Praveen K. Sharma

Serum CXCL13 positively correlates with prostatic disease, prostate-specific antigen and mediates prostate cancer cell invasion, integrin clustering and cell adhesion

Chemokines and their corresponding receptor interactions have been shown to be involved in prostate cancer (PCa) progression and organ-specific metastasis. We have recently shown that PCa cell lines and primary prostate tumors express CXCR5, which correlates with PCa grade. In this study, we present the first evidence that CXCL13, the only ligand for CXCR5, and IL-6 were significantly elevated in PCa patient serum compared to serum from subjects with benign prostatic hyperplasia (BPH), or high-grade prostatic intraepithelial neoplasia (HGPIN) as well as normal healthy donors (NHD). Serum CXCL13 levels significantly (p<0.0001) correlated with serum prostate-specific antigen (PSA), whereas serum IL-6 levels significantly (p<0.0003) correlated with CXCL13 serum levels. CXCL13 was found to be a better predictor of PCa than PSA. CXCL13 was highly expressed by human bone marrow endothelial (HBME) cells and osteoblasts (OBs), but not osteoclasts (OCs), following treatment with physiologically relevant levels of interleukin-6 (IL-6). We further demonstrate that CXCL13, produced by IL-6-treated HBME cells, was able to induce PCa cell invasion in a CXCR5-dependent manner. CXCL13-mediated PCa cell adhesion to HBME cells and alpha(v)beta(3)-integrin clustering was abrogated by CXCR5 blockade. These results demonstrate that the CXCL13-CXCR5 axis is significantly associated with PCa progression.

CXCL13 mediates prostate cancer cell proliferation through JNK signalling and invasion through ERK activation.

Objectives:  The focus of this study was to determine the dedicator of cytokinesis 2 (DOCK2), extracellular signal‐regulated kinase 1/2 (ERK1/2), c‐Jun N‐terminal kinase‐1 (JNK) and Akt signals involved in CXCL13‐mediated prostate cancer (PCa) cell invasion and proliferation.

CCR9 mediates PI3K/AKT-dependent antiapoptotic signals in prostate cancer cells and inhibition of CCR9-CCL25 interaction enhances the cytotoxic effects of etoposide

Despite recent advances in treatment and management of prostate cancer (PCa), it remains the second leading cause of cancer‐related deaths among men in the US. Chemotherapy is one of the treatment alternatives for hormone refractory metastatic PCa. However, current chemotherapeutic regimens provide palliative benefit but relatively modest survival advantage primarily due to chemo‐resistance and upregulated antiapoptotic machineries in PCa cells. Therefore, blocking the mechanisms responsible for suppression of apoptosis might improve current chemotherapeutic regimens. In this study, we show that CC chemokine receptor‐9 (CCR9) and its natural ligand CCL25 interaction upregulates antiapoptotic proteins (i.e., PI3K, AKT, ERK1/2 and GSK‐3β) and downregulate activation of caspase‐3 in PCa cells. Significant downregulation of these CCR9‐mediated antiapoptotic proteins in the presence of a PI3K inhibitor (wortmannin), further suggests that the antiapoptotic action of CCR9 is primarily regulated through PI3K. Furthermore, the cytotoxic effect of etoposide was significantly inhibited in the presence of CCL25, and this inhibitory effect of CCL25 was abrogated when CCR9‐CCL25 interaction was blocked using anti‐CCR9 monoclonal antibodies. In conformation to these in vitro studies, significant reduction in tumor burden was found in mice receiving CCL25 neutralizing antibodies and etoposide together as compared to both as a single agent. These results suggest that the CCR9‐CCL25 axis mediates PI3K/AKT‐dependent antiapoptotic signals in PCa cells and could be a possible reason for low apoptosis and modest chemotherapeutic response. Therefore, targeting CCR9‐CCL25 axis with cytotoxic agents may provide better therapeutic outcomes than using cytotoxic agents alone.

PI3Kp110-, Src-, FAK-dependent and DOCK2-independent migration and invasion of CXCL13-stimulated prostate cancer cells.

BackgroundMost prostate cancer (PCa)-related deaths are due to metastasis, which is mediated in part by chemokine receptor and corresponding ligand interaction. We have previously shown that PCa tissue and cell lines express high levels of the chemokine receptor CXCR5, than compared to their normal counterparts, and interaction of CXCR5 with its specific ligand (CXCL13) promoted PCa cell invasion, migration, and differential matrix metalloproteinase (MMP) expression. This study dissects some of the molecular mechanisms following CXCL13-CXCR5 interaction that mediate PCa cell migration and invasion.ResultsUsing Western blot analysis, kinase-specific cell-based ELISAs, and migration and invasion assays, we show that PCa cell lines differentially express phosphoinositide-3 kinase (PI3K) catalytic subunit isoforms and dedicator of cytokinesis 2 (DOCK2). Specifically, we show that PC3 and normal prostatic epithelial (RWPE-1), but not LNCaP cell lines expressed DOCK2, while RWPE, PC3, and LNCaP cell lines expressed PI3K-p110α and -p110β. Moreover, PC3 selectively expressed PI3K-p110γ, but LNCaP and RWPE cell lines expressed PI3Kp110δ. CXCL13 caused CXCR5-dependent activation of the PI3Kp85α in LNCaP cells, and p85α as well as -p101 in PC3 cells. CXCL13-CXCR5 interaction regulated LNCaP and PC3 cell migration and invasion through extracellular signal-regulated kinase 1/2 (ERK1/2) activation that was primarily dependent on the PI3Kp110 isoform(s), Src, and focal adhesion kinase (FAK), but not DOCK2.ConclusionsWhile additional studies will be needed to determine the PI3K-independent (i.e., DOCK2-mediated) and -dependent events that dictate PCa cell responsiveness to CXCL13, these data provide evidence of the existence of cell type- and stimulus-specific signaling events that support migration and invasion of PCa cells.

Vibrio cholerae Porin OmpU Induces Pro-Inflammatory Responses, but Down-Regulates LPS-Mediated Effects in RAW 264.7, THP-1 and Human PBMCs

Vibrio cholerae porin OmpU plays a crucial role in the survival of the organism in the human gut. Various observations suggest critical involvement of OmpU in V. cholerae pathogenesis. However, OmpU is poorly characterized in terms of its ability to evoke cellular responses, particularly in the context of host immune system. Therefore, towards characterizing V. cholerae OmpU for its host immunomodulatory functions, we have studied the ability of OmpU to elicit pro-inflammatory responses in a range of immune cells which include, mouse RAW 264.7 macrophages, human THP-1 monocytes and human PBMCs. We have observed that purified OmpU induces pro-inflammatory responses in terms of production of NO, TNFα and IL-6. Interestingly, pre-treatment of the cells with OmpU suppresses the production of NO, TNFα, IL-6 as well as IL-12 upon subsequent activation with LPS. Our results therefore suggest that V. cholerae OmpU may have a differential regulatory role in terms of host immunomodulatory function: it can induce pro-inflammatory responses in target host immune cells, whereas it can also exert suppressive effect on LPS-induced pro-inflammatory responses. In addition, our study indicates that purified OmpU may have the ability to skew the Th1 response towards the Th2 response, presumably via suppression of IL-12 production.

Helper T Cell Epitope-Mapping Reveals MHC-Peptide Binding Affinities That Correlate with T Helper Cell Responses to Pneumococcal Surface Protein A

Understanding the requirements for protection against pneumococcal carriage and pneumonia will greatly benefit efforts in controlling these diseases. Several proteins and polysaccharide capsule have recently been implicated in the virulence of and protective immunity against Streptococcus pneumonia. Pneumococcal surface protein A (PspA) is highly conserved among S. pneumonia strains, inhibits complement activation, binds lactoferrin, elicits protective systemic immunity against pneumococcal infection, and is necessary for full pneumococcal virulence. Identification of PspA peptides that optimally bind human leukocyte antigen (HLA) would greatly contribute to global vaccine efforts, but this is hindered by the multitude of HLA polymorphisms. Here, we have used an experimental data set of 54 PspA peptides and in silico methods to predict peptide binding to HLA and murine major histocompatibility complex (MHC) class II. We also characterized spleen- and cervical lymph node (CLN)-derived helper T lymphocyte (HTL) cytokine responses to these peptides after S. pneumonia strain EF3030-challenge in mice. Individual, yet overlapping peptides, 15 amino acids in length revealed residues 199 to 246 of PspA (PspA199–246) consistently caused the greatest IFN-γ, IL-2, IL-5 and proliferation as well as moderate IL-10 and IL-4 responses by ex vivo stimulated splenic and CLN CD4+ T cells isolated from S. pneumonia strain EF3030-challeged F1 (B6×BALB/c) mice. IEDB, RANKPEP, SVMHC, MHCPred, and SYFPEITHI in silico analysis tools revealed peptides in PspA199–246 also interact with a broad range of HLA-DR, -DQ, and -DP allelles. These data suggest that predicted MHC class II-peptide binding affinities do not always correlate with T helper (Th) cytokine or proliferative responses to PspA peptides, but when used together with in vivo validation can be a useful tool to choose candidate pneumococcal HTL epitopes.

Prediction and characterization of helper T‐cell epitopes from pneumococcal surface adhesin A

Pneumococcal surface adhesin A (PsaA) is a multifunctional lipoprotein known to bind nasopharyngeal epithelial cells, and is significantly involved in bacterial adherence and virulence. Identification of PsaA peptides that optimally bind human leucocyte antigen (HLA) and elicit a potent immune response would be of great importance to vaccine development. However, this is hindered by the multitude of HLA polymorphisms in humans. To identify the conserved immunodominant epitopes, we used an experimental dataset of 28 PsaA synthetic peptides and in silico methods to predict specific peptide‐binding to HLA and murine MHC class II molecules. We also characterized spleen and cervical lymph node (CLN) ‐derived T helper (Th) lymphocyte cytokine responses to these peptides after Streptococcus pneumoniae strain EF3030 challenge in mice. Individual, yet overlapping, peptides 15 amino acids in length revealed residues of PsaA that consistently caused the highest interferon‐γ, interleukin‐2 (IL‐2), IL‐5 and IL‐17 responses and proliferation as well as moderate IL‐10 and IL‐4 responses by ex vivo re‐stimulated splenic and CLN CD4+ T cells isolated from S. pneumoniae strain EF3030‐challenged F1 (B6 × BALB/c) mice. In silico analysis revealed that peptides from PsaA may interact with a broad range of HLA‐DP, ‐DQ and ‐DR alleles, due in part to regions lacking β‐turns and asparagine endopeptidase sites. These data suggest that Th cell peptides (7, 19, 20, 22, 23 and 24) screened for secondary structures and MHC class II peptide‐binding affinities can elicit T helper cytokine and proliferative responses to PsaA peptides.

Association of Brain-derived Neurotrophic Factor(BDNF) Gene SNPs G196A and C270T with Parkinson’sdisease: A Meta- Analysis

Parkinson’s disease (PD) is the most common neurodegenerative disorder, clinically characterized by resting tremor, rigidity, gait abnormalities, postural imbalance and bradykinesia [1]. These underlying pathological events in PD result from the death of dopamine-generating cells in the region of the midbrain [2]. Although the etiology of PD is not fully known, the studies had already shown the role of both genetic factors [3,4] and environmental factors [5] in the pathogenesis of PD [6]. The brain-derived neurotrophic factor (BDNF) gene, encoding a nerve growth factor, and promoting the survival of dopaminergic neurons in the substantia nigra, is highly expressed in the nervous system [7,8]. Decreased BDNF mRNA expression and protein have been observed in the substantia nigra of PD patients [9,10] making BDNF, an important candidate gene for PD risk. Based on these observations, several molecular epidemiological studies have investigated the association of BDNF G196A (rs6265) and C270T (rs56164415) Single Nucleotide Polymorphisms (SNPs) with PD risk in different populations (Table 1). However, the findings from these studies for the probable association between these two SNPs on the susceptibility of PD remain inconsistent.

Abstract 5215: CXCR6-mediated cellular and molecular events involved in breast cancer cell migration and invasion

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Optimizing standard treatment modalities for breast cancer has improved the outlook for women afflicted with it. However, 40% breast cancer patients still die from the disease, primarily due to metastasis, which highlights the need for new therapies and better understanding of the mechanisms involved in disease progression. The reason(s) why breast cancer cells preferentially metastasize to lymph nodes, bone, brain, and lung is not fully understood. In this regard, chemokines and their corresponding receptor interactions in metastasis is an area of intense pre-clinical and clinical research. Analysis of breast cancer tissue micro-array in our lab demonstrated expression of CXCR6 by the breast cancer tissue. In confirmation with the TMA, breast cancer cell lines (MCF-7 and MDA-MB-231) also showed higher expression of CXCR6 in comparison to the normal transformed memory epithelial cell line (MCF-10A). We further investigated the role of CXCR6 in breast cancer cell migration and invasion, which are the key steps used by the cancer cells to achieve metastatic goals. Interestingly, breast cancer cells showed higher migratory and invasive potential towards the chemotactic gradients of CXCL16, which is the only natural ligand of CXCR6. This invasive and migratory potential was significantly inhibited after CXCR6 blockade. Our antibody array data show phosphorylation Src, FAK and ERK1/2 in breast cancer cells following CXCL16 treatment as compared to normal breast cancer cell. To determine the molecular mechanisms by which the CXCR6-CXCL16 axis mediates migration and invasion, inhibitors against Src kinase, FAK and ERK1/2 were used during the migration and invasion assay, which resulted in decreased migration and invasion. This suggests that Src, FAK, and ERK1/2 are required for CXCR6-mediated migration and invasion. Furthermore, an increase in F-actin polymerization was observed after CXCL16 treatment, which was significantly inhibited by the pharmacological inhibitors against Src, FAK, and ERK1/2, suggesting their potential role in CXCR6-mediated cytoskeletal rearrangement during breast cancer cell migration and invasion. Taken together, these results indicate clinical and biological significance of CXCR6-CXCL16 axis in breast cancer and targeting this axis may provide new therapeutic option. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5215.

Abstract 3377: CXCL13-directed therapy against bone metastasis in hormone-refractory prostate cancer

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Metastasis is responsible for most prostate cancer (PCa) related deaths; therefore, therapies designed to prevent or abrogate the spread of cancer cells and prognostic tests to better predict outcomes are greatly needed. Chemokines and their receptors are thought to play key roles in this multi-factorial process. We have recently reported, chemokine receptor CXCR5 and its ligand CXCL13 expression positively correlates with prostate tumor staging. We have also noted differential expression of CXCR5 by PCa cell lines (LNCaP, C4-2B, DU145, and PC3). By virtue of the presence of elevated CXCL13 levels in patient serum, CXCL13 secretion by bone marrow endothelium, and expression of CXCR5 on the surface of PCa cells, we hypothesized that gradient-dependent CXCR5-CXCL13 interactions are responsible in part for PCa cell bone metastases and inhibition of this axis may prove beneficial for prevention of bone metastasis and tumor progression. Luciferase-expressing PC3 cells were injected into the right carotid artery or directly into the tibia of nude mice. Before and after the development of bone metastases, groups were treated with control or anti-CXCL13 antibodies every third day and monitored for tumor progression or regression by non-invasive bioluminescent and microCT in vivo imaging. We demonstrate CXCL13 is present in bone marrow of xenograft tumor-bearing mice following intra-cardiac /intra-tibial injection of PC3 cells. This hormone-refractory PCa cell line established skeletal (osteolytic) metastases and tumor growth, but CXCL13 blockade significantly delayed prostate tumor formation, osteolysis, and spread to bones as well as growth, than compared to mice treated with control antibody. In conclusion, our study shows that the CXCL13-CXCR5 axis supports PCa bone metastasis and growth. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3377.