation and impair T cell function. The Affymetrix Genechip microarray assay reveals over-activation of the mTOR signaling pathway in lal-/- MDSCs. By cross breeding of immortomouse (simian virus 40 large T antigen transgenic mice) with wild type and lal-/- mice, we have established wild type (HD1A) and lal-/- (HD1B) myeloid cell lines. Compared with HD1A cells, HD1B cells demonstrated many characteristics similar to lal-/- MDSCs. HD1B cells exhibited increased lysosomes around perinuclear areas. HD1B cells showed increased glycolytic metabolism during blockage of fatty acid metabolism to fuel the energy need. Compared with HD1A cells, HD1B cells showed increased glucose concentration, suggesting the enhanced glycolytic metabolic pathway, in which glucose converts into pyruvate. Indeed, the pyruvate concentration was increased in HD1B cells compared with that in HD1A cells. Glycolysis occurs in the cytosol of the cell. Pyruvic acid supplies energy to living cells through the citric acid cycle (TCA) in the mitochondria, which generates NADH for the oxidative phosphorylation (OXPHOS, electron transport pathway) to produce ATP. Aconitase is the rate-limiting enzyme in the TCA cycle. Its activity was doubled in HD1B cells compared with HD1A cells. GLUT (SLC2) family members are the major membrane transporters. Among them, GLUT 1-5 have been well characterized as glucose and/or fructose transporters in various tissues and cell types. Thirteen GLUT proteins have been reported to be expressed in mice (14 in humans). Using the Real-time PCR method, expression of all GLUT members was assessed in HD1A and HD1B cells, in which GLUT3, GLUT6, GLUT8, GLUT12, and CLUT13 were upregulated, while GLUT 5 and GLUT 9 were downregulated in HD1B cells. This supports a concept that the LAL metabolic pathway controls the balance of glucose transportation to fuel the energy need in HD1B cells. In addition, dysfunction of mitochondria in HD1B cells skews toward fission structure, damaged membrane potential, and increased ROS production. Similar to lal-/- MDSCs, the mTOR signal pathway in HD1B cells is overly activated. Rapamycin treatment of HD1B cells reduced ROS production and restored the mitochondrial membrane potential. HD1B cells showed much stronger immunosuppression on CD4+ T cell proliferation and function in vitro, and enhanced cancer cell proliferation. Knockdown of mTOR with siRNA reduced the HD1B cell ability to immunosuppress T cells and stimulate cancer cell proliferation. Therefore, the HD1B myeloid cell line is an “MDSC-like” cell line. HD1B cells can be used as an alternative in vitro system to study how the lipid metabolic signaling pathway regulates myeloid cell functions. Both HD1A and HD1B cell lines will be useful for identifying pharmacological drugs to suppress MDSCs expansion in multiple inflammation-induced diseases that involve MDSCs. Grant support: This work was supported by National Institutes of Health Grants HL087001 (to H. D.), and CA138759, CA152099 (to C. Y.). Citation Format: Cong Yan, Xinchun Ding, Lingyan Wu, Hong Du. Establishment of myeloid lineage cell line that resembles myeloid-derived suppressive cells. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A12.

Abstract A11: Myeloid-derived suppressor cells suppress immune surveillance and stimulate cancer cell proliferation/metastasis through activation of mTOR pathway in lal-/- mice

Inflammation critically contributes to cancer metastasis, in which myeloid-derived suppressor cells (MDSCs) are an important participant. Although MDSCs are known to suppress immune surveillance, their roles in directly stimulating cancer cell proliferation and metastasis currently remain unclear. MDSCs development and homeostasis is controlled by lysosomal acid lipase (LAL), a critical enzyme in the metabolic signaling pathway that hydrolyzes cholesteryl esters (CE) and triglycerides (TG) in lysosomes. Lysosomal acid lipase (LAL) deficiency causes systemic expansion and infiltration of MDSCs in multiple organs. LAL-deficient (lal-/-) MDSCs arise from dysregulated production of progenitor cells in the bone marrow. In humans, increased CD14+CD16+ and CD14+CD33+ MDSC subsets have been reported with heterozygote carriers of LAL mutations. Patients with mutations in the LAL gene has been reported to be associated with carcinogenesis. In the LAL-deficient (lal-/-) mouse model, melanoma metastasized massively in allogeneic lal-/- mice, which was suppressed in allogeneic lal+/+ mice due to immune rejection. Here we report for the first time that MDSCs isolated from lal-/- mice directly stimulated B16 melanoma cell proliferation in vitro, and growth and metastasis in vivo. Cytokines i.e., IL-1β and TNFα from MDSCs are required for B16 melanoma cell proliferative function in vitro. Myeloid-specific expression of human LAL (hLAL) in lal-/- mice rescues these malignant phenotypes in vitro and in vivo. The tumor-promoting function of lal-/- MDSCs is mediated, at least in part, through over-activation of the mammalian target of rapamycin (mTOR) pathway. mTOR is a serine/threonine protein kinase that regulates cell growth, proliferation, migration, survival, protein synthesis, and transcription in response to growth factors and mitogens. mTOR is the catalytic subunit of two distinctive complexes: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Unique accessory proteins, regulatory-associated protein of mTOR (RAPTOR), and rapamycin-insensitive companion of mTOR (RICTOR) define the TORC1 and TORC2 complexes, respectively. Affymetrix GeneChip microarray analysis and Ingenuity Pathway Analysis of gene transcripts revealed up-regulation of multiple genes in the mTOR signaling pathway in lal-/- MDSCs. We have shown that inhibition of mTOR in lal-/- MDSCs increased apoptosis, decreased ATP synthesis, decreased ROS production, and recovered impairment of mitochondrial membrane potential. As a result, lal-/- MDSCs reversed the increased cell proliferation, corrected enhanced lal-/- MDSCs development from lineage negative progenitor cells, and reduced systemic MDSC expansion. More importantly, knockdown of mTOR, Raptor or Rictor in lal-/- MDSCs reversed the immune suppression on T cell proliferation and function, as well as suppressed their stimulation on proliferation of cancer cells (including B16 melanoma, LLC and Tramp-C2 cancer cells). Our results indicate that LAL plays a critical role in regulating MDSCs through modulation of the mTOR pathway. MDSCs possess dual functions to overcome immune rejection and facilitate cancer metastasis: 1) suppress immune surveillance, and 2) stimulate cancer cell proliferation, growth, and metastasis. Grant support: This work was supported by National Institutes of Health Grants CA138759, CA152099 (to C. Y.) and HL087001 (to H. D.). Citation Format: Hong Du, Ting Zhao, Xinchun Ding, Katlin Walls, Cong Yan. Myeloid-derived suppressor cells suppress immune surveillance and stimulate cancer cell proliferation/metastasis through activation of mTOR pathway in lal-/- mice. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A11.