IU Indianapolis ScholarWorks :: Browsing by Author "Ding, Xinchun"

Browsing by Author "Ding, Xinchun"

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Activation of mTOR pathway in myeloid-derived suppressor cells stimulates cancer cell proliferation and metastasis in lal(-/-) mice
(Springer Nature, 2015-04-09) Zhao, Ting; Du, Hong; Ding, Xinchun; Walls, Katlin; Yan, Cong; Department of Pathology & Laboratory Medicine, IU School of Medicine
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.
CAMKK2 is Upregulated in Primary Human Osteoarthritis and its Inhibition Protects Against Chondrocyte Apoptosis
(Elsevier, 2023) Dilley, Julian E.; Seetharam, Abhijit; Ding, Xinchun; Bello, Margaret A.; Shutter, Jennifer; Burr, David B.; Natoli, Roman M.; McKinley, Todd O.; Sankar, Uma; Anatomy, Cell Biology and Physiology, School of Medicine
Objective: To investigate the role of calcium/calmodulin-dependent protein kinase kinase 2 (CAMKK2) in human osteoarthritis. Materials and methods: Paired osteochondral plugs and articular chondrocytes were isolated from the relatively healthier (intact) and damaged portions of human femoral heads collected from patients undergoing total hip arthroplasty for primary osteoarthritis (OA). Cartilage from femoral plugs were either flash frozen for gene expression analysis or histology and immunohistochemistry. Chondrocyte apoptosis in the presence or absence of CAMKK2 inhibition was measured using flow cytometry. CAMKK2 overexpression and knockdown in articular chondrocytes were achieved via Lentivirus- and siRNA-mediated approaches respectively, and their effect on pro-apoptotic and cartilage catabolic mechanisms was assessed by immunoblotting. Results: CAMKK2 mRNA and protein levels were elevated in articular chondrocytes from human OA cartilage compared to paired healthier intact samples. This increase was associated with elevated catabolic marker matrix metalloproteinase 13 (MMP-13), and diminished anabolic markers aggrecan (ACAN) and type II collagen (COL2A1) levels. OA chondrocytes displayed enhanced apoptosis, which was suppressed following pharmacological inhibition of CAMKK2. Levels of MMP13, pSTAT3, and the pro-apoptotic marker BAX became elevated when CAMKK2, but not its kinase-defective mutant was overexpressed, whereas knockdown of the kinase decreased the levels of these proteins. Conclusions: CAMKK2 is upregulated in human OA cartilage and is associated with elevated levels of pro-apoptotic and catabolic proteins. Inhibition or knockdown of CAMKK2 led to decreased chondrocyte apoptosis and catabolic protein levels, whereas its overexpression elevated them. CAMKK2 may be a therapeutic target to prevent or mitigate human OA.
Critical Role of the mTOR Pathway in Development and Function of Myeloid-Derived Suppressor Cells in lal−/− Mice
(Elsevier B.V., 2014-02) Ding, Xinchun; Du, Hong; Yoder, Mervin C.; Yan, Cong; Department of Pathology and Laboratory Medicine, IU School of Medicine
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.
Endothelial Rab7 GTPase mediates tumor growth and metastasis in lysosomal acid lipase-deficient mice
(American Society for Biochemistry and Molecular Biology, 2017-11-24) Zhao, Ting; Ding, Xinchun; Yan, Cong; Du, Hong; Pathology and Laboratory Medicine, School of Medicine
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.
Establishment of lal-/- myeloid lineage cell line that resembles myeloid-derived suppressive cells
(PLoS, 2015-03-25) Ding, Xinchun; Wu, Lingyan; Yan, Cong; Du, Hong; Department of Pathology and Laboratory Medicine, IU School of Medicine
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.
Hepatocyte-Specific Expression of Human Lysosome Acid Lipase Corrects Liver Inflammation and Tumor Metastasis in lal(-/-) Mice
(Elsevier, 2015-09) Du, Hong; Zhao, Ting; Ding, Xinchun; Yan, Cong; Department of Pathology and Laboratory Medicine, IU School of Medicine
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.
LAL deficiency induced myeloid-derived suppressor cells as targets and biomarkers for lung cancer
(BMJ, 2023) Zhao, Ting; Liu, Sheng; Hanna, Nasser H.; Jalal, Shadia; Ding, Xinchun; Wan, Jun; Yan, Cong; Du, Hong; Pathology and Laboratory Medicine, School of Medicine
Background: Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells in tumor microenvironment, which suppress antitumor immunity. Expansion of various MDSC subpopulations is closely associated with poor clinical outcomes in cancer. Lysosomal acid lipase (LAL) is a key enzyme in the metabolic pathway of neutral lipids, whose deficiency (LAL-D) in mice induces the differentiation of myeloid lineage cells into MDSCs. These Lal -/- MDSCs not only suppress immune surveillance but also stimulate cancer cell proliferation and invasion. Understanding and elucidating the underlying mechanisms of MDSCs biogenesis will help to facilitate diagnosis/prognosis of cancer occurrence and prevent cancer growth and spreading. Methods: Single-cell RNA sequencing (scRNA-seq) was performed to distinguish intrinsic molecular and cellular differences between normal versus Lal -/- bone marrow-derived Ly6G+ myeloid populations in mice. In humans, LAL expression and metabolic pathways in various myeloid subsets of blood samples of patients with non-small cell lung cancer (NSCLC) were assessed by flow cytometry. The profiles of myeloid subsets were compared in patients with NSCLC before and after the treatment of programmed death-1 (PD-1) immunotherapy. Results: scRNA-seq of Lal -/- CD11b+Ly6G+ MDSCs identified two distinctive clusters with differential gene expression patterns and revealed a major metabolic shift towards glucose utilization and reactive oxygen species (ROS) overproduction. Blocking pyruvate dehydrogenase (PDH) in glycolysis reversed Lal -/- MDSCs' capabilities of immunosuppression and tumor growth stimulation and reduced ROS overproduction. In the blood samples of human patients with NSCLC, LAL expression was significantly decreased in CD13+/CD14+/CD15+/CD33+ myeloid cell subsets. Further analysis in the blood of patients with NSCLC revealed an expansion of CD13+/CD14+/CD15+ myeloid cell subsets, accompanied by upregulation of glucose-related and glutamine-related metabolic enzymes. Pharmacological inhibition of the LAL activity in the blood cells of healthy participants increased the numbers of CD13+ and CD14+ myeloid cell subsets. PD-1 checkpoint inhibitor treatment in patients with NSCLC reversed the increased number of CD13+ and CD14+ myeloid cell subsets and PDH levels in CD13+ myeloid cells. Conclusion: These results demonstrate that LAL and the associated expansion of MDSCs could serve as targets and biomarkers for anticancer immunotherapy in humans.
Lung Epithelial Cell–Specific Expression of Human Lysosomal Acid Lipase Ameliorates Lung Inflammation and Tumor Metastasis in Lipa−/− Mice
(Elsevier, 2016-08) Zhao, Ting; Ding, Xinchun; Du, Hong; Yan, Cong; Pathology and Laboratory Medicine, School of Medicine
Lysosomal acid lipase (LAL), a key enzyme in the metabolic pathway of neutral lipids, has a close connection with inflammation and tumor progression. One major manifestation in LAL-deficient (Lipa−/−) mice is an increase of tumor growth and metastasis associated with expansion of myeloid-derived suppressor cells. In the lung, LAL is highly expressed in alveolar type II epithelial cells. To assess how LAL in lung epithelial cells plays a role in this inflammation-related pathogenic process, lung alveolar type II epithelial cell–specific expression of human LAL (hLAL) in Lipa−/− mice was established by crossbreeding of CCSP-driven rtTA transgene and (TetO)7-CMV-hLAL transgene into Lipa−/− mice (CCSP-Tg/KO). hLAL expression in lung epithelial cells not only reduced tumor-promoting myeloid-derived suppressor cells in the lung, but also down-regulated the synthesis and secretion of tumor-promoting cytokines and chemokines into the bronchoalveolar lavage fluid of Lipa−/− mice. hLAL expression reduced the immunosuppressive functions of bronchoalveolar lavage fluid cells, inhibited bone marrow cell transendothelial migration, and inhibited endothelial cell proliferation and migration in Lipa−/− mice. As a result, hLAL expression in CCSP-Tg/KO mice corrected pulmonary damage, and inhibited tumor cell proliferation and migration in vitro, and tumor metastasis to the lung in vivo. These results support a concept that LAL is a critical metabolic enzyme in lung epithelial cells that regulates lung homeostasis, immune response, and tumor metastasis.
Lysosomal Acid Lipase Deficiency Controls T- and B-Regulatory Cell Homeostasis in the Lymph Nodes of Mice with Human Cancer Xenotransplants
(Elsevier, 2021) Ding, Xinchun; Zhao, Ting; Lee, Chih-Chun; Yan, Cong; Du, Hong; Pathology and Laboratory Medicine, School of Medicine
Utilization of proper preclinical models accelerates development of immunotherapeutics and the study of the interplay between human malignant cells and immune cells. Lysosomal acid lipase (LAL) is a critical lipid hydrolase that generates free fatty acids and cholesterol. Ablation of LAL suppresses immune rejection and allows growth of human lung cancer cells in lal-/- mice. In the lal-/- lymph nodes, the percentages of both T- and B-regulatory cells (Tregs and Bregs, respectively) are increased, with elevated expression of programmed death-ligand 1 and IL-10, and decreased expression of interferon-γ. Levels of enzymes in the glucose and glutamine metabolic pathways are elevated in Tregs and Bregs of the lal-/- lymph nodes. Pharmacologic inhibitor of pyruvate dehydrogenase, which controls the transition from glycolysis to the citric acid cycle, effectively reduces Treg and Breg elevation in the lal-/- lymph nodes. Blocking the mammalian target of rapamycin or reactivating peroxisome proliferator-activated receptor γ, an LAL downstream effector, reduces lal-/- Treg and Breg elevation and PD-L1 expression in lal-/- Tregs and Bregs, and improves human cancer cell rejection. Treatment with PD-L1 antibody also reduces Treg and Breg elevation in the lal-/- lymph nodes and improves human cancer cell rejection. These observations conclude that LAL-regulated lipid metabolism is essential to maintain antitumor immunity.
Lysosomal acid lipase, CSF1R, and PD-L1 determine functions of CD11c+ myeloid-derived suppressor cells
(The American Society for Clinical Investigation, 2022-09-08) Zhao, Ting; Liu, Sheng; Ding, Xinchun; Johnson, Erica M.; Hanna, Nasser H.; Singh, Kanhaiya; Sen, Chandan K.; Wan, Jun; Du, Hong; Yan, Cong; Pathology and Laboratory Medicine, School of Medicine
Lysosomal acid lipase (LAL) is a key enzyme in the metabolic pathway of neutral lipids. In the blood of LAL-deficient (Lal-/-) mice, increased CD11c+ cells were accompanied by upregulated programmed cell death ligand 1 (PD-L1) expression. Single-cell RNA sequencing of Lal-/- CD11c+ cells identified 2 distinctive clusters with a major metabolic shift toward glucose utilization and reactive oxygen species overproduction. Pharmacologically blocking pyruvate dehydrogenase in glycolysis not only reduced CD11c+ cells and their PD-L1 expression but also reversed their capabilities of T cell suppression and tumor growth stimulation. Colony-stimulating factor 1 receptor (CSF1R) played an essential role in controlling Lal-/- CD11c+ cell homeostasis and function and PD-L1 expression. Pharmacological inhibition of LAL activity increased CD11c, PD-L1, and CSF1R levels in both normal murine myeloid cells and human blood cells. Tumor-bearing mice and human patients with non-small cell lung cancer also showed CD11c+ cell expansion with PD-L1 and CSF1R upregulation and immunosuppression. There were positive correlations among CD11c, PD-L1, and CSF1R expression and negative correlations with LAL expression in patients with lung cancer or melanoma using The Cancer Genome Atlas database and patient samples. Therefore, CD11c+ cells switched their functions to immune suppression and tumor growth stimulation through CSF1R/PD-L1 upregulation and metabolic reprogramming.

Browsing by Author "Ding, Xinchun"

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