INTERACTION BETWEEN BRK AND HER2 IN BREAST CANCER

INTERACTION BETWEEN BRK AND HER2 IN BREAST CANCER Midan Ai, Ph.D. Supervisory Professor: Zhen Fan, M.D. Breast tumor kinase (Brk) is a nonreceptor protein-tyrosine kinase that is highly expressed in approximately two thirds of breast cancers but is not detectable or is expressed at very low levels in normal mammary epithelium. Brk plays important roles in promoting proliferation, survival, invasion, and metastasis of breast cancer cells, but the mechanism(s) of which remain largely unknown. Recent studies showed that Brk is frequently co-overexpressed with human epidermal growth factor receptor-2 (HER2) and is physically associated with HER2 in breast cancer. The mechanism needs to be determined. In my studies, I found that high expression of HER2 is correlated with high expression of Brk in breast cancer cell lines. Silencing HER2 expression via RNA interference in HER2 over-expressed breast cancer cells resulted in Brk protein decrease and overexpression of HER2 in HER2 low-expressed breast cancer cells up-regulated Brk expression. The mechanism study indicated that overexpression of HER2 increased Brk protein stability. Brk was degraded through a Ca2+-dependent protease pathway involving calpain and HER2 stimulated Brk expression via inhibiting calpain activity. Calpastatin is a calpain endogenous inhibitor and the calpain-calpastatin system has been implicated in a number of cell physiological functions. HER2 restrained calpain activation via up-regulating calpastatin expression and HER2 downstream signaling, MAPK pathway, was involved in the regulation. Furthermore, silencing of Brk expression by RNA interference in HER2-overexpressing breast cancer cells decreased HER2-mediated cell proliferation, survival, invasion/metastasis potential and increased cell sensitivity to HER2 kinase inhibitor, lapatinib, treatment, indicating that Brk plays important roles in regulating and mediating the oncogenic functions of HER2. The Stat3 pathway played important roles in Brk mediated cell survival and invasion/metastasis in the context of HER2-overexpressing breast cancer cells. However, transgenic mice with inducible expression of constitutively active Brk (CA) in the mammary epithelium failed to develop malignant change in the mammary glands after Brk induction for 15 months which indicated that expression of Brk protein alone was not sufficiently to induce spontaneous breast tumor. Bitransgenic mice with co-expression of HER2/neu and inducible expression of Brk in the mammary epithelium developed multifocal mammary tumors, but there were no significant difference in the tumor occurring time, tumor size, tumor weight and tumor multiplicity between the mouse group with co-expression of Brk and HER2/neu and the mouse group with HER2/neu expression only

HSV-tk/GCV gene therapy mediated by EBV-LMP1 for EBV-associated cancer

<p>Abstract</p> <p>Background</p> <p>To investigate the feasibility of gene therapy in treating Epstein-Barr virus (EBV)-associated cancer by employing the suicide gene, herpes simplex virus thymidine kinase/ganciclovir (HSV-tk/GCV), which uses the signaling pathway through the HIV-long terminal repeat (LTR) gene which is expressed from a nuclear factor-κB (NF-κB)-binding motif-containing promoter that is regulated by EBV-latent membrane protein 1 (LMP1) via NF-κB.</p> <p>Methods</p> <p>First, we constructed the plasmid pVLTR-tk, which was regulated by EBV-LMP1 via NF-κB, and then investigated the cytotoxic effect of the pVLTR-tk/GCV on cancer cells, using MTT assays, clonogenic assays, flow cytometry, and animal experiments.</p> <p>Results</p> <p>The activation of TK was increased after transfection of the pVLTR-tk into the EBV-LMP1 positive cells. After GCV treatment, the clonogenicity and survival of the cells substantially declined, and a bystander effect was also observed. The LMP1 positive cells exhibited remarkable apoptosis following pVLTR-tk/GCV treatment, and the pVLTR-tk/GCV restrained tumor growth in vivo for EBV-LMP1 positive cancers.</p> <p>Conclusion</p> <p>The pVLTR-tk/GCV suicide gene system may be used as a new gene targeting strategy for EBV-associated cancer.</p

A Novel Synthetic Analog of 5, 8-Disubstituted Quinazolines Blocks Mitosis and Induces Apoptosis of Tumor Cells by Inhibiting Microtubule Polymerization

Many mitosis inhibitors are powerful anticancer drugs. Tremendous efforts have been made to identify new anti-mitosis compounds for developing more effective and less toxic anti-cancer drugs. We have identified LJK-11, a synthetic analog of 5, 8-disubstituted quinazolines, as a novel mitotic blocker. LJK-11 inhibited growth and induced apoptosis of many different types of tumor cells. It prevented mitotic spindle formation and arrested cells at early phase of mitosis. Detailed in vitro analysis demonstrated that LJK-11 inhibited microtubule polymerization. In addition, LJK-11 had synergistic effect with another microtubule inhibitor colchicine on blocking mitosis, but not with vinblastine or nocodazole. Therefore, LJK-11 represents a novel anti-microtubule structure. Understanding the function and mechanism of LJK-11 will help us to better understand the action of anti-microtubule agents and to design better anti-cancer drugs

Effect of LJK-11 on the growth and death of different tumor cells.

<p>A549, Hela, HGC-27, or MDA-MB-453 cells were incubated with the indicated concentrations of LJK-11 for 48 hours. The effect of LJK-11 on cell growth and death was evaluated by MTT assay. The cell viability is expressed as a percentage of the compound-treated viable cells divided by the viable cells of the untreated control. The data are the means of triplicates ±SD.</p

Synergistic effect of LJK-11 and colchicine on blocking mitosis.

<p><b>A</b>. Flow cytometry analysis of the effects of LJK-11 (10 µM), colchicines (20 nM), or the combination of the two on cell cycle distribution. A549 cells were incubated with 10 µM LJK-11, 20 nM colchicine, or combination of 10 µM LJK-11 and 20 nM colchicine for 24 hours. The cells were then fixed and stained with PI, and analyzed by flow cytometry. <b>B</b>. Percentage of cells in G2/M phase after 24 hours treatment with 10 µM LJK-11, 20 nM colchicine, or combination of 10 µM LJK-11 and 20 nM colchicine. <b>C</b>. Concentration dependent G2/M arrest by treatment of colchicines or LJK-11 for 24 h. Also indicated in the figures are the percentages of G2/M arrest induced by the combination of 10 µM LJK-11 and 20 nM colchicine. Data are the means of triplicates ± SD.</p

Effects of LJK-11 on mitotic spindle formation.

<p>A549 cells were incubated on glass coverslips with different reagents for 16 hours, and then fixed and stained with α-tubulin antibody to visualize microtubules (green) and with DAPI to visualize chromosomes (blue). The cells were visualized by indirect immunofluorescent microscopy. A: control cells treated with equal volume of DMSO (0.1%). B: cells treated with 100 µM LJK-11. C: cells treated with 5 nM nocodazole. D: cells treated with 100 nM colchicine. E: cells treated with 50 nM Taxol.</p