Elizabeth A. Lipscomb

Integrin (α6β4) regulation of eIF-4E activity and VEGF translation: a survival mechanism for carcinoma cells

We define a novel mechanism by which integrins regulate growth factor expression and the survival of carcinoma cells. Specifically, we demonstrate that the α6β4 integrin enhances vascular endothelial growth factor (VEGF) translation in breast carcinoma cells. The mechanism involves the ability of this integrin to stimulate the phosphorylation and inactivation of 4E-binding protein (4E-BP1), a translational repressor that inhibits the function of eukaryotic translation initiation factor 4E (eIF-4E). The regulation of 4E-BP1 phosphorylation by α6β4 derives from the ability of this integrin to activate the PI-3K–Akt pathway and, consequently, the rapamycin-sensitive kinase mTOR that can phosphorylate 4E-BP1. Importantly, we show that this α6β4-dependent regulation of VEGF translation plays an important role in the survival of metastatic breast carcinoma cells by sustaining a VEGF autocrine signaling pathway that involves activation of PI-3K and Akt. These findings reveal that integrin-mediated activation of PI-3K–Akt is amplified by integrin-stimulated VEGF expression and they provide a mechanism that substantiates the reported role of α6β4 in carcinoma progression.

Mobilization and activation of a signaling competent alpha6beta4integrin underlies its contribution to carcinoma progression.

This review examines the hypothesis that the function of the α 6β 4 integrin is altered substantially as normal epithelia undergo malignant transformation and progress to invasive carcinoma and that the functions of this integrin contribute to the behavior of aggressive carcinoma cells. Specifically, α 6β 4 functions primarily as an adhesion receptor in normal epithelia, often as a component of hemidesmosomes and associated with intermediate filaments. Factors in the host-tumor microenvironment have the potential to mobilize α 6β 4 from hemidesmosomes and promote its association with F-actin in lamellae and filopodia, a process that is mediated by PKC-dependent phosphorylation of the β 4 cytoplasmic domain. Importantly, this altered localization of α 6β 4 appears to be coupled to an activation of its signaling potential, which may occur through its association with growth factor receptors or lipid rafts, possibilities that are not mutually exclusive. The primal signaling event triggered by α 6β 4 appears to be activation of PI3-K and this activation has profound consequences on the migration, invasion and survival of carcinoma cells. Arguably, the ability of α 6β 4 to stimulate the PI3-K-dependent translation of VEGF and possibly other growth factors may be the most significant contribution of this integrin to carcinoma because of the potential autocrine and paracrine effects of these factors.

SM-20 Is a Novel Mitochondrial Protein That Causes Caspase-dependent Cell Death in Nerve Growth Factor-dependent Neurons

Sympathetic neurons undergo protein synthesis-dependent apoptosis when deprived of nerve growth factor (NGF). Expression of SM-20 is up-regulated in NGF-deprived sympathetic neurons, and ectopic SM-20 is sufficient to promote neuronal death in the presence of NGF. We now report that SM-20 is a mitochondrial protein that promotes cell death through a caspase-dependent mechanism. SM-20 immunofluorescence was present in the cytoplasm in a punctate pattern that colocalized with cytochrome oxidase I and with mitochondria-selective dyes. Analysis of SM-20/dihydrofolate reductase fusion proteins revealed that the first 25 amino acids of SM-20 contain a functional mitochondrial targeting sequence. An amino-terminal truncated form of SM-20 was not restricted to mitochondria but instead localized throughout the cytosol and nucleus. Nevertheless, the truncated SM-20 retained the ability to induce neuronal death, similar to the wild type protein. SM-20-induced death was accompanied by caspase-3 activation and was blocked by a general caspase inhibitor. Additionally, overexpression of SM-20, under conditions where cell death is blocked by a general caspase inhibitor, did not result in widespread release of cytochrome c from mitochondria. These results indicate that SM-20 is a novel mitochondrial protein that may be an important mediator of neurotrophin-withdrawal-mediated cell death.

Expression of the SM-20 Gene Promotes Death in Nerve Growth Factor-Dependent Sympathetic Neurons

Abstract: Sympathetic neurons undergo apoptosis when deprived of nerve growth factor (NGF). Inhibitors of RNA or protein synthesis block this death, suggesting that gene expression is important for apoptosis in this system. We have identified SM‐20 as a new gene that increases in expression in sympathetic neurons after NGF withdrawal. Expression of SM‐20 also increases during neuronal death caused by cytosine arabinoside or the phosphatidylinositol 3‐kinase inhibitor LY294002. In addition, SM‐20 protein synthesis is elevated in NGF‐deprived neurons compared with neurons maintained with NGF. Importantly, expression of SM‐20 in sympathetic neurons causes cell death in the presence of NGF. These results suggest that SM‐20 may function to regulate cell death in neurons.

Non-Angiogenic Functions of VEGF in Breast Cancer

This review advances the hypothesis that the function of vascular endothelial growth factor (VEGF) in breast cancer is not limited to angiogenesis, and that VEGF signaling in breast carcinoma cells is important for the ability of these cells to evade apoptosis and progress towards invasive and metastatic disease. In other terms, VEGF signaling provides a selective advantage for the survival and dissemination of breast carcinoma cells that may be independent of angiogenesis. The key component of this hypothesis is that breast carcinoma cells express specific VEGF receptors and that these receptors respond to autocrine VEGF, resulting in the activation of signaling pathways that impede apoptosis and promote cell migration. A related hypothesis, which is developed in this review, is that the α6β4 integrin, which has been implicated in the survival and motility of breast cancer cells, can stimulate the translation of VEGF mRNA and, consequently, autocrine VEGF signaling. These findings imply that VEGF and VEGF receptor-based therapeutics, in addition to targeting angiogenesis, may also target tumor cells directly.

Use of RNA interference to inhibit integrin (alpha6beta4)-mediated invasion and migration of breast carcinoma cells

The application of small interfering RNA (siRNA) oligonucleotides to silence gene expression has profound implications for the intervention of human diseases including cancer. Using this technique, we explored the possibility that the α6β4 integrin, a laminin adhesion receptor with a recognized role in the invasive phenotype of many carcinomas, represents a potential therapeutic target to inhibit the migration and invasion of carcinoma cells. We found that siRNA oligonucleotides targeted to either subunit of the α6β4 integrin reduced cell surface expression of this integrin and resulted in decreased invasion of MDA-MB-231 breast carcinoma cells. Interestingly, reduced α6β4 expression also promoted decreased migration on non-laminin substrata indicating that this integrin can function in a ligand-independent manner. In addition, the absence of β4 expression in these cells augmented the formation of α6β1 heterodimers and increased adhesion to laminin-1. Taken together, these results substantiate the importance of the α6β4 integrin in invasion and migration that has been demonstrated previously by expression of the β4 subunit in β4-deficient cell lines and by function blocking antibodies. Furthermore, these data suggest that the utilization of siRNA oligonucleotides to reduce the expression of the α6β4 integrin may be a useful approach to prevent carcinoma cell progression.

A Novel Mechanism for Integrin-Mediated Ras Activation in Breast Carcinoma Cells: The α6β4 Integrin Regulates ErbB2 Translation and Transactivates Epidermal Growth Factor Receptor/ErbB2 Signaling

ErbB2 (HER2, Neu) and Ras play key roles in tumor invasion and metastasis. We identified a novel mechanism by which integrin alpha(6)beta(4) regulates ErbB2 expression, Ras activation, and the invasion of breast carcinoma cells. Here we show that integrin alpha(6)beta(4) regulates Ras activity especially in serum-depleted condition. Down-regulation of beta(4) integrin by beta(4) short hairpin RNA (shRNA) decreased Ras activity and carcinoma invasion whereas reexpression of this integrin restored Ras activity. ErbB2, a binding partner of epidermal growth factor receptor (EGFR), and EGFR modulated Ras activity, and integrin alpha(6)beta(4) regulated phospho-EGFR level without affecting EGFR expression. We also found that integrin alpha(6)beta(4) is involved in ErbB2 expression. Depletion of beta(4) by shRNA reduced ErbB2 protein level without affecting ErbB2 mRNA level and reexpression of beta(4) increased ErbB2 protein level. Reduction of eukaryotic initiation factor 4E, a rate-limiting factor for cap-dependent translation, decreased ErbB2 protein level, and beta(4) shRNA cells exhibited a shift in ErbB2 mRNA to light polysomes compared with control cells. These results show that integrin alpha(6)beta(4) regulates ErbB2 through translational control. In summary, we propose a novel mechanism for ErbB2 up-regulation and Ras activation in serum-depleted breast cancer cells; integrin alpha(6)beta(4) regulates the expression of ErbB2 and the subsequent phosphorylation of EGFR and activation of Ras. These findings provide a mechanism that substantiates the reported role of alpha(6)beta(4) in carcinoma invasion.

Induction of SM-20 in PC12 cells leads to increased cytochrome c levels, accumulation of cytochrome c in the cytosol, and caspase-dependent cell death

Sympathetic neurons deprived of nerve growth factor (NGF) release cytochrome c into the cytosol and undergo caspase‐dependent cell death through a process that requires de novo gene expression. Expression of the SM‐20 gene increases after NGF withdrawal, and ectopic SM‐20 expression induces cell death in NGF‐maintained neurons. To further evaluate the mechanism by which SM‐20 promotes cell death, we developed a PC12‐derived cell line in which SM‐20 expression can be induced by addition of doxycycline to the culture medium. Induction of SM‐20 in either undifferentiated or NGF‐differentiated cells resulted in cell death. Cell death was accompanied by an increase in caspase activity and was inhibited by the caspase inhibitor zVAD‐FMK. Analysis of cytochrome c in cytosolic and mitochondria‐enriched subcellular fractions revealed that induction of SM‐20 led to the accumulation of cytochrome c in the cytosol. Surprisingly, SM‐20 expression also resulted in a selective increase in the total amount of cytochrome c protein. Thus, induction of SM‐20 expression appears to affect both the amount and subcellular localization of cytochrome c in PC12 cells. These results suggest that SM‐20 promotes caspase‐dependent cell death through a mechanism involving cytochrome c.