Knock-Out of the Five Lysyl-Oxidase Family Genes Enables Identification of Lysyl-Oxidase Pro-Enzyme Regulated Genes.

The five lysyl-oxidase genes share similar enzymatic activities and contribute to tumor progression. We have knocked out the five lysyl-oxidase genes in MDA-MB-231 breast cancer cells using CRISPR/Cas9 in order to identify genes that are regulated by LOX but not by other lysyl-oxidases and in order to study such genes in more mechanistic detail in the future. Re-expression of the full-length cDNA encoding LOX identified four genes whose expression was downregulated in the knock-out cells and rescued following LOX re-expression but not re-expression of other lysyl-oxidases. These were the AGR2, STOX2, DNAJB11 and DNAJC3 genes. AGR2 and STOX2 were previously identified as promoters of tumor progression. In addition, we identified several genes that were not downregulated in the knock-out cells but were strongly upregulated following LOX or LOXL3 re-expression. Some of these, such as the DERL3 gene, also promote tumor progression. There was very little proteolytic processing of the re-expressed LOX pro-enzyme in the MDA-MB-231 cells, while in the HEK293 cells, the LOX pro-enzyme was efficiently cleaved. We introduced point mutations into the known BMP-1 and ADAMTS2/14 cleavage sites of LOX. The BMP-1 mutant was secreted but not cleaved, while the LOX double mutant dmutLOX was not cleaved or secreted. However, even in the presence of the irreversible LOX inhibitor ß-aminoproprionitrile (BAPN), these point-mutated LOX variants induced the expression of these genes, suggesting that the LOX pro-enzyme has hitherto unrecognized biological functions.

The role of CCR5 in directing the mobilization and biological function of CD11b+Gr1+Ly6Clow polymorphonuclear myeloid cells in cancer.

Bone marrow (BM) cells of the hematopoietic system, also known as BM-derived leukocytes (BMD), are mobilized from the BM to the blood and then colonize tumor sites. These cells then become key players in either promoting or regulating the development and progression of tumors. Among the cells that suppress anti-tumor immunity are regulatory T cells (Tregs), tumor-associated macrophages (TAMS) and myeloid-derived suppressor cells (MDSC). MDSC comprise CD11b+Gr1+Ly6Clow polymorphonuclear myeloid cells (PMN-MDSC), and CD11b+Gr1+Ly6Chigh monocytic myeloid cells (Mo-MDSC). Several studies including ours have identified the CCR2-CCL2 axis as the key driver of the mobilization of monocytic cells from the BM to the blood and later their colonization at the tumor site. The current review focuses on the mechanisms by which PMN-MDSC are mobilized from the BM to the blood and later to the tumor site, and their clinical implications.

Chemokines beyond chemo-attraction: CXCL10 and its significant role in cancer and autoimmunity.

Chemokines are mostly known for their chemotactic properties, and less for their ability to direct the biological function of target cells, including T cells. The current review focuses on a key chemokine named CXCL10 and its role in directing the migratory propertied and biological function of CD4+ and CD8+ T cells in the context of cancer and inflammatory autoimmunity. CXCR3 is a chemokine receptor that is abundant on CD4+ T cells, CD8+ T cells and NK cells. It has three known ligands: CXCL9, CXCL10 and CXCL11. Different studies, including those coming form our laboratory, indicated that aside of attracting CD8+ and CD4+ effector T cells to tumor sites and sites of inflammation CXCL10 directs the polarization and potentiates the biological function of these cells. This makes CXCL10 a "key driver chemokine" and a valid target for therapy of autoimmune diseases such as Inflammatory Bowl's Disease, Multiple Sclerosis, Rheumatoid arthritis and others. As for cancer this motivated different groups, including our group to develop CXCL10 based therapies for cancer due to its ability to enhance T-dependent anti cancer immunity. The current review summarizes these findings and their potential translational implication.

CCR5+ Myeloid-Derived Suppressor Cells Are Enriched and Activated in Melanoma Lesions.

Accumulation of myeloid-derived suppressor cells (MDSC) in melanoma microenvironment is supported by chemokine receptor/chemokine signaling. Although different chemokines were suggested to be involved in this process, the role of CCR5 and its ligands is not established. Using a Ret transgenic mouse melanoma model, we found an accumulation of CCR5+ MDSCs in melanoma lesions associated with both increased concentrations of CCR5 ligands and tumor progression. Tumor-infiltrating CCR5+ MDSCs displayed higher immunosuppressive activity than their CCR5- counterparts. Upregulation of CCR5 expression on CD11b+Gr1+ myeloid cells was induced in vitro by CCR5 ligands and other inflammatory factors. In melanoma patients, CCR5+ MDSCs were enriched at the tumor site and correlated with enhanced production of CCR5 ligands. Moreover, they exhibited a stronger immunosuppressive pattern compared with CCR5- MDSCs. Blocking CCR5/CCR5 ligand interactions increased survival of tumor-bearing mice and was associated with reduced migration and immunosuppressive potential of MDSCs in tumor lesions. Our findings define a critical role for CCR5 in recruitment and activation of MDSCs, suggesting a novel strategy for melanoma treatment.Significance: These findings validate the importance of the CCR5/CCR5 ligand axis not only for MDSC recruitment but also for further activation of their immunosuppressive functions in the tumor microenvironment, with potentially broad therapeutic implications, given existing clinically available inhibitors of this axis. Cancer Res; 78(1); 157-67. ©2017 AACR.

CCR5 Directs the Mobilization of CD11b+Gr1+Ly6Clow Polymorphonuclear Myeloid Cells from the Bone Marrow to the Blood to Support Tumor Development.

Cells of hematopoietic origin can be subdivided into cells of the lymphoid lineage and those of the myeloid lineage, among which are myeloid-derived suppressor cells (MDSCs). The MDSCs can be further divided into CD11b+Ly6G-Ly6Chi monocytic (Mo) MDSCs and CD11b+Ly6G+Ly6Clow polymorphonuclear (PMN) MDSCs. Both subtypes support tumor growth and suppress anti-tumor immunity. Their accumulation at the tumor site includes mobilization from the bone marrow to the blood followed by colonization at the tumor site. The present study examines the mechanism by which PMN-MDSCs are mobilized from the BM to the blood to later accumulate at the tumor site. We show that the chemokine receptor CCR5 is a key driver of this event. We also show that, beyond chemoattraction, the interaction between CCR5 and its ligands promotes the proliferation of CCR5+ PMN-MDSCs at the BM and, later, potentiates their immune-suppressive activities at the tumor site in part by inducing arginase-1.

Effect of alpha-naphthylisothiocyanate-induced liver injury on intestinal adaptation in a rat model of short bowel syndrome.

BACKGROUND/PURPOSE: Progressive hyperbilirubinemia and end-stage liver failure are among the most serious complications of short bowel syndrome (SBS), representing the principle cause of death in a majority of fatal cases. In the current study, we examined the effects of alpha-naphthylisothiocyanate (ANIT)-induced liver injury on intestinal adaptation in a rat model of SBS. METHODS: Male rats were divided into four groups: Sham rats underwent bowel transection (n = 8), Sham liver-injury rats underwent bowel transection and IP injection of ANIT (100 mg/kg, n = 8), SBS rats underwent a 75% bowel resection, and SBS-ANIT rats underwent bowel resection and liver injury similar to group sham-ANIT (n = 8). Fourteen days after intervention, liver biopsies and intestinal samples were obtained and evaluated for liver damage and measures of intestinal adaptation. Real time PCR and Western blotting were used to determine the level of bax and bcl-2 mRNA and protein, and p-ERK protein levels. Statistical analysis was performed using the one-way ANOVA test, with p < 0.05 considered statistically significant. RESULTS: All ANIT-treated animals exhibited histological evidence of liver damage that was associated with the expansion of atypical ductal proliferation near the periportal areas, intense neutrophil infiltration in the liver, increased mitotic activity, Kupfer cells hyperplasia and fatty liver degeneration. ANIT-induced liver damage in bowel resected animals was associated with a significant decrease in all parameters of intestinal adaptation including bowel and mucosal weight in jejunum (twofold decrease) and ileum (twofold decrease), mucosal DNA in jejunum (fourfold decrease), mucosal protein in jejunum (threefold decrease) and ileum (threefold decrease), villus height in jejunum (38%) and ileum (34%), and crypt depth in jejunum (24%) and ileum (30%) compared to SBS animals. Both Sham-ANIT and SBS-ANIT rats demonstrated decreased enterocyte proliferation rates that were accompanied by decreased p-ERK protein levels. Lower apoptotic rates in jejunum (40%) and ileum (52%) in SBS-ANIT rats (vs. SBS) coincided with decreased bax mRNA and protein levels. CONCLUSIONS: In a rat model of SBS, ANIT-induced liver injury was associated with decreased enterocyte proliferation and inhibited intestinal adaptation.