Nywana Sizemore

The AKT|[sol]|I|[kappa]|B kinase pathway promotes angiogenic|[sol]|metastatic gene expression in colorectal cancer by activating nuclear factor-|[kappa]|B and |[beta]|-catenin

Our laboratory has delineated that the phosphatidylinositol 3′ kinase (PI3K)/AKT/IκB kinase (IKK) pathway positively regulates NFκB and β-catenin, both important transcriptional regulators in colorectal cancer (CRC). Therefore, we investigated the effect of inhibiting the PI3K/AKT/IKKα pathway in regulating the inappropriate constitutive activation of NFκB and β-catenin in CRC cell lines. SW480 and RKO CRC cell lines demonstrate constitutive activation of AKT as well as both NFκB- and β-catenin-dependent transcription. The constitutive activation of NFκB- and β-catenin-dependent transcription is inhibited by transiently transfecting either kinase dead (KD) IKKα, which blocks IKKα kinase activity, KD AKT, which blocks AKT activity, or wildtype (WT) PTEN, which inhibits PI3K and AKT activity. The ability of KD IKKα, KD AKT or WT PTEN to decrease β-catenin-dependent transcription is independent of their effects on NFκB. Inducible expression of either KD IKKα or WT PTEN strongly inhibits both the constitutive NFκB- and β-catenin-dependent promoter and endogenous gene activation. Targeted array-based gene expression analysis of this inducible system reveals that many of the genes downregulated upon inhibition of this pathway are involved in tumor angiogenesis and metastasis. The activation of this pathway and the expression of the three most repressed genes was further analysed in samples of CRC. These results indicate a role of this pathway in controlling gene expression important in tumor progression and metastasis.

A Raf-independent Epidermal Growth Factor Receptor Autocrine Loop Is Necessary for Ras Transformation of Rat Intestinal Epithelial Cells*

We recently have shown that activated Ras, but not Raf, causes transformation of intestinal (RIE-1, IEC-6) epithelial cells, whereas both activated Ras and Raf transform NIH 3T3 fibroblasts (Oldham, S. M., Clark, G. J., Gangarosa, L. M., Coffey, R. J., and Der, C. J. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 6924–6928). The observations that conditioned medium from Ras-, but not Raf-, transfected RIE-1 cells, as well as exogenous transforming growth factor α (TGFα), promoted morphological transformation of parental RIE-1 cells prompted us to identify epidermal growth factor (EGF) receptor (EGFR) ligands produced by Ras-transformed RIE-1 cells responsible for this autocrine effect. Since studies in fibroblasts have shown that v-Src is transforming, we also determined if v-Src could transform RIE-1 cells. H- or K-Ras-transformed cells secreted significant amounts of TGFα protein, and mRNA transcripts for TGFα, amphiregulin (AR), and heparin-binding EGF-like growth factor (HB-EGF) were induced. Like Ras, v-Src caused morphological and growth transformation of parental RIE-1 cells. However, TGFα protein was not secreted by RIE-1 cells stably expressing v-Src or activated Raf, and only minor increases in EGFR ligand mRNA expression were detected in these cells. A selective EGFR tyrosine kinase inhibitor PD153035 attenuated the Ras-, but not Src-, transformed phenotype. Taken together, these observations provide a mechanistic and biochemical basis for the ability of activated Ras, but not activated Raf, to cause transformation of RIE-1 cells. Finally, we suggest that an EGFR-dependent mechanism is necessary for Ras, but not Src, transformation of these intestinal epithelial cells.

Breast Cancer Metastasis Suppressor 1 Inhibits Gene Expression by Targeting Nuclear Factor-κB Activity

Breast cancer metastasis suppressor 1 ( BRMS1 ) functions as a metastasis suppressor gene in breast cancer and melanoma cell lines, but the mechanism of BRMS1 suppression remains unclear. We determined that BRMS1 expression was inversely correlated with that of urokinase-type plasminogen activator ( uPA ), a prometastatic gene that is regulated at least in part by nuclear factor-κB (NF-κB). To further investigate the role of NF-κB in BRMS1-regulated gene expression, we examined NF-κB binding activity and found an inverse correlation between BRMS1 expression and NF-κB binding activity in MDA-MB-231 breast cancer and C8161.9 melanoma cells stably expressing BRMS1. In contrast, BRMS1 expression had no effect on activation of the activator protein-1 transcription factor. Further, we showed that suppression of both constitutive and tumor necrosis factor-α–induced NF-κB activation by BRMS1 may be due to inhibition of IκBα phosphorylation and degradation. To examine the relationship between BRMS1 and uPA expression in primary breast tumors, we screened a breast cancer dot blot array of normalized cDNA from 50 breast tumors and corresponding normal breast tissues. There was a significant reduction in BRMS1 mRNA expression in breast tumors compared with matched normal breast tissues (paired t test, P uPA gene expression ( P

Podocalyxin Increases the Aggressive Phenotype of Breast and Prostate Cancer Cells In vitro through Its Interaction with Ezrin

Podocalyxin is an anti-adhesive transmembrane sialomucin that has been implicated in the development of more aggressive forms of breast and prostate cancer. The mechanism through which podocalyxin increases cancer aggressiveness remains poorly understood but may involve the interaction of podocalyxin with ezrin, an established mediator of metastasis. Here, we show that overexpression of podocalyxin in MCF7 breast cancer and PC3 prostate cancer cell lines increased their in vitro invasive and migratory potential and led to increased expression of matrix metalloproteases 1 and 9 (MMP1 and MMP9). Podocalyxin expression also led to an increase in mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) activity. To determine the role of ezrin in these podocalyxin-dependent phenotypic events, we first confirmed that podocalyxin formed a complex with ezrin in MCF7 and PC3 cells. Furthermore, expression of podocalyxin was associated with a changed ezrin subcellular localization and increased ezrin phosphorylation. Transient knockdown of ezrin protein abrogated MAPK and PI3K signaling as well as MMP expression and invasiveness in cancer cells overexpressing podocalyxin. These findings suggest that podocalyxin leads to increased in vitro migration and invasion, increased MMP expression, and increased activation of MAPK and PI3K activity in MCF7 and PC3 cells through its ability to form a complex with ezrin.

Ras, but not Src, transformation of RIE-1 epithelial cells is dependent on activation of the mitogen-activated protein kinase cascade

Src transformation of NIH3T3 mouse fibroblasts has been shown to be dependent on Ras function. Since we recently showed that the signaling pathways that mediate Ras transformation of RIE-1 rat intestinal epithelial cells are distinct from those that cause Ras transformation of fibroblasts, we utilized three approaches to determine if Src transformation of RIE-1 cells is dependent on Ras. First, although both Ras and Src cause upregulation of an epidermal growth factor (EGF) receptor-dependent autocrine growth loop, only Ras transformation required this activity. Second, whereas both Src and Ras caused upregulation of the p42 and p44 mitogen-activated protein kinases (MAPKs), only Ras transformation was blocked by the inhibition of MAPK activation by treatment with the PD 98059 MEK inhibitor. Third, treatment with the farnesyltransferase inhibitor FTI-277 blocked Ras, but not Src, transformation. Taken together, these observations suggest that Src transformation of RIE-1 cells is not dependent on Ras. Finally, we determined that Ras activation of Raf-independent pathways alone is sufficient to cause growth transformation of RIE-1 cells. Thus, both Ras and Src cause transformation of RIE-1 cells via pathways distinct from those required to cause transformation of NIH3T3 cells.

Pharmacological inhibition of Ras-transformed epithelial cell growth is linked to down-regulation of epidermal growth factor–related peptides

BACKGROUND & AIMS Posttranslational farnesylation is required for Ras activation. Farnesyl transferase inhibitors (FTIs) selectively block protein farnesylation and reduce the growth of many Ras-transformed cells in vitro and in vivo. Activated Ras transforms rat intestinal epithelial (RIE-1) cells by a mechanism distinct from NIH 3T3 fibroblasts in that an epidermal growth factor receptor (EGFR) autocrine loop contributes significantly to the Ras-transformed RIE-1 phenotype. METHODS The ability of FTIs to block growth of Ras-transformed RIE-1 cells was evaluated, and these results were correlated with decreased EGFR ligand production. RESULTS FTI L744,832 caused a selective, dose-dependent, reversible blockade in proliferation of H-Ras-transformed RIE-1 cells, whereas control cell lines, K-Ras-transformed cells, and activated raf-transfected RIE cells were unaffected. The growth-inhibitory effects of L744,832 correlated with loss of farnesylated H-Ras protein and a marked reduction in transforming growth factor (TGF)-alpha and amphiregulin expression. Inhibition of proliferation of H-Ras RIE-1 cells by L744,832 was overcome by exogenous TGF-alpha, and enhanced growth inhibition was achieved by EGFR blockade in combination with L744,832. + CONCLUSIONS These data suggest that one mechanism by which FTIs inhibit growth of H-Ras-transformed epithelial cells is by reducing Ras-induced EGFR ligand production.

Development and Validation of Two Solid-Phase Enzyme Immunoassays (ELISA) for Quantitation of Human Epidermal Growth Factors (hEGFs)

AbstractPurpose. The purpose of the present investigation was to develop and validate two separate enzyme-linked immunosorbent assays (ELISA) for quantitation of exogenous human epidermal growth factor (hEGFl -53) and its truncated fragment (hEGFl-48) in rat plasma. Methods. The present assay systems were based on the sandwiching of the antigen between a monoclonal mouse anti-hEGFl-53 antibody, pre-coated on a 96-well polystyrene plate, and a polyclonal rabbit anti-hEGFl-48 antibody, which is then detected with a peroxidase-labeled goat anti-rabbit antibody. Results. The calibration curves for hEGFl-48 and hEGFl -53 in plasma were validated over a concentration range of 7.8–250 and 62.5–1000 pg/ml, respectively. Determined from replicate assays of hEGFl-48 quality control samples, the intra-assay precision and accuracy were ≤8.8% RSD and within ± 9.8%; and the inter-assay precision and accuracy were ≤14.8% RSD and within ± 9.7% RE, respectively. Determined from replicate assays of hEGFl-53 quality control samples, the intra-assay precision and accuracy were ≤10.0% RSD and within ± 8.5%; and the inter-assay precision and accuracy were ≤ 10.0% RSD and within ± 5.7% RE, respectively. The limit of quantitation of the hEGFl-48 and hEGFl-53 assay using 200 µL plasma per well is 7.8 and 62.5 pg/ml, respectively. These two ELISA methods are specific to hEGFs and do not cross-react with mouse EGF or other growth factors (TGFα, TGFβ, PDGF, and FGF) or lymphokines (IL1β and TNFα). These validated methods have been routinely applied to assay of plasma samples from various pharmacokinetic studies in rats receiving intravenous hEGFs. Both assay methods were also adapted to assay endogenous hEGFs in biological fluids of different animal species. Conclusions. Two sensitive ELISA methods have been validated for quantitation of hEGFl–53 and hEGFl–48 in rat plasma. Their utility has been demonstrated in the application of assaying immunoreactive concentrations of exogenous and endogenous epidermal growth factors.