9th International conference on biosience, biochemistry & bioinformatics 2018

Transient receptor potential cation channel subfamily M member 4 (TRPM4) is overexpressed in activated B-cell-like subtype of diffuse large B-cell lymphoma (ABC-DLBCL) associated with poor survival. In this study, its functions in the disease and the potency of its inhibitor 9-phenanthrol were investigated. The biological functions associated with TR.PM4 mRNA expression were examined through Gene Set Enrichment Analysis (GSEA) in ABC-DLBCL cases (n=15). The cytotoxicity of 9-phenanthrol in three ABC-DLBCL cell lines (SUDHL2, OCI-LY3, OCI-LYIO) was tested at six different concentrations (0.0InM, 0.1 nM, InM, lOnM, 25nM, 50nM). GSEA results showed that cell cycle gene sets conferred the highest number of gene sets representing 42% (n=21/50) of the top 50 most significantly enriched gene sets ranked according to false discovery rate (FDR; all 50 gene sets had FDRO.OI), followed by DNA replication (n=8/50; 16%) and RNA processing (n=8/50; 16%), suggesting the roles of TRPM4 in cell cycle progression and cellular division of ABC-DLBCL. In terms of the cytotoxicity effects of 9-phenanthrol, the resulting GI50 for all ABC-DLBCL cell lines ranged from 19nM-41,88nM. In conclusion, TRPM4 is potentially involved in the cell cycle progression and cellular division of ABC-DLBCL cells, and the TRPM4 inhibitor 9-phenanthrol was cytotoxic against ABC-DLBCL cells

Molecular mechanism of the actin-associated protein hip1r on malignant 8-cell migration

Cancer research at Universiti Sains Malaysia (USM) has been increasingly active in recent years. Many young researchers who recently started their service in USM work on cancer. The inaugural Next Generation Researchers Cancer Symposium organized by the Centre for Research Initiative - Clinical and Health Sciences (CRI-CHS) is thus initiated to bring together these young researchers with diverse expertise from various faculties in USM, to share and discuss various aspects of cancer research and new research tools. The meeting is aimed to provide an active platform for communication, knowledge-sharing and collaboration among these young researchers and also to initiate research interactions with senior academic members. a. To provide a platform for meaningful and active interactions between researchers working on cancer b. To address on types of research funding available c. To discuss the role and expectations of a researcher in an academic institution

FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures.

FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL

Gold nanoparticles conjugated with anti-CD133 monoclonal antibody and 5-fluorouracil chemotherapeutic agent as nanocarriers for cancer cell targeting

The enhanced permeability and retention effect allows for passive targeting of solid tumours by nanoparticles carrying anticancer drugs. However, active targeting by incorporation of various ligands onto nanoparticles can provide for a more selective and enhanced chemotherapeutic effect and complement the deficiencies of the passive targeting approach. Here we report on the design of the carboxyl-terminated PEGylated gold nanoparticles (AuNPs), their functionalization with anti-CD133 monoclonal antibody (mAb) via a crosslinking reaction, and subsequent 5-fluorouracil (5-FU) drug loading. The synthesized products in the form of stable colloids were characterised using a range of physicochemical techniques, including X-ray diffraction (XRD), UV-Vis spectroscopy, transmission electron microscopy (TEM), and dynamic light scattering (DLS). Conjugation of anti-CD133 mAb onto PEGylated AuNPs was confirmed with the use of UV-Vis, BCA protein assay and fluorescence microscopy. HCT116 colorectal cancer cells abundantly expressed CD133: 92.4 ± 1.3%, as measured by flow cytometry. Whereas PEGylated AuNPs not conjugated with anti-CD133 mAb accumulated mainly at the cellular membrane, nanoparticles conjugated with anti-CD133 mAb were contained within the nuclear region of the cells. Anti-CD133 mAb conjugation facilitated the specific intracellular uptake due to specific antigen–antibody binding interaction. In vitro cytotoxicity studies on HCT116 cells showed that PEGylated AuNPs and PEGylated AuNPs-CD133 did not elicit any toxicity at any of the tested concentrations. Meanwhile, 5-FU-PEGylated AuNPs-CD133 significantly reduced the cell viability relative to the treatment with 5-FU-PEGylated AuNPs without anti-CD133 mAb conjugates (p < 0.0001). This study shows that the conjugation of nanocarriers with the anti-CD133 antibody improves the specific targeting of 5-FU against colorectal cancer cells. These results demonstrate that simultaneous functionalisation of PEGylated AuNPs with antibodies and chemotherapeutic drugs is a viable strategy to combat cancer through targeted drug delivery

Validation of lymphoma-associated antigens identified using autoantibody profiling and protein arrays

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma subtype with heterogeneous clinical outcome and a significant number of patients still die of their disease. Characterising lymphoma patients’ autoantibody repertoires represent one approach to improve the understanding of their disease biology. Our hypothesis was that characterisation of antigens eliciting humoural immune responses in lymphoma patients may provide insights into mechanisms of lymphomagenesis and identify novel diagnostic/therapeutic targets.HIP1R was validated as a novel B-NHL autoantigen by immunoblotting with patients’ sera. No response was identified to the related HIP1 protein. Consistent with this finding, more widespread expression of HIP1R, compared to HIP1, was observed in lymphoma cell lines. Expression studies, at both transcript (qRT-PCR and analysis of microarray datasets) and protein levels (blotting and immunolabelling), identified abundant HIP1R in normal B cells and low level expression in a poor prognosis activated B-cell (ABC)-like DLBCL subtype. Upregulation of HIP1R expression was observed in activated non-malignant B cells at both transcript and protein levels, suggesting that downregulation of HIP1R expression in ABC-DLBCL might be a disease-related process. Despite its potential for DLBCL subtyping, HIP1R protein expression was not statistically significantly associated with patients’ survival in a series of 256 DLBCL. Short FOXP1 isoforms were identified as one mechanism repressing HIP1R transcription in an ABC-DLBCL cell line. Phenotypic analysis of HIP1R-depleted B-cell lymphoma cells indicated that HIP1R silencing could increase the surface levels of B-cell receptor (BCR) components such as IgM and CD79b.HIP1R represents a novel lymphoma autoantigen and cell-of-origin marker for distinguishing germinal centre- versus ABC-DLBCL subtypes. As ABC-DLBCL survival is dependent on chronic BCR signalling, future studies will address whether HIP1R silencing plays a fundamental role in disease pathogenesis by promoting BCR signalling.</p

Expression profile of HIP1R in B-cell subsets and in silico prediction of its functions in diffuse large B-cell lymphoma

Huntingtin-interacting protein 1 (HIP1R) is an endocytic protein involved in endocytosis of surface receptors by regulating actin polymerization. We have previously shown that HIP1R was expressed in lymphoid B cells and diffuse large B-cell lymphoma (DLBCL) associated with better survival. Herein, we examined the expression profile of HIP1R in different immune cell populations and its potential functions in DLBCL. By utilizing a validated anti-HIP1R monoclonal antibody (clone 44), we examined whether the following immune cells in human reactive tonsils expressed HIP1R through double immunostaining: T cells (CD3 + ), macrophages (CD68'), mantle zone (MZ) B cells (PAX5 + ), germinal centre (GC) B cells (BCL6 + ) and plasma cells (IRF4/MUM1 + ). HIP1R was strongly expressed in PAX5 + MZ B cells, moderately expressed in BCL6 + GC B cells, but absent in CD3 + T cells, CD68 + macrophages and IRF4/MUM1 + plasma cells. In particular, we observed that HIP1R was absent in IRF4/MUM1 + plasma cells residing within the GC or non-GC interfollicular regions, suggesting that IRF4/MUM1 might downregulate HIP1R expression in activated B cells. We have previously shown that HIP1R expression is directly suppressed by the transcription factor FOXP1 in activated B-cell-like diffuse large B-cell lymphoma (ABC-DLBCL) cells, however FOXP1 is absent in normal plasma cells, suggesting the presence of other regulators. Our previous immunostaining results in a series of DLBCL patient cases (n=155) showed a significant inverse correlation between HIP1R and IRF4/MUM1 (Pearson r = -0.495; p <; 0.001). Indeed, knockdown of IRF4/MUM1 expression in the ABC-DLBCL cell line OCI-LY3 by two independent IRF4 siRNA constructs increased HIP1R expression at both transcript and protein levels. In terms of functional relevance, the bioinformatics approach Gene Set Enrichment Analysis (GSEA) was adopted to examine gene sets positively-associated with HIP1R transcript expression profile in three independent gene expression profiling (GEP) datasets of DLBCL patient cases derived from Gene Expression Omnibus database i.e. GSE10846 (n=233), GSE23501 (n=63), and GSE19246 (n=59). Our GSEA results showed that the gene set 'Rho GTPase Activator Activity' (GO ID:0005100) was significantly positively-associated with HIP1R expression profile across all three GEP datasets GSE10846 (p = 0.0016), GSE23501 (p <; 0.0001) and GSE19246 (p = 0.0167). These results suggest that HIP1R is involved in the activation of Rho GTPase signaling pathway, which has been documented to inhibit migration of DLBCL cells, and HIP1R expression is suppressed by transcription factors involved in B-cell activation including FOXP1 and IRF4/MUM1

Validation of lymphoma-associated antigens identified using autoantibody profiling and protein arrays

Diffuse large B-cell lymphoma (DLBCL) is an aggressive lymphoma subtype with heterogeneous clinical outcome and a significant number of patients still die of their disease. Characterising lymphoma patients’ autoantibody repertoires represent one approach to improve the understanding of their disease biology. Our hypothesis was that characterisation of antigens eliciting humoural immune responses in lymphoma patients may provide insights into mechanisms of lymphomagenesis and identify novel diagnostic/therapeutic targets. HIP1R was validated as a novel B-NHL autoantigen by immunoblotting with patients’ sera. No response was identified to the related HIP1 protein. Consistent with this finding, more widespread expression of HIP1R, compared to HIP1, was observed in lymphoma cell lines. Expression studies, at both transcript (qRT-PCR and analysis of microarray datasets) and protein levels (blotting and immunolabelling), identified abundant HIP1R in normal B cells and low level expression in a poor prognosis activated B-cell (ABC)-like DLBCL subtype. Upregulation of HIP1R expression was observed in activated non-malignant B cells at both transcript and protein levels, suggesting that downregulation of HIP1R expression in ABC-DLBCL might be a disease-related process. Despite its potential for DLBCL subtyping, HIP1R protein expression was not statistically significantly associated with patients’ survival in a series of 256 DLBCL. Short FOXP1 isoforms were identified as one mechanism repressing HIP1R transcription in an ABC-DLBCL cell line. Phenotypic analysis of HIP1R-depleted B-cell lymphoma cells indicated that HIP1R silencing could increase the surface levels of B-cell receptor (BCR) components such as IgM and CD79b. HIP1R represents a novel lymphoma autoantigen and cell-of-origin marker for distinguishing germinal centre- versus ABC-DLBCL subtypes. As ABC-DLBCL survival is dependent on chronic BCR signalling, future studies will address whether HIP1R silencing plays a fundamental role in disease pathogenesis by promoting BCR signalling.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

TRPM4 is overexpressed in breast cancer associated with estrogen response and epithelial-mesenchymal transition gene sets.

Ion channels form an important class of drug targets in malignancies. Transient receptor potential cation channel subfamily M member 4 (TRPM4) plays oncological roles in various solid tumors. Herein, we examined TRPM4 protein expression profile by immunohistochemistry (IHC) in breast cancer cases compared with normal breast ducts, its association with clinico-demographical parameters, and its potential function in breast cancers by Gene Set Enrichment Analysis (GSEA). Data-mining demonstrated that TRPM4 transcript levels were significantly higher in The Cancer Genome Atlas series of breast cancer cases (n = 1,085) compared with normal breast tissues (n = 112) (p = 1.03 x 10-11). Our IHC findings in tissue microarrays showed that TRPM4 protein was overexpressed in breast cancers (n = 83/99 TRPM4+; 83.8%) compared with normal breast ducts (n = 5/10 TRPM4+; 50%) (p = 0.022). Higher TRPM4 expression (median frequency cut-off) was significantly associated with higher lymph node status (N1-N2 vs N0; p = 0.024) and higher stage (IIb-IIIb vs I-IIa; p = 0.005). GSEA evaluation in three independent gene expression profiling (GEP) datasets of breast cancer cases (GSE54002, n = 417; GSE20685, n = 327; GSE23720, n = 197) demonstrated significant association of TRPM4 transcript expression with estrogen response and epithelial-mesenchymal transition (EMT) gene sets (p<0.01 and false discovery rate<0.05). These gene sets were not enriched in GEP datasets of normal breast epithelium cases (GSE10797, n = 5; GSE9574, n = 15; GSE20437, n = 18). In conclusion, TRPM4 protein expression is upregulated in breast cancers associated with worse clinico-demographical parameters, and TRPM4 potentially regulates estrogen receptor signaling and EMT progression in breast cancer