In Vitro and In Vivo Study of Vitamin E TPGs Coated Immunoliposomes for Sustained and Targeted Delivery of Docetaxel

Master'sMASTER OF ENGINEERIN

Dependency of NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis.

Cancer cells undergo transcriptional reprogramming to drive tumor progression and metastasis. Using cancer cell lines and patient-derived tumor organoids, we demonstrate that loss of the negative elongation factor (NELF) complex inhibits breast cancer development through downregulating epithelial-mesenchymal transition (EMT) and stemness-associated genes. Quantitative multiplexed Rapid Immunoprecipitation Mass spectrometry of Endogenous proteins (qPLEX-RIME) further reveals a significant rewiring of NELF-E-associated chromatin partners as a function of EMT and a co-option of NELF-E with the key EMT transcription factor SLUG. Accordingly, loss of NELF-E leads to impaired SLUG binding on chromatin. Through integrative transcriptomic and genomic analyses, we identify the histone acetyltransferase, KAT2B, as a key functional target of NELF-E-SLUG. Genetic and pharmacological inactivation of KAT2B ameliorate the expression of EMT markers, phenocopying NELF ablation. Elevated expression of NELF-E and KAT2B is associated with poorer prognosis in breast cancer patients, highlighting the clinical relevance of our findings. Taken together, we uncover a crucial role of the NELF-E-SLUG-KAT2B epigenetic axis in breast cancer carcinogenesis

An intravascular MRI contrast agent based on Gd(DO3A-Lys) for tumor angiography

An intravascular MRI contrast agent Gd(DO3A-Lys), Gadolinium(III) (2,2',2″-(10-(3-(5-benzamido-6-methoxy-6-oxohexylamino)-3-oxopropyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate), has been studied for tumor angiography based on its high relaxivity and long blood half-life. The preparation procedures of the contrast agent have been modified in order to achieve higher yield and improve the synthetic reproducibility. High relaxivity of Gd(DO3A-Lys) has been confirmed by measurements at 3T, 7T and 9.4T magnetic fields. The relaxivity-dependent albumin binding study indicated that Gd(DO3A-Lys) partially bound to albumin protein. Invitro cell viability in HK2 cell indicated low cytotoxicity of Gd(DO3A-Lys) up to 1.2mm [Gd] concentration. Invivo toxicity studies demonstrated no toxicity of Gd(DO3A-Lys) on kidney tissues up to 0.2mm [Gd]. While the toxicity on liver tissue was not observed at low dosage (1.0mm [Gd]), Gd(DO3A-Lys) cause certain damage on hepatic tissue at high dosage (2.0mm [Gd]). The DO3A-Lys has been labeled with Ga radioisotope for biodistribution studies. Ga(DO3A-Lys) has high uptake in both HT1080 and U87MG xenograft tumors, and has high accumulation in blood. Contrast-enhanced MR angiography (CE-MRA) in mice bearing U87MG xenograft tumor demonstrated that Gd(DO3A-Lys) could enhance vascular microenvironment around the tumor, and displays promising characteristics of an MRI contrast agent for tumor angiography

Agrin as a Mechanotransduction Signal Regulating YAP through the Hippo Pathway

The Hippo pathway effectors YAP and TAZ act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. Here, we uncover a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 signaling and negating the functions of Merlin and LATS1/2. We further show that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. We propose that Agrin acts as a mechanotransduction signal in the ECM

Two-Photon Dye Cocktail for Dual-Color 3D Imaging of Pancreatic Beta and Alpha Cells in Live Islets

Insulin-secreting beta cells together with glucagon-producing alpha cells play an essential role in maintaining the optimal blood glucose level in the body, so the development of selective probes for imaging of these cell types in live islets is highly desired. Herein we report the development of a 2glucosamine-based two-photon fluorescent probe, TP-beta, that is suitable for imaging of beta cells in live pancreatic islets from mice. Flow cytometry studies confirmed that TP-beta is suitable for isolation of primary beta cells. Moreover, two-photon imaging of TP-beta-stained pancreatic islets showed brightly stained beta cells in live islets. Insulin enzyme-linked immunosorbent assays revealed that 'TP-beta has no effect on glucose-stimulated insulin secretion from the stained islet. Finally, to develop a more convenient islet imaging application, we combined our recently published alpha-cell-selective probe TP-alpha with TP-beta to make a "TP islet cocktail". This unique dye cocktail enabled single excitation (820 nm) and simultaneous dual-color imaging of alpha cells (green) and beta cells (red) in live pancreatic islets. This robust TP islet cocktail may serve as a valuable tool for basic diabetic studies.114sciescopu

A role of Agrin in maintaining the stability of vascular endothelial growth factor receptor-2 during tumor angiogenesis

Endothelial cell (EC) recruitment is central to the vascularization of tumors. Although several proteoglycans have been implicated in cancer and angiogenesis, their roles in EC recruitment and vascularization during tumorigenesis remain poorly understood. Here, we reveal that Agrin, which is secreted in liver cancer, promotes angiogenesis by recruiting ECs within tumors and metastatic lesions and facilitates adhesion of cancer cells to ECs. In ECs, Agrin-induced angiogenesis and adherence to cancer cells are mediated by Integrin-β1, Lrp4-MuSK pathways involving focal adhesion kinase. Mechanistically, we uncover that Agrin regulates VEGFR2 levels that sustain the angiogenic property of ECs and adherence to cancer cells. Agrin attributes an ECM stiffness-based stabilization of VEGFR2 by enhancing interactions with Integrin-β1-Lrp4 and additionally stimulates endothelial nitric-oxide synthase (e-NOS) signaling. Therefore, we propose that cross-talk between Agrin-expressing cancer and ECs favor angiogenesis by sustaining the VEGFR2 pathway.ASTAR (Agency for Sci., Tech. and Research, S’pore)NMRC (Natl Medical Research Council, S’pore)Published versio

GREB1 : an evolutionarily conserved protein with a glycosyltransferase domain links ERα glycosylation and stability to cancer

What covalent modifications control the temporal ubiquitination of ERα and hence the duration of its transcriptional activity remain poorly understood. We show that GREB1, an ERα-inducible enzyme, catalyzes O-GlcNAcylation of ERα at residues T553/S554, which stabilizes ERα protein by inhibiting association with the ubiquitin ligase ZNF598. Loss of GREB1-mediated glycosylation of ERα results in reduced cellular ERα levels and insensitivity to estrogen. Higher GREB1 expression in ERα+ve breast cancer is associated with greater survival in response to tamoxifen, an ERα agonist. Mice lacking Greb1 exhibit growth and fertility defects reminiscent of phenotypes in ERα-null mice. In summary, this study identifies GREB1, a protein with an evolutionarily conserved domain related to DNA-modifying glycosyltransferases of bacteriophages and kinetoplastids, as the first inducible and the only other (apart from OGT) O-GlcNAc glycosyltransferase in mammalian cytoplasm and ERα as its first substrate.National Research Foundation (NRF)Published versionThe V.T. laboratory is funded by the National Research Foundation, Singapore (NRF-CRP17-2017-02). V.T.H. is supported by SINGA program. L.M.I. and L.A. were supported by the Intramural Research Program of the NIH, National Library of Medicine

H3K27me3-rich genomic regions can function as silencers to repress gene expression via chromatin interactions

10.1038/s41467-021-20940-yNature Communications12171

Two-Photon Dye Cocktail for Dual-Color 3D Imaging of Pancreatic Beta and Alpha Cells in Live Islets

Insulin-secreting beta cells together with glucagon-producing alpha cells play an essential role in maintaining the optimal blood glucose level in the body, so the development of selective probes for imaging of these cell types in live islets is highly desired. Herein we report the development of a 2-glucosamine-based two-photon fluorescent probe, <b>TP-β</b>, that is suitable for imaging of beta cells in live pancreatic islets from mice. Flow cytometry studies confirmed that <b>TP-β</b> is suitable for isolation of primary beta cells. Moreover, two-photon imaging of <b>TP-β</b>-stained pancreatic islets showed brightly stained beta cells in live islets. Insulin enzyme-linked immunosorbent assays revealed that <b>TP-β</b> has no effect on glucose-stimulated insulin secretion from the stained islet. Finally, to develop a more convenient islet imaging application, we combined our recently published alpha-cell-selective probe <b>TP-α</b> with <b>TP-β</b> to make a “TP islet cocktail”. This unique dye cocktail enabled single excitation (820 nm) and simultaneous dual-color imaging of alpha cells (green) and beta cells (red) in live pancreatic islets. This robust TP islet cocktail may serve as a valuable tool for basic diabetic studies

Non-canonical NF-κB signalling and ETS1/2 cooperatively drive C250T mutant TERT promoter activation

Transcriptional reactivation of TERT, the catalytic subunit of telomerase, is necessary for cancer progression in about 90% of human cancers. The recent discovery of two prevalent somatic mutations - C250T and C228T - in the TERT promoter in various cancers has provided insight into a plausible mechanism of TERT reactivation. Although the two hotspot mutations create a similar binding motif for E-twenty-six (ETS) transcription factors, we show that they are functionally distinct, in that the C250T unlike the C228T TERT promoter is driven by non-canonical NF-κB signalling. We demonstrate that binding of ETS to the mutant TERT promoter is insufficient in driving its transcription but this process requires non-canonical NF-κB signalling for stimulus responsiveness, sustained telomerase activity and hence cancer progression. Our findings highlight a previously unrecognized role of non-canonical NF-κB signalling in tumorigenesis and elucidate a fundamental mechanism for TERT reactivation in cancers, which if targeted could have immense therapeutic implications