Alexey A. Leontovich

Characterization of PKD Protein-Positive Exosome-Like Vesicles

Proteins associated with autosomal dominant and autosomal recessive polycystic kidney disease (polycystin-1, polycystin-2, and fibrocystin) localize to various subcellular compartments, but their functional site is thought to be on primary cilia. PC1+ vesicles surround cilia in Pkhd1(del2/del2) mice, which led us to analyze these structures in detail. We subfractionated urinary exosome-like vesicles (ELVs) and isolated a subpopulation abundant in polycystin-1, fibrocystin (in their cleaved forms), and polycystin-2. This removed Tamm-Horsfall protein, the major contaminant, and subfractionated ELVs into at least three different populations, demarcated by the presence of aquaporin-2, polycystin-1, and podocin. Proteomic analysis of PKD ELVs identified 552 proteins (232 not yet in urinary proteomic databases), many of which have been implicated in signaling, including the molecule Smoothened. We also detected two other protein products of genes involved in cystic disease: Cystin, the product of the mouse cpk locus, and ADP-ribosylation factor-like 6, the product of the human Bardet-Biedl syndrome gene (BBS3). Our proteomic analysis confirmed that cleavage of polycystin-1 and fibrocystin occurs in vivo, in manners consistent with cleavage at the GPS site in polycystin-1 and the proprotein convertase site in fibrocystin. In vitro, these PKD ELVs preferentially interacted with primary cilia of kidney and biliary epithelial cells in a rapid and highly specific manner. These data suggest that PKD proteins are shed in membrane particles in the urine, and these particles interact with primary cilia.

Evidence of systemic Th2 driven chronic inflammation in patients with metastatic melanoma

Purpose: Immunotherapeutic modalities are commonly used for treatment of patients with melanoma. The therapeutic success in preclinical models has not yielded the expected clinical results. To understand this discrepancy, we attempted to define immune homeostasis of 209 patients with melanoma across stages of disease relative to normal controls. Experimental Design: Peripheral blood mononuclear cells (PBMC) and plasma were collected from patients and healthy donors. PBMC were analyzed for frequencies of natural killer, dendritic, and T cells and their functional status. Matched plasma samples were analyzed for the concentrations of 27 cytokines, chemokines, and growth factors. RNA was isolated from 24 metastatic melanoma tumor biopsies and profiled by microarray analysis. Results: The frequency of natural killer, T, and dendritic cells in patients does not significantly change across stages of melanoma. However, plasma concentrations of Th2 cytokines [interleukin (IL)-4, IL-5, IL-10, and IL-13] in tumor-bearing patients were significantly higher than those with resected melanoma. Expression array analysis of metastatic melanoma revealed that the malignant melanocytes were not the source of the Th2 cytokines but did highly up-regulate vascular endothelial growth factor (VEGF) transcripts, consistent with plasma VEGF concentrations. In vitro VEGF exposure of normal PBMC lead to repolarization from Th1 to Th2 emulating the state of metastatic melanoma. Conclusions: Patients with metastatic melanoma exist in a state of Th2-mediated “chronic inflammation” as a result of at least VEGF overproduction by malignant tumors. These data support prior observations regarding the effect of VEGF on immune cell function and suggests consideration of VEGF inhibitors in future cancer immunotherapy clinical studies in metastatic melanoma.

Loss of CBP causes T cell lymphomagenesis in synergy with p27Kip1 insufficiency

CBP can function as a tumor suppressor, but the mechanisms that govern oncogenesis in its absence are unknown. Here we show that CBP inactivation in mouse thymocytes leads to lymphoma. Although CBP has been implicated in the transactivation functions of p53, development of these tumors does not seem to involve loss of p53 activity. CBP-null tumors show reduced levels of p27Kip1 and increased levels of cyclin E and Skp2, two oncoproteins that can promote p27Kip1 proteolysis. Reduction of p27Kip1 by introduction of a p27Kip1-null allele into CBP knockout mice accelerates lymphomagenesis and seems to obviate the requirement for Skp2 and cyclin E upregulation. These data suggest that CBP loss mediates lymphomagenesis in cooperation with a mechanism that reduces p27Kip1 abundance.

Heritable Transmission of Diabetic Metabolic Memory in Zebrafish Correlates With DNA Hypomethylation and Aberrant Gene Expression

Metabolic memory (MM) is the phenomenon whereby diabetes complications persist and progress after glycemic recovery is achieved. Here, we present data showing that MM is heritable and that the transmission correlates with hyperglycemia-induced DNA hypomethylation and aberrant gene expression. Streptozocin was used to induce hyperglycemia in adult zebrafish, and then, following streptozocin withdrawal, a recovery phase was allowed to reestablish a euglycemic state. Blood glucose and serum insulin returned to physiological levels during the first 2 weeks of the recovery phase as a result of pancreatic β-cell regeneration. In contrast, caudal fin regeneration and skin wound healing remained impaired to the same extent as in diabetic fish, and this impairment was transmissible to daughter cell tissue. Daughter tissue that was never exposed to hyperglycemia, but was derived from tissue that was, did not accumulate AGEs or exhibit increased levels of oxidative stress. However, CpG island methylation and genome-wide microarray expression analyses revealed the persistence of hyperglycemia-induced global DNA hypomethylation that correlated with aberrant gene expression for a subset of loci in this daughter tissue. Collectively, the data presented here implicate the epigenetic mechanism of DNA methylation as a potential contributor to the MM phenomenon.

MicroRNA expression in ileal carcinoid tumors: downregulation of microRNA-133a with tumor progression

MicroRNAs (miRNAs) are involved in cell proliferation, differentiation, and apoptosis and can function as tumor suppressor genes or oncogenes. The role of miRNAs in neuroendocrine tumors such as ileal carcinoids is largely unknown. We examined the differential expression of 95 miRNAs by RT–PCR using the QuantiMir System in eight matching primary and metastatic carcinoid tumors from the ileum. All miRNAs chosen for the QuantiMir System array were based on their potential functions related to cancer biology, cell development, and apoptosis. The expression of miRNAs for the samples was normalized to miRNA-197, and the matching primary and metastatic tumors were compared. There was downregulation of miRNA-133a, -145, -146, -222, and -10b in all samples between the primary and matching metastatic tumors and upregulation of miRNA-183, -488, and -19a+b in six of eight metastatic carcinoids compared to the primary tumors. miRNA-133a was further analyzed by TaqMan real-time RT–PCR and northern hybridization using six additional matching primary and metastatic samples, which supported the PCR array findings. There were significant differences in miRNA-133a expression with downregulation in the metastasis compared to the primary in the eight original cases (P<0.009) and in the six additional cases used for validation (P<0.014). Laser capture microdissection and real-time RT–PCR analysis using normal ileum found miRNA-133a expression in normal enterochromaffin cells. In situ hybridization in normal ileum showed that some of the mucosal endocrine cells expressed miRNA-133a. Both primary and metastatic ileal carcinoid tumors expressed miRNA-133a by in situ hybridization. These results provide information about novel marker miRNAs that may be used as biomarkers and/or therapeutic targets in intestinal carcinoid tumors.

Proteomic Analysis of Waldenstrom Macroglobulinemia

To better understand the molecular changes that occur in Waldenstrom macroglobulinemia (WM), we employed antibody-based protein microarrays to compare patterns of protein expression between untreated WM and normal bone marrow controls. Protein expression was defined as a >2-fold or 1.3-fold change in at least 67% of the tumor samples. Proteins up-regulated by >2-fold included Ras family proteins, such as Rab-4 and p62DOK, and Rho family proteins, such as CDC42GAP and ROKalpha. Other proteins up-regulated by >1.3-fold included cyclin-dependent kinases, apoptosis regulators, and histone deacetylases (HDAC). We then compared the samples of patients with symptomatic and asymptomatic WM and showed similar protein expression signatures, indicating that the dysregulation of signaling pathways occurs early in the disease course. Three proteins were different by >2-fold in symptomatic versus asymptomatic, including the heat shock protein HSP90. Elevated protein expression was confirmed by immunohistochemistry and immunoblotting. Functional significance was validated by the induction of apoptosis and inhibition of proliferation using specific HDAC and HSP90 inhibitors. This study, therefore, identifies, for the first time, multiple novel proteins that are dysregulated in WM, which both enhance our understanding of disease pathogenesis and represent targets of novel therapeutics.

Intranasal exposure to staphylococcal enterotoxin B elicits an acute systemic inflammatory response.

ABSTRACT Staphylococcus aureus produces a variety of superantigen exotoxins, including staphylococcal enterotoxin B (SEB). Little is known regarding the pathogenesis of SEB entering through the intranasal route. Intranasal exposure to SEB might occur because of nasal packing following surgical procedure, biologic warfare, or even S. aureus colonization. We evaluated the local and systemic effects of intranasally delivered SEB using a series of human leukocyte antigen (HLA) class II transgenic mice as conventional mice expressing endogenous class II molecules mount a poor immune response to SEB. Gene expression profiling using microarrays showed robust up-regulation of genes involved in several proinflammatory pathways as early as 3 h post-intranasal challenge with SEB in HLA class II transgenic mice. This was accompanied by a several hundred-fold increase in serum levels of pro-inflammatory cytokines such as IL-12, IL-6, TNF-&agr;, IFN-&ggr;, as well as MCP-1 in HLA class II transgenic mice but not in C57BL/6 mice; CD4+ or CD8+ T-cells independently contributed to the systemic cytokine response. Defective IL-12 or IL-4 receptor signaling significantly decreased or increased serum IFN-&ggr;, respectively. Intranasal exposure to SEB resulted in neutrophil influx into bronchoalveolar lavage fluid and caused expansion of both CD4+ and CD8+ T-cells expressing TCR V&bgr;8 in the spleen. This was accompanied by mononuclear cell infiltration in the liver reminiscent of the systemic inflammatory response syndrome. Thus, we have shown, for the first time, that intranasal administration of SEB can cause systemic immune activation.ABBREVIATIONS-BAL, bronchoalveolar lavage, SEB, staphylococcal enterotoxin B, STAT, signal transducer and activator of transcription

The mitotic kinase Aurora-A promotes distant metastases by inducing epithelial-to-mesenchymal transition in ERα + breast cancer cells

In this study, we demonstrate that constitutive activation of Raf-1 oncogenic signaling induces stabilization and accumulation of Aurora-A mitotic kinase that ultimately drives the transition from an epithelial to a highly invasive mesenchymal phenotype in estrogen receptor α-positive (ERα+) breast cancer cells. The transition from an epithelial- to a mesenchymal-like phenotype was characterized by reduced expression of ERα, HER-2/Neu overexpression and loss of CD24 surface receptor (CD24−/low). Importantly, expression of key epithelial-to-mesenchymal transition (EMT) markers and upregulation of the stemness gene SOX2 was linked to acquisition of stem cell-like properties such as the ability to form mammospheres in vitro and tumor self-renewal in vivo. Moreover, aberrant Aurora-A kinase activity induced phosphorylation and nuclear translocation of SMAD5, indicating a novel interplay between Aurora-A and SMAD5 signaling pathways in the development of EMT, stemness and ultimately tumor progression. Importantly, pharmacological and molecular inhibition of Aurora-A kinase activity restored a CD24+ epithelial phenotype that was coupled to ERα expression, downregulation of HER-2/Neu, inhibition of EMT and impaired self-renewal ability, resulting in the suppression of distant metastases. Taken together, our findings show for the first time the causal role of Aurora-A kinase in the activation of EMT pathway responsible for the development of distant metastases in ERα+ breast cancer cells. Moreover, this study has important translational implications because it highlights the mitotic kinase Aurora-A as a novel promising therapeutic target to selectively eliminate highly invasive cancer cells and improve the disease-free and overall survival of ERα+ breast cancer patients resistant to conventional endocrine therapy.

Early cutaneous gene transcription changes in adult atopic dermatitis and potential clinical implications.

Abstract:  Atopic dermatitis (AD) is a common pruritic dermatitis with macroscopically non‐lesional skin that is often abnormal. Therefore, we used high‐density oligonucleotide arrays to identify cutaneous gene transcription changes associated with early AD inflammation as potential disease control targets. Skin biopsy specimens analysed included normal skin from five healthy non‐atopic adults and both minimally lesional skin and nearby or contralateral non‐lesional skin from six adult AD patients. Data were analysed on an individual gene basis and to identify biologically relevant gene networks. Transcription levels of selected genes were also analysed by quantitative PCR. Differential transcription occurring early in AD skin was indicated for (i) individual genes such as C‐C chemokine ligand (CCL)18, CCL13, and interferon‐α2 (IFNα2), (ii) genes associated with peroxisome proliferator‐activated receptor (PPAR)α‐ and PPARγ‐regulated transcription, and possibly for (iii) immunoglobulin J‐chain and heavy chain isotype transcripts. These data suggest that local changes in immunoglobulin‐associated transcription may favour IgE over secretory immunoglobulin (multimeric IgM and IgA) expression in AD skin. Decreased PPAR activity appears common to both AD and psoriasis, and reduced cutaneous IFNα2 transcription also appears characteristic of AD. Identification of these genes and pathways will direct future research towards controlling AD.

Patterns of gene expression in pituitary carcinomas and adenomas analyzed by high-density oligonucleotide arrays, reverse transcriptase-quantitative PCR, and protein expression

Very few of the genes that are important in pituitary tumor initiation, progression, and metastasis have been identified to date. To identify potential genes that may be important in pituitary tumor progression and carcinoma development, we used AffymetrixTM GeneChip HGU-133A-oligonucleotide arrays, which contain more than 15,000 characterized genes from the human genome to study gene expression in an ACTH pituitary carcinoma metastatic to the liver and four pituitary adenomas. Reverse-transcriptase real-time quantitative-PCR (RT-qPCR) was then used to analyze 4 nonneoplastic pituitaries, 19 adenomas, and the ACTH carcinoma. A larger series of pituitary adeno mas and carcinomas were also analyzed for protein expression using tissue microarrays (TMA) (n=233) and by Western blotting (n=18). There were 4298 genes that were differentially expressed among the adenomas compared to the carcinoma, with 2057 genes overexpressed and 2241 genes underexpressed in the adenomas. The betagalacioside binding protein galactin-3 was underexpressed in some adenomas compared to the carcinomas. Prolactin (PRL) and ACTH tumors had the highest levels of expression of galectin-3. The human achaetescute homolog-1 ASCL1 (hASH-1) gene was also underexpressed in some adenomas compared to the carcinoma. Prolactin and ACTH tumors had the highest levels of expression of hASH-1. ID2, which has an important role in cell development and tumorigenesis, was underexpressed in some adenomas compared to the carcinomas. Transducin-like enhancer of split four/Croucho (TLE-4) was over-expressed in adenomas compared to the ACTH carcinoma. The differential expression of these genes was validated by RT-qPCR, by immunohistochemistry using TMA and by Western blotting. These results indicate that the LGALS3, hASH1, ID2, and TLE-4 genes may have important roles in the development of pituitary carcinomas.