Characterization of citB in the methylcitric acid cycle of Bacillus subtilis 168 ; and, characterization of antimicrobial activity in the mucosal epithelial layer and gill tissue of largemouth bass (Micropterus salmoides)

Bacillus subtilis 168 is a gram-positive, aerobic, rod shaped bacteria that has the ability to undergo sporulation when under nutritional stress. There is an incomplete understanding of the metabolic pathways in B. subtilis 168 during sporulation. Characterizing the metabolic pathways of B. subtilis is important for understanding the sporulation process in pathogenic bacteria including B. anthraces (anthrax), for understanding cell differentiation of stem cells, and for maximizing biotechnological applications of B. subtilis 168. In this study, we hypothesize that citB, the only know aconitase in B. subtilis 168, can convert 2-methylaconitate to 2-methylisocitrate in the methylcitric acid cycle. This is an important step missing from the methylcitric acid cycle encoded by the mother cell metabolic gene (mmg) operon. In this study we utilized an overall approach involving purified citB protein, HPLC coupled to UV-VIS, and LC-MS to show that the citB protein can convert 2-methylaconitate to 2-methylisocitrate. We have also shown that a previously uncharacterized 2-methylisocitrate lyase (yqiQ) from B. subtilis 168 can convert the citB product 2-methylisocitrate to succinate and pyruvate, thus completing the methylcitric acid cycle. This study confirmed our hypotheses regarding citB and yqiQ and also achieved the first in vitro reconstitution of a complete methylcitric acid cycle from B. subtilis. There is little known about the biochemistry of largemouth bass (Micropterus psalmodies), especially the antimicrobial properties that may be present in the outer mucosal layer of the skin and gills as an initial defense against bacteria, protozoa and fungi. Understanding the antimicrobial properties of the small antimicrobial peptides in largemouth bass is important for the development of new antibiotics for potential use in humans and for decreasing the mortality rates of largemouth bass handled in recreational and tournament fishing, as well as those captured and released for studies by state wildlife management agencies. It is hypothesized that the skin secretions and gills of largemouth bass contain a small, cationic, amphipathic peptide that prevents initial infection from microorganisms present in the environment. In this study, we have tested the hypothesis with in vitro experiments through the use of disc diffusion assays on Escherichia coli K12 and Bacillus subtilis 168 bacteria and liquid growth assays using Staphylococcus aureus. Results of this study show that there is antimicrobial activity in the gills of largemouth bass. We will also report ongoing efforts toward isolating and characterizing the components responsible for this antimicrobial activity

TGLI1 transcription factor mediates breast cancer brain metastasis via activating metastasis-initiating cancer stem cells and astrocytes in the tumor microenvironment

Mechanisms for breast cancer metastasis remain unclear. Whether truncated glioma-associated oncogene homolog 1 (TGLI1), a transcription factor known to promote angiogenesis, migration and invasion, plays any role in metastasis of any tumor type has never been investigated. In this study, results of two mouse models of breast cancer metastasis showed that ectopic expression of TGLI1, but not GLI1, promoted preferential metastasis to the brain. Conversely, selective TGLI1 knockdown using antisense oligonucleotides led to decreased breast cancer brain metastasis (BCBM) in vivo. Immunohistochemical staining showed that TGLI1, but not GLI1, was increased in lymph node metastases compared to matched primary tumors, and that TGLI1 was expressed at higher levels in BCBM specimens compared to primary tumors. TGLI1 activation is associated with a shortened time to develop BCBM and enriched in HER2-enriched and triple-negative breast cancers. Radioresistant BCBM cell lines and specimens expressed higher levels of TGLI1, but not GLI1, than radiosensitive counterparts. Since cancer stem cells (CSCs) are radioresistant and metastasis-initiating cells, we examined TGLI1 for its involvement in breast CSCs and found TGLI1 to transcriptionally activate stemness genes CD44, Nanog, Sox2, and OCT4 leading to CSC renewal, and TGLI1 outcompetes with GLI1 for binding to target promoters. We next examined whether astrocyte-priming underlies TGLI1-mediated brain tropism and found that TGLI1-positive CSCs strongly activated and interacted with astrocytes in vitro and in vivo. These findings demonstrate, for the first time, that TGLI1 mediates breast cancer metastasis to the brain, in part, through promoting metastasis-initiating CSCs and activating astrocytes in BCBM microenvironment

The gain-of-function GLI1 transcription factor TGLI1 enhances expression of VEGF-C and TEM7 to promote glioblastoma angiogenesis

We recently discovered that truncated glioma-associated oncogene homolog 1 (TGLI1) is highly expressed in glioblastoma (GBM) and linked to increased GBM vascularity. The mechanisms underlying TGLI1-mediated angiogenesis are unclear. In this study, we compared TGLI1- with GLI1-expressing GBM xenografts for the expression profile of 84 angiogenesis-associated genes. The results showed that expression of six genes were upregulated and five were down-regulated in TGLI1-carrying tumors compared to those with GLI1. Vascular endothelial growth factor-C (VEGF-C) and tumor endothelial marker 7 (TEM7) were selected for further investigations because of their significant correlations with high vascularity in 135 patient GBMs. TGLI1 bound to both VEGF-C and TEM7 gene promoters. Conditioned medium from TGLI1-expressing GBM cells strongly induced tubule formation of brain microvascular endothelial cells, and the induction was prevented by VEGF-C/TEM7 knockdown. Immunohistochemical analysis of 122 gliomas showed that TGLI1 expression was positively correlated with VEGF-C, TEM7 and microvessel density. Analysis of NCBI Gene Expression Omnibus datasets with 161 malignant gliomas showed an inverse relationship between tumoral VEGF-C, TEM7 or microvessel density and patient survival. Together, our findings support an important role that TGLI1 plays in GBM angiogenesis and identify VEGF-C and TEM7 as novel TGLI1 target genes of importance to GBM vascularity

An FDA-Approved Antifungal, Ketoconazole, and Its Novel Derivative Suppress tGLI1-Mediated Breast Cancer Brain Metastasis by Inhibiting the DNA-Binding Activity of Brain Metastasis-Promoting Transcription Factor tGLI1

The goal of this study is to identify pharmacological inhibitors that target a recently identified novel mediator of breast cancer brain metastasis (BCBM), truncated glioma-associated oncogene homolog 1 (tGLI1). Inhibitors of tGLI1 are not yet available. To identify compounds that selectively kill tGLI1-expressing breast cancer, we screened 1527 compounds using two sets of isogenic breast cancer and brain-tropic breast cancer cell lines engineered to stably express the control, GLI1, or tGLI1 vector, and identified the FDA-approved antifungal ketoconazole (KCZ) to selectively target tGLI1-positive breast cancer cells and breast cancer stem cells, but not tGLI1-negative breast cancer and normal cells. KCZ’s effects are dependent on tGLI1. Two experimental mouse metastasis studies have demonstrated that systemic KCZ administration prevented the preferential brain metastasis of tGLI1-positive breast cancer and suppressed the progression of established tGLI1-positive BCBM without liver toxicities. We further developed six KCZ derivatives, two of which (KCZ-5 and KCZ-7) retained tGLI1-selectivity in vitro. KCZ-7 exhibited higher blood–brain barrier penetration than KCZ/KCZ-5 and more effectively reduced the BCBM frequency. In contrast, itraconazole, another FDA-approved antifungal, failed to suppress BCBM. The mechanistic studies suggest that KCZ and KCZ-7 inhibit tGLI1’s ability to bind to DNA, activate its target stemness genes Nanog and OCT4, and promote tumor proliferation and angiogenesis. Our study establishes the rationale for using KCZ and KCZ-7 for treating and preventing BCBM and identifies their mechanism of action