Ern Yu Tan

Selective Susceptibility of Human Skin Antigen Presenting Cells to Productive Dengue Virus Infection

Dengue is a growing global concern with 390 million people infected each year. Dengue virus (DENV) is transmitted by mosquitoes, thus host cells in the skin are the first point of contact with the virus. Human skin contains several populations of antigen-presenting cells which could drive the immune response to DENV in vivo: epidermal Langerhans cells (LCs), three populations of dermal dendritic cells (DCs), and macrophages. Using samples of normal human skin we detected productive infection of CD14+ and CD1c+ DCs, LCs and dermal macrophages, which was independent of DC-SIGN expression. LCs produced the highest viral titers and were less sensitive to IFN-β. Nanostring gene expression data showed significant up-regulation of IFN-β, STAT-1 and CCL5 upon viral exposure in susceptible DC populations. In mice infected intra-dermally with DENV we detected parallel populations of infected DCs originating from the dermis and migrating to the skin-draining lymph nodes. Therefore dermal DCs may simultaneously facilitate systemic spread of DENV and initiate the adaptive anti-viral immune response.

IRAK1 is a therapeutic target that drives breast cancer metastasis and resistance to paclitaxel

Metastatic tumour recurrence due to failed treatments remains a major challenge of breast cancer clinical management. Here we report that interleukin-1 receptor-associated kinase 1 (IRAK1) is overexpressed in a subset of breast cancers, in particular triple-negative breast cancer (TNBC), where it acts to drive aggressive growth, metastasis and acquired resistance to paclitaxel treatment. We show that IRAK1 overexpression confers TNBC growth advantage through NF-κB-related cytokine secretion and metastatic TNBC cells exhibit gain of IRAK1 dependency, resulting in high susceptibility to genetic and pharmacologic inhibition of IRAK1. Importantly, paclitaxel treatment induces strong IRAK1 phosphorylation, an increase in inflammatory cytokine expression, enrichment of cancer stem cells and acquired resistance to paclitaxel treatment. Pharmacologic inhibition of IRAK1 is able to reverse paclitaxel resistance by triggering massive apoptosis at least in part through inhibiting p38-MCL1 pro-survival pathway. Our study thus demonstrates IRAK1 as a promising therapeutic target for TNBC metastasis and paclitaxel resistance.

RASAL2 activates RAC1 to promote triple-negative breast cancer progression

Patients with triple-negative breast cancer (TNBC) have a high incidence of early relapse and metastasis; however, the molecular basis for recurrence in these individuals remains poorly understood. Here, we demonstrate that RASAL2, which encodes a RAS-GTPase-activating protein (RAS-GAP), is a functional target of anti-invasive microRNA-203 and is overexpressed in a subset of triple-negative or estrogen receptor-negative (ER-negative) breast tumors. As opposed to luminal B ER-positive breast cancers, in which RASAL2 has been shown to act as a RAS-GAP tumor suppressor, we found that RASAL2 is oncogenic in TNBC and drives mesenchymal invasion and metastasis. Moreover, high RASAL2 expression was predictive of poor disease outcomes in patients with TNBC. RASAL2 acted independently of its RAS-GAP catalytic activity in TNBC; however, RASAL2 promoted small GTPase RAC1 signaling, which promotes mesenchymal invasion, through binding and antagonizing the RAC1-GAP protein ARHGAP24. Together, these results indicate that activation of a RASAL2/ARHGAP24/RAC1 module contributes to TNBC tumorigenesis and identify a context-dependent role of RASAL2 in breast cancer.

Protein tyrosine phosphatase UBASH3B is overexpressed in triple-negative breast cancer and promotes invasion and metastasis

Efforts to improve the clinical outcome of highly aggressive triple-negative breast cancer (TNBC) have been hindered by the lack of effective targeted therapies. Thus, it is important to identify the specific gene targets/pathways driving the invasive phenotype to develop more effective therapeutics. Here we show that ubiquitin-associated and SH3 domain-containing B (UBASH3B), a protein tyrosine phosphatase, is overexpressed in TNBC, where it supports malignant growth, invasion, and metastasis largely through modulating epidermal growth factor receptor (EGFR). We also show that UBASH3B is a functional target of anti-invasive microRNA200a (miR200a) that is down-regulated in TNBC. Importantly, the oncogenic potential of UBASH3B is dependent on its tyrosine phosphatase activity, which targets CBL ubiquitin ligase for dephosphorylation and inactivation, leading to EGFR up-regulation. Thus, UBASH3B may function as a crucial node in bridging multiple invasion-promoting pathways, thereby providing a potential therapeutic target for TNBC.

ALDH1 expression is enriched in breast cancers arising in young women but does not predict outcome

Background:Tumours arising in younger women appear to be biologically more aggressive and tend to have a poorer outcome. Being relatively resistant to conventional treatments, breast cancer stem cells (CSCs) have been postulated as a possible cause of disease recurrence after treatment. In this study, we used ALDH1 as a CSC marker and determined whether ALDH1 expression correlated with clinical outcome in young women with breast cancer.Methods:The expression of ALDH1 was evaluated through immunohistochemistry on microarrayed cores obtained from 141 consecutive patients up to 35 years of age.Results:The expression of ALDH1 was observed in 25% (35 of 141) of tumours, in a median of 5% of cells. Younger women were 14 times more likely to have ALDH1-positive tumours (P<0.01, OR 14.4, 95% CI 4.34–48.09). The ALDH1 correlated independently with ER negativity (P=0.01, OR 0.33, 95% CI 0.15–0.77). There was no correlation with disease recurrence or breast cancer-related deaths.Conclusion:In younger women, ALDH1 was more highly expressed, and it correlated with ER negativity. It, however, did not predict survival in this study.

Delivery of doxorubicin and paclitaxel from double-layered microparticles: The effects of layer thickness and dual-drug vs. single-drug loading.

UNLABELLED Double-layered microparticles composed of poly(d,l-lactic-co-glycolic acid, 50:50) (PLGA) and poly(l-lactic acid) (PLLA) were loaded with doxorubicin HCl (DOX) and paclitaxel (PCTX) through a solvent evaporation technique. DOX was localized in the PLGA shell, while PCTX was localized in the PLLA core. The aim of this study was to investigate how altering layer thickness of dual-drug, double-layered microparticles can influence drug release kinetics and their antitumor capabilities, and against single-drug microparticles. PCTX-loaded double-layered microparticles with denser shells retarded the initial release of PCTX, as compared with dual-drug-loaded microparticles. The DOX release from both DOX-loaded and dual-drug-loaded microparticles were observed to be similar with an initial burst. Through specific tailoring of layer thicknesses, a suppressed initial burst of DOX and a sustained co-delivery of two drugs can be achieved over 2months. Viability studies using spheroids of MCF-7 cells showed that controlled co-delivery of PCTX and DOX from dual-drug-loaded double-layered microparticles were better in reducing spheroid growth rate. This study provides mechanistic insights into how by tuning the layer thickness of double-layered microparticles the release kinetics of two drugs can be controlled, and how co-delivery can potentially achieve better anticancer effects. STATEMENT OF SIGNIFICANCE While the release of multiple drugs has been reported to achieve successful apoptosis and minimize drug resistance, most conventional particulate systems can only deliver a single drug at a time. Recently, although a number of formulations (e.g. micellar nanoparticles, liposomes) have been successful in delivering two or more anticancer agents, sustained co-delivery of these agents remains inadequate due to the complex agent loading processes and rapid release of hydrophilic agents. Therefore, the present work reports the multilayered particulate system that simultaneously hosts different drugs, while being able to tune their individual release over months. We believe that our findings would be of interest to the readers of Acta Biomaterialia because the proposed system could open a new avenue on how two drugs can be released, through rate-controlling carriers, for combination chemotherapy.

Development of a magnetic 3D spheroid platform with potential application for high-throughput drug screening.

Three-dimensional (3D) cell culture has become increasingly adopted as a more accurate model of the complex in vivo microenvironment compared to conventional two-dimensional (2D) cell culture. Multicellular spheroids are important 3D cell culture models widely used in biological studies and drug screening. To facilitate simple spheroid manipulation, magnetic spheroids were generated from magnetically labeled cells using a scaffold-free approach. This method is applicable to a variety of cell types. The spheroids generated can be targeted and immobilized using magnetic field gradients, allowing media change or dilution to be performed with minimal disruption to the spheroids. Cells in magnetic spheroids showed good viability and displayed typical 3D morphology. Using this platform, a 28 day study was carried out using doxorubicin on magnetic MCF-7 spheroids. The results provided a proof-of-principle for using magnetic tumor spheroids in therapeutic studies. They can offer beneficial insights that help to bridge the gap between in vitro and in vivo models. Furthermore, this platform can be adapted for high-throughput screening in drug discovery.

Manipulating magnetic 3D spheroids in hanging drops for applications in tissue engineering and drug screening.

Magnetic spheroid manipulation can be carried out in hanging drops to generate distinctly structured heterotypic microtissues through sequential addition of cells or spheroid to homotypic spheroids. These spheroids can also be incorporated in a droplet-based assay to screen for therapeutic efficacy in prolonged studies. This simple and versatile technique can offer potential benefits in tissue engineering and drug screening applications.

Inhibition of 3‐D Tumor Spheroids by Timed‐Released Hydrophilic and Hydrophobic Drugs from Multilayered Polymeric Microparticles

First-line cancer chemotherapy necessitates high parenteral dosage and repeated dosing of a combination of drugs over a prolonged period. Current commercially available chemotherapeutic agents, such as Doxil and Taxol, are only capable of delivering single drug in a bolus dose. The aim of this study is to develop dual-drug-loaded, multilayered microparticles and to investigate their antitumor efficacy compared with single-drug-loaded particles. Results show hydrophilic doxorubicin HCl (DOX) and hydrophobic paclitaxel (PTX) localized in the poly(dl-lactic-co-glycolic acid, 50:50) (PLGA) shell and in the poly(l-lactic acid) (PLLA) core, respectively. The introduction of poly[(1,6-bis-carboxyphenoxy) hexane] (PCPH) into PLGA/PLLA microparticles causes PTX to be localized in the PLLA and PCPH mid-layers, whereas DOX is found in both the PLGA shell and core. PLGA/PLLA/PCPH microparticles with denser shells allow better control of DOX release. A delayed release of PTX is observed with the addition of PCPH. Three-dimensional MCF-7 spheroid studies demonstrate that controlled co-delivery of DOX and PTX from multilayered microparticles produces a greater reduction in spheroid growth rate compared with single-drug-loaded particles. This study provides mechanistic insights into how distinctive structure of multilayered microparticles can be designed to modulate the release profiles of anticancer drugs, and how co-delivery can potentially provide better antitumor response.

Predicting Survival of De Novo Metastatic Breast Cancer in Asian Women: Systematic Review and Validation Study

Background In Asia, up to 25% of breast cancer patients present with distant metastases at diagnosis. Given the heterogeneous survival probabilities of de novo metastatic breast cancer, individual outcome prediction is challenging. The aim of the study is to identify existing prognostic models for patients with de novo metastatic breast cancer and validate them in Asia. Materials and Methods We performed a systematic review to identify prediction models for metastatic breast cancer. Models were validated in 642 women with de novo metastatic breast cancer registered between 2000 and 2010 in the Singapore Malaysia Hospital Based Breast Cancer Registry. Survival curves for low, intermediate and high-risk groups according to each prognostic score were compared by log-rank test and discrimination of the models was assessed by concordance statistic (C-statistic). Results We identified 16 prediction models, seven of which were for patients with brain metastases only. Performance status, estrogen receptor status, metastatic site(s) and disease-free interval were the most common predictors. We were able to validate nine prediction models. The capacity of the models to discriminate between poor and good survivors varied from poor to fair with C-statistics ranging from 0.50 (95% CI, 0.48–0.53) to 0.63 (95% CI, 0.60–0.66). Conclusion The discriminatory performance of existing prediction models for de novo metastatic breast cancer in Asia is modest. Development of an Asian-specific prediction model is needed to improve prognostication and guide decision making.