Chung-Hsuan Chen

EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2

MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at the post-transcriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in the regulation of cellular response to a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumour. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumour cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here we show that epidermal growth factor receptor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific tumour-suppressor-like miRNAs in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. The association between EGFR and AGO2 is enhanced by hypoxia, leading to elevated AGO2-Y393 phosphorylation, which in turn reduces the binding of Dicer to AGO2 and inhibits miRNA processing from precursor miRNAs to mature miRNAs. We also identify a long-loop structure in precursor miRNAs as a critical regulatory element in phospho-Y393-AGO2-mediated miRNA maturation. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients. Our study reveals a previously unrecognized function of EGFR in miRNA maturation and demonstrates how EGFR is likely to function as a regulator of AGO2 through novel post-translational modification. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells and has potential clinical implications.

The Crosstalk of mTOR/S6K1 and Hedgehog Pathways

Esophageal adenocarcinoma (EAC) is the most prevalent esophageal cancer type in the United States. The TNF-α/mTOR pathway is known to mediate the development of EAC. Additionally, aberrant activation of Gli1, downstream effector of the Hedgehog (HH) pathway, has been observed in EAC. In this study, we found that an activated mTOR/S6K1 pathway promotes Gli1 transcriptional activity and oncogenic function through S6K1-mediated Gli1 phosphorylation at Ser84, which releases Gli1 from its endogenous inhibitor, SuFu. Moreover, elimination of S6K1 activation by an mTOR pathway inhibitor enhances the killing effects of the HH pathway inhibitor. Together, our results established a crosstalk between the mTOR/S6K1 and HH pathways, which provides a mechanism for SMO-independent Gli1 activation and also a rationale for combination therapy for EAC.

Multiple Lineages of Human Breast Cancer Stem/Progenitor Cells Identified by Profiling with Stem Cell Markers

Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vitro soft agar colony formation assay and the ability to form tumors in NOD/SCID mice. We found that the expression of stem cell markers varied greatly among breast cancer cell lines. In MDA-MB-231 cells, PROCR and ESA, instead of the widely used breast cancer stem cell markers CD44+/CD24-/low and ALDH, could be used to highly enrich cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically. Furthermore, the PROCR+/ESA+ cells expressed epithelial-mesenchymal transition markers. PROCR could also be used to enrich cells with colony forming ability from MB-361 cells. Moreover, consistent with the marker profiling using cell lines, the expression of stem cell markers differed greatly among primary tumors. There was an association between metastasis status and a high prevalence of certain markers including CD44+/CD24−/low, ESA+, CD133+, CXCR4+ and PROCR+ in primary tumor cells. Taken together, these results suggest that similar to leukemia, several stem/progenitor cell-like subpopulations can exist in breast cancer.

Biocompatibility of Fe(3)O(4) nanoparticles evaluated by in vitro cytotoxicity assays using normal, glia and breast cancer cells.

In order to reveal the biocompatibility of Fe(3)O(4) nanoparticles and bipolar surfactant tetramethylammonium 11-aminoundecanoate cytotoxicity tests were performed as a function of concentration from low (0.1 microg ml(-1)) to higher concentration (100 microg ml(-1)) using various human glia, human breast cancer and normal cell lines. Cytotoxicity tests for human glia (D54MG, G9T, SF126, U87, U251, U373), human breast cancer (MB157, SKBR3, T47D) and normal (H184B5F5/M10, WI-38, SVGp12) cell lines exhibited almost nontoxicity and reveal biocompatibility of Fe(3)O(4) nanoparticles in the concentration range of 0.1-10 microg ml(-1), while accountable cytotoxicity can be seen at 100 microg ml(-1). The results of our studies suggest that Fe(3)O(4) nanoparticles coated with bipolar surfactant tetramethylammonium 11-aminoundecanoate are biocompatible and promising for bio-applications such as drug delivery, magnetic resonance imaging and magnetic hyperthermia.

Nuclear translocation of epidermal growth factor receptor by Akt-dependent phosphorylation enhances breast cancer-resistant protein expression in gefitinib-resistant cells

Epidermal growth factor receptor (EGFR), an aberrantly overexpressed or activated receptor-tyrosine kinase in many cancers, plays a pivotal role in cancer progression and has been an attractive target for cancer therapy. Gefitinib and erlotinib, two EGFR-tyrosine kinase inhibitors, have been approved for non-small cell lung cancer. However, durable clinical efficacy of these EGFR inhibitors is severely limited by the emergence of acquired resistance. For example, the expression of breast cancer-resistant protein (BCRP/ABCG2) has been shown to confer acquired resistance of wild-type EGFR (wtEGFR)-expressing cancer cells to gefitinib. However, the underlying molecular mechanisms still remain unclear. Here, we show that wtEGFR expression is elevated in the nucleus of acquired gefitinib-resistant cancer cells. Moreover, nuclear translocation of EGFR requires phosphorylation at Ser-229 by Akt. In the nucleus, EGFR then targets the proximal promoter of BCRP/ABCG2 and thereby enhances its gene transcription. The nuclear EGFR-mediated BCRP/ABCG2 expression may contribute at least in part to the acquired resistance of wtEGFR-expressing cancer cells to gefitinib. Our findings shed light on the role of nuclear EGFR in the sensitivity of wtEGFR-expressing cancer cells to EGFR tyrosine kinase inhibitors and also deciphered a putative molecular mechanism contributing to gefitinib resistance through BCRP/ABCG2 expression.

Seedless, silver-induced synthesis of star-shaped gold/silver bimetallic nanoparticles as high efficiency photothermal therapy reagent

This work demonstrates a simple method for synthesizing a shape-controllable bimetallic gold/silver nanostructured material. Spiky star-shaped gold/silver nanoparticles are obtained by mixing HAuCl4, AgNO3 and ascorbic acid with shaking for 20 s. The wide range of star shapes and irregular quasi-spherical nanoparticles is tailored by tuning the ratio of metal precursors. The wavelengths absorbed by the nanoparticles can be tuned from visible light to near infrared by controlling their shape. To maintain the morphology of the nanoparticles, enhance their thermal stability and support their application in biological systems, modified chitosan was utilized for the properties and to keep the material well dispersed in solution in deionized water. The moderate concentration of modified chitosan capped bimetallic star-shaped nanoparticles not only ensured non-toxicity to normal cells and cancer cells, but also promoted high efficiency photothermal ablation of cancer cells. Ultimately, this nanotechnology-driven assay has huge potential for application in rapid synthesis, tunable absorption and non-cytotoxic photothermal therapy for the effective treatment of cancer.

Review of a current role of mass spectrometry for proteome research.

This review is intended to give readers a snapshot of current mass spectrometry for proteomics research. It covers a brief history of mass spectrometry proteomic research, peptidomics and proteomics for biomarker search, quantitative proteomics, proteomics with post-translational modification and future perspective of proteomics.

Iron Oxide/Gold Core/Shell Nanoparticles for Ultrasensitive Detection of Carbohydrate−Protein Interactions

Changes in the expression of cell surface glycan are often associated with malignant metastasis. The expression level may be dramatically enhanced during tumor progression. A highly sensitive assay that is capable of detecting low levels of cancer-associated carbohydrate antigens can be a powerful tool for early diagnosis. In this work, an ultrasensitive glycans array using iron oxide/gold core/shell nanoparticles conjugated with antibodies or proteins is developed. A magnetic field is applied to quickly bring nanoparticle labeled proteins or antibodies from a solution to an array of carbohydrates immobilized on glass slides and to help them to encounter the carbohydrates at very low concentration. The gold shell provides a well established platform for conjugation of biomolecules. Well-defined recognition systems, namely, mannose derivatives (Man1, Man4, and Man9) with a mannose binding lectin (Concanavalin A) and a stage-specific embryonic antigens-3 (SSEA-3) with a monoclonal antibody (anti-SSEA-3) were chosen to establish this detection tool. Array systems were conducted to determine their surface dissociation constant (K(D,surface)) and their binding specificity for qualitative and quantitative analysis of carbohydrate-protein and carbohydrate-antibody interactions. When coupled with a signal amplification method based on nanoparticle-promoted reduction of silver, the sensitivity of an iron oxide/gold core/shell nanoparticle-based assay reached to subattomole level in carbohydrate detection.

RNA helicase A is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus

EGF induces the translocation of EGF receptor (EGFR) from the cell surface to the nucleus where EGFR activates gene transcription through its binding to an AT-rich sequence (ATRS) of the target gene promoter. However, how EGFR, without a DNA-binding domain, can bind to the gene promoter is unclear. In the present study, we show that RNA helicase A (RHA) is an important mediator for EGFR-induced gene transactivation. EGF stimulates the interaction of EGFR with RHA in the nucleus of cancer cells. The EGFR/RHA complex then associates with the target gene promoter through binding of RHA to the ATRS of the target gene promoter to activate its transcription. Knockdown of RHA expression in cancer cells abrogates the binding of EGFR to the target gene promoter, thereby reducing EGF/EGFR-induced gene expression. In addition, interruption of EGFR–RHA interaction decreases the EGFR-induced promoter activity. Consistently, we observed a positive correlation of the nuclear expression of EGFR, RHA, and cyclin D1 in human breast cancer samples. These results indicate that RHA is a DNA-binding partner for EGFR-mediated transcriptional activation in the nucleus.

Differential Receptor Binding Affinities of Influenza Hemagglutinins on Glycan Arrays

A library of 27 sialosides, including seventeen 2,3-linked and ten 2,6-linked glycans, has been prepared to construct a glycan array and used to profile the binding specificity of different influenza hemagglutinins (HA) subtypes, especially from the 2009 swine-originated H1N1 and seasonal influenza viruses. It was found that the HAs from the 2009 H1N1 and the seasonal Brisbane strain share similar binding profiles yet different binding affinities toward various α2,6 sialosides. Analysis of the binding profiles of different HA subtypes indicate that a minimum set of 5 oligosaccharides can be used to differentiate influenza H1, H3, H5, H7, and H9 subtypes. In addition, the glycan array was used to profile the binding pattern of different influenza viruses. It was found that most binding patterns of viruses and HA proteins are similar and that glycosylation at Asn27 is essential for receptor binding.