David D. Chang

Rack1, a receptor for activated protein kinase C, interacts with integrin beta subunit

The integrin β subunit cytoplasmic domains are important for activation-dependent cell adhesion and adhesion-dependent signaling events. We report an interaction between integrin β subunit cytoplasmic domain and Rack1, a Trp-Asp (WD) repeat protein that has been shown to bind activated protein kinase C. The Rack1-binding site on integrin β2subunit resides within a conserved, membrane-proximal region. In the yeast two-hybrid assay, WD repeats five to seven of Rack1 (Rack1-WD5/7) interact with integrin β1, β2, and β5 cytoplasmic domain. In eukaryotic cells, Rack1 co-immunoprecipitates with at least two different β integrins, β1 integrins in 293T cells and β2 integrins in JY lymphoblastoid cells. Whereas Rack1-WD5/7 binds integrins constitutively, the association of full-length Rack1 to integrinsin vivo requires a treatment with phorbol esters, which promotes cell spreading and adhesion. These findings suggest that Rack1 may link protein kinase C directly to integrins and participate in the regulation of integrin functions.

EPLIN regulates actin dynamics by cross-linking and stabilizing filaments

Epithelial protein lost in neoplasm (EPLIN) is a cytoskeleton-associated protein encoded by a gene that is down-regulated in transformed cells. EPLIN increases the number and size of actin stress fibers and inhibits membrane ruffling induced by Rac. EPLIN has at least two actin binding sites. Purified recombinant EPLIN inhibits actin filament depolymerization and cross-links filaments in bundles. EPLIN does not affect the kinetics of spontaneous actin polymerization or elongation at the barbed end, but inhibits branching nucleation of actin filaments by Arp2/3 complex. Side binding activity may stabilize filaments and account for the inhibition of nucleation mediated by Arp2/3 complex. We propose that EPLIN promotes the formation of stable actin filament structures such as stress fibers at the expense of more dynamic actin filament structures such as membrane ruffles. Reduced expression of EPLIN may contribute to the motility of invasive tumor cells.

Imatinib mesylate (Gleevec) inhibits ovarian cancer cell growth through a mechanism dependent on platelet-derived growth factor receptor α and Akt inactivation

Purpose: We identified the platelet-derived growth factor receptor α (PDGFRα) as an ovarian cancer-specific gene by microarray hybridization using primary cultures. The purpose of this study is to evaluate whether disruption of the platelet-derived growth factor-regulated growth pathway by Imatinib mesylate (Gleevec), a partially selective PDGFR inhibitor, inhibits growth of ovarian cancer cells expressing PDGFR. Experimental Design: To investigate the effects of Imatinib mesylate in ovarian cancer, we established an in vitro model by immortalizing primary ovarian cells, which express endogenous PDGFR, and we evaluated the effects of Imatinib on cell proliferation. In addition, we investigated the involvement of Akt in mediating Imatinib-inhibited cell growth inhibition. Results: We found that 39% of ovarian tumors express PDGFR by immunohistochemistry. We showed that Imatinib inhibits the growth of ovarian cancer cells in a PDGFR-specific manner, at clinically relevant concentrations (IC50 < 1 μm). Imatinib inhibits the growth of three primary ovarian cultures and two immortalized cultures (PDGFR positive), but has no effects on SkOv3 and CaOv3 cell lines (PDGFR negative). Imatinib exerts antiproliferative effects by arresting cells at G0-G1 and preventing progression through S phase. Imatinib inhibits both PDGFRα and Akt phosphorylation at a concentration of 1 μm. Stable expression of constitutively active Akt induces partial resistance to PDGFR inhibition in ovarian cancer cells, as demonstrated by cell proliferation assay and cell cycle analysis. Conclusions: Our data indicate that Imatinib mesylate inhibits the growth of ovarian cancer cells through PDGFR inactivation. In addition, our results suggest that constitutive Akt activation modulates sensitivity to Imatinib in ovarian cancer cells.

Gene expression in epithelial ovarian carcinoma

We analysed the mRNA levels corresponding to 12 600 transcripts in primary cultures of ovarian epithelial cells derived from nine normal ovaries and 21 epithelial ovarian carcinoma. The class distinction and hierarchical clustering of expression data revealed a clear distinction in gene expression between normal and carcinoma-derived ovarian epithelial cells. Comparison of expression levels revealed 111 genes with mean expression values of >2.5-fold higher in carcinoma cells. Similarly, 62 genes were expressed at >2.5-fold higher levels in normal ovarian epithelial cells. For a few selected genes, we demonstrate that the pattern of differential expression observed in cultured epithelial cells is present in the normal ovaries and epithelial ovarian carcinoma. Use of cultured epithelial cells represents a novel strategy to study gene expression in a cell-type specific manner.

EPLIN, epithelial protein lost in neoplasm.

We have identified a novel cytoskeletal protein, EPLIN (Epithelial Protein Lost In Neoplasm), that is preferentially expressed in human epithelial cells. Two EPLIN isoforms, a 600 amino acid EPLIN-α and a 759 amino acid EPLIN-β, are detected in primary epithelial cells of oral mucosa, prostate and mammary glands. The expression of EPLIN-α is either down-regulated or lost in the majority of oral cancer cell lines (8/8), prostate cancer cell lines (4/4) and xenograft tumors (3/3), and breast cancer cell lines (5/6). The amino acid sequence of EPLIN is characterized by the presence of a single centrally located LIM domain. Both EPLIN isoforms localize to filamentous actin and suppress cell proliferation when overexpressed. These findings indicate that the loss of EPLIN seen in cancer cells may play a role in cancer progression.

Characterization of transformation related genes in oral cancer cells.

A cDNA representational difference analysis (cDNA-RDA) and an arrayed filter technique were used to characterize transformation-related genes in oral cancer. From an initial comparison of normal oral epithelial cells and a human papilloma virus (HPV)-immortalized oral epithelial cell line, we obtained 384 differentially expressed gene fragments and arrayed them on a filter. Two hundred and twelve redundant clones were identified by three rounds of back hybridization. Sequence analysis of the remaining clones revealed 99 unique clones corresponding to 69 genes. The expression of these transformation related gene fragments in three non-tumorigenic HPV-immortalized oral epithelial cell lines and three oral cancer cell lines were simultaneously monitored using a cDNA array hybridization. Although there was a considerable cell line-to-cell line variability in the expression of these clones, a reliable prediction of their expression could be made from the cDNA array hybridization. Our study demonstrates the utility of combining cDNA-RDA and arrayed filters in high-throughput gene expression difference analysis. The differentially expressed genes identified in this study should be informative in studying oral epithelial cell carcinogenesis.

Neutralizing Monoclonal Antibody to Periostin Inhibits Ovarian Tumor Growth and Metastasis

Periostin, an extracellular matrix protein, is reported to be overexpressed in a variety of human cancers and its functions seem to be linked to tumor metastasis. Our previous results show that engineered periostin overexpression promotes ovarian tumor growth and dissemination in vivo. In this study, we developed a neutra-lizing monoclonal antibody to periostin, named MZ-1, and investigated its effects on human ovarian tumor growth and metastasis. Our in vivo studies showed significant growth inhibition by MZ-1 on both subcutaneous and intraperitoneal (i.p.) tumors derived from the periostin-expressing ovarian cancer cell line A2780. In addition, MZ-1 treatment led to a reduction of the metastatic potential of these A2780 i.p. tumors. The in vivo antitumor effects of MZ-1 were linked to its specific inhibition of anchorage-independent growth and survival of periostin-expressing cells, as well as its neutralizing effects on periostin-induced cancer cell migration and invasion. The data suggest that blocking periostin expression may be a novel approach for treating the subset of invasive ovarian tumors that overexpress periostin protein. Mol Cancer Ther; 10(8); 1500–8. ©2011 AACR.

Overexpression of wild-type RhoA produces growth arrest by disrupting actin cytoskeleton and microtubules.

We have investigated the role of Rho GTPase in cell growth by generating stable cells that express the wild‐type RhoA (RhoAwt) under the control of an inducible promoter. Induction of RhoAwt had a biphasic effect on the actin cytoskeleton. At low levels of expression, RhoAwt stimulated the assembly of actin stress fibers without affecting cell growth. At high levels, there was a paradoxical disruption of the actin cytoskeleton accompanied by a growth arrest. Cell cycle analysis revealed a dual block at the G1/S and G2/M checkpoints. The G1/S arrest correlated with the accumulation of p21Cip1, resulting in the inhibition of cdk2 activity, whereas the G2/M block correlated with the loss of microtubules. The cyclin B level and the cdc2 kinase activity, however, were increased, suggesting that the progression through mitosis rather than entry into the G2/M is defective when RhoAwt is overexpressed. Similar cell cycle defects and the loss of microtubules were observed after a cytochalasin D treatment, indicating that the ability of RhoA to regulate the integrity of actin cytoskeleton may be critical for the cell cycle transition through both the G1/S and M phase checkpoints. J. Cell. Biochem. 80:229–240, 2000.

Characterization of mouse epithelial protein lost in neoplasm (EPLIN) and comparison of mammalian and zebrafish EPLIN.

EPLIN is a cytoskeleton-associated protein that was initially identified as the product of a gene that is transcriptionally down-regulated in cancer cells. In human, there are two known isoforms, EPLIN-alpha and -beta, generated by alternative promoter usage from a single gene. With the exception of a single LIM (lin-11, isl-1, and mec-3) domain, the sequence of EPLIN is unique and does not provide any clues to its function. To identify conserved regions of EPLIN that may be important for its function, we have characterized mouse (m) and zebrafish (zf) EPLIN. As in human, two isoforms, the 593 aa mEPLIN-alpha (77% identity; 83% similarity) and 753 aa mEPLIN-beta (75% identity; 83% similarity), were present in mouse. mEPLIN-alpha is highly expressed in embryonic tissue and adult lung and spleen, whereas mEPLIN-beta is preferentially expressed in kidney, testis, lung and liver. The analysis of mEPLIN gene revealed that the overall organization of the exons in mouse and human are conserved. In zebrafish, there was only one form, the 629 aa zfEPLIN, corresponding to the mammalian EPLIN-beta. Like its mammalian counterparts, ectopically expressed zfEPLIN is co-localized to the actin cytoskeleton. While the overall homology between mammalian and zebrafish EPLIN was not striking (37% identity; 50% similarity), there were seven highly conserved regions, which should be useful in structure-function studies of this novel protein.

Abstract 4407: Neutralizing monoclonal antibody to periostin (PN) inhibits ovarian tumor metastatic growth in animal models

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Periostin (PN), an extracellular matrix protein, is reported to be overexpressed in a variety of human cancers and its functions appear to be linked to tumor angiogenesis and metastasis. Our previous results show that engineered PN overexpression promotes ovarian tumor growth and dissemination in vivo. In the present study, we developed a neutralizing monoclonal antibody to PN, named MZ-1, and have investigated its effects on human ovarian tumor cell metastasis and progression. MZ-1 markedly reduced tumor metastatic growth in orthotopic ovarian tumor models derived from two PN-expressing ovarian cancer cell lines A2780 and TOV-112D. These in vivo antitumor effects of MZ-1 were attributed to its specific inhibition of anchorage-independent growth and survival of PN-expressing cells, as well as its neutralization on PN-induced migration. The data suggests that blocking PN expression may be a novel approach for treatment of ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4407.