Germline E-cadherin mutations in familial lobular breast cancer.

BACKGROUND: The cell surface glycoprotein E-cadherin (CDH1) is a key regulator of adhesive properties in epithelial cells. Germline mutations in CDH1 are well established as the defects underlying hereditary diffuse gastric cancer (HDGC) syndrome, and an increased risk of lobular breast cancer (LBC) has been described in HDGC kindreds. However, germline CDH1 mutations have not been described in patients with LBC in non-HDGC families. This study aimed to investigate the frequency of germline CDH1 mutations in patients with LBC with early onset disease or family histories of breast cancer without DGC. METHODS: Germline DNA was analysed in 23 women with invasive lobular or mixed ductal and lobular breast cancers who had at least one close relative with breast cancer or had themselves been diagnosed before the age of 45 years, had tested negative for a germline BRCA1 or BRCA2 mutation, and reported no personal or family history of diffuse gastric cancer. The full coding sequence of CDH1 including splice junctions was amplified using PCR and screened for mutations using DHPLC and sequencing. RESULTS: A novel germline CDH1 truncating mutation in the extracellular portion of the protein (517insA) was identified in one woman who had LBC at the age of 42 years and a first degree relative with invasive LBC. CONCLUSIONS: Germline CDH1 mutations can be associated with invasive LBC in the absence of diffuse gastric cancer. The finding, if confirmed, may have implications for management of individuals at risk for this breast cancer subtype. Clarification of the cancer risks in the syndrome is essential.

Inhibition of tumor cell invasion and angiogenesis by motuporamines.

Tissue invasion is an important determinant of angiogenesis and metastasis and constitutes an attractive target for cancer therapy. We have developed an assay to identify agents that inhibit invasion by mechanisms other than inhibition of cell attachment or cytotoxicity. A screen of marine sponge extracts identified motuporamines as micromolar inhibitors of invasion of basement membrane gels by MDA-231 breast carcinoma, PC-3 prostate carcinoma, and U-87 and U-251 glioma cells. Motuporamine C inhibits cell migration in monolayer cultures and impairs actin-mediated membrane ruffling at the leading edge of lamellae. Motuporamine C also reduces beta1-integrin activation, raising the possibility that it interferes with "inside-out" signaling to integrins. In addition, motuporamine C inhibits angiogenesis in an in vitro sprouting assay with human endothelial cells and an in vivo chick chorioallantoic membrane assay. The motuporamines show little or no toxicity or inhibition of cell proliferation, and they are structurally simple and easy to synthesize, making them attractive drug candidates.

Tumor suppressor PTEN inhibits nuclear accumulation of beta-catenin and T cell/lymphoid enhancer factor 1-mediated transcriptional activation.

beta-Catenin is a protein that plays a role in intercellular adhesion as well as in the regulation of gene expression. The latter role of beta-catenin is associated with its oncogenic properties due to the loss of expression or inactivation of the tumor suppressor adenomatous polyposis coli (APC) or mutations in beta-catenin itself. We now demonstrate that another tumor suppressor, PTEN, is also involved in the regulation of nuclear beta-catenin accumulation and T cell factor (TCF) transcriptional activation in an APC-independent manner. We show that nuclear beta-catenin expression is constitutively elevated in PTEN null cells and this elevated expression is reduced upon reexpression of PTEN. TCF promoter/luciferase reporter assays and gel mobility shift analysis demonstrate that PTEN also suppresses TCF transcriptional activity. Furthermore, the constitutively elevated expression of cyclin D1, a beta-catenin/TCF-regulated gene, is also suppressed upon reexpression of PTEN. Mechanistically, PTEN increases the phosphorylation of beta-catenin and enhances its rate of degradation. We define a pathway that involves mainly integrin-linked kinase and glycogen synthase kinase 3 in the PTEN-dependent regulation of beta-catenin stability, nuclear beta-catenin expression, and transcriptional activity. Our data indicate that beta-catenin/TCF-mediated gene transcription is regulated by PTEN, and this may represent a key mechanism by which PTEN suppresses tumor progression.

The integrin linked kinase (ILK) induces an invasive phenotype via AP-1 transcription factor-dependent upregulation of matrix metalloproteinase 9 (MMP-9).

Overexpression of Integrin Linked Kinase (ILK) in intestinal and mammary epithelial cells results in a highly invasive phenotype, associated with increased levels of expression of the matrix metalloproteinase MMP-9. This increase was at the transcriptional level as determined by MMP-9 promoter-CAT reporter assays. Mutations in the two AP-1 binding sites within the MMP-9 promoter completely inhibited the reporter activity. We have previously shown that ILK inhibits glycogen synthase kinase-3 (GSK-3) activity. Transient transfection of wild-type GSK-3beta in ILK-overexpressing cells decreased MMP-9 promoter activity and AP-1 activity, indicating that ILK can stimulate MMP-9 expression via GSK-3beta and AP-1 transcription factor. A small molecule inhibitor of the ILK kinase reduced the in vitro invasiveness of ILK-overexpressing cells as well as the invasiveness of several human brain tumor cell lines. Furthermore, both MMP-9 promoter and AP-1 activities were inhibited by the ILK inhibitor. Invasiveness of ILK-overexpressing cells was also reduced by inhibition of MMP-9. These data demonstrate that ILK can induce an invasive phenotype via AP-1-dependent upregulation of MMP-9.

The integrin-linked kinase regulates the cyclin D1 gene through glycogen synthase kinase 3beta and cAMP-responsive element-binding protein-dependent pathways.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the pRB tumor suppressor protein. Cyclin D1 is overexpressed in 20-30% of human breast tumors and is induced both by oncogenes including those for Ras, Neu, and Src, and by the beta-catenin/lymphoid enhancer factor (LEF)/T cell factor (TCF) pathway. The ankyrin repeat containing serine-threonine protein kinase, integrin-linked kinase (ILK), binds to the cytoplasmic domain of beta(1) and beta(3) integrin subunits and promotes anchorage-independent growth. We show here that ILK overexpression elevates cyclin D1 protein levels and directly induces the cyclin D1 gene in mammary epithelial cells. ILK activation of the cyclin D1 promoter was abolished by point mutation of a cAMP-responsive element-binding protein (CREB)/ATF-2 binding site at nucleotide -54 in the cyclin D1 promoter, and by overexpression of either glycogen synthase kinase-3beta (GSK-3beta) or dominant negative mutants of CREB or ATF-2. Inhibition of the PI 3-kinase and AKT/protein kinase B, but not of the p38, ERK, or JNK signaling pathways, reduced ILK induction of cyclin D1 expression. ILK induced CREB transactivation and CREB binding to the cyclin D1 promoter CRE. Wnt-1 overexpression in mammary epithelial cells induced cyclin D1 mRNA and targeted overexpression of Wnt-1 in the mammary gland of transgenic mice increased both ILK activity and cyclin D1 levels. We conclude that the cyclin D1 gene is regulated by the Wnt-1 and ILK signaling pathways and that ILK induction of cyclin D1 involves the CREB signaling pathway in mammary epithelial cells.

Inhibition of integrin-linked kinase (ILK) suppresses activation of protein kinase B/Akt and induces cell cycle arrest and apoptosis of PTEN-mutant prostate cancer cells.

PTEN is a tumor suppressor gene located on chromosome 10q23 that encodes a protein and phospholipid phosphatase. Somatic mutations of PTEN are found in a number of human malignancies, and loss of expression, or mutational inactivation of PTEN, leads to the constitutive activation of protein kinase B (PKB)/Akt via enhanced phosphorylation of Thr-308 and Ser-473. We recently have demonstrated that the integrin-linked kinase (ILK) can phosphorylate PKB/Akt on Ser-473 in a phosphoinositide phospholipid-dependent manner. We now demonstrate that the activity of ILK is constitutively elevated in a serum- and anchorage-independent manner in PTEN-mutant cells, and transfection of wild-type (WT) PTEN into these cells inhibits ILK activity. Transfection of a kinase-deficient, dominant-negative form of ILK or exposure to a small molecule ILK inhibitor suppresses the constitutive phosphorylation of PKB/Akt on Ser-473, but not on Thr-308, in the PTEN-mutant prostate carcinoma cell lines PC-3 and LNCaP. Transfection of dominant-negative ILK and WT PTEN into these cells also results in the inhibition of PKB/Akt kinase activity. Furthermore, dominant-negative ILK or WT PTEN induces G(1) phase cycle arrest and enhanced apoptosis. Together, these data demonstrate a critical role for ILK in PTEN-dependent cell cycle regulation and survival and indicate that inhibition of ILK may be of significant value in PTEN-mutant tumor therapy.

Cell-extracellular matrix interactions stimulate the AP-1 transcription factor in an integrin-linked kinase- and glycogen synthase kinase 3-dependent manner.

Integrin-mediated interactions of cells with components of the extracellular matrix regulate cell survival, cell proliferation, cell differentiation, and cell migration. Some of these physiological responses are regulated via activation of transcription factors such as activator protein 1 (AP-1). Integrin-linked kinase (ILK) is an ankyrin repeat containing serine-threonine protein kinase whose activity is rapidly and transiently stimulated by cell-fibronectin interactions as well as by insulin stimulation. ILK activates protein kinase B and inhibits the glycogen synthase kinase 3 (GSK-3) activity in a phosphatidylinositol-3-kinase (PI 3-kinase)-dependent manner. We now show that cell adhesion to fibronectin results in a rapid and transient stimulation of AP-1 activity. At the same time, the kinase activity of ILK is stimulated whereas that of GSK-3 is inhibited. This fibronectin-dependent activation of AP-1 activity is inhibited in a dose-dependent manner if the cells are transfected with wild-type GSK-3, and also by inhibitors of PI 3-kinase. Stable or transient overexpression of ILK results in a stimulation of AP-1 activity which is inhibited by cotransfection with wild-type GSK-3 and kinase-deficient ILK. Transient transfection of ILK in HEK-293 cells stimulates complex formation between an AP-1 consensus oligonucleotide and nuclear proteins containing c-jun. The formation of this complex is inhibited by cotransfection with active GSK-3 or kinase-deficient ILK, suggesting that ILK may regulate AP-1 activation by inhibiting GSK-3, which has previously been shown to be a negative regulator of AP-1. In the presence of serum, ILK has no effect on the phosphorylation of Ser-73 in the N-terminal transactivation domain of c-jun. These results demonstrate a novel signaling pathway for the adhesion-mediated stimulation of AP-1 transcriptional activity involving ILK and GSK-3 and the subsequent regulation of the c-jun-DNA interaction.

Inhibitory effects of specific apolipoprotein C-III isoforms on the binding of triglyceride-rich lipoproteins to the lipolysis-stimulated receptor.

ApoC-III overexpression in mice results in severe hypertriglyceridemia due primarily to a delay in the clearance of triglyceride-rich lipoproteins. We have, in primary cultures of rat hepatocytes, characterized a lipolysis-stimulated receptor (LSR). The apparent number of LSR that are available on rat liver plasma membranes is negatively correlated with plasma triglyceride concentrations measured in the fed state. We therefore proposed that the primary physiological role of the LSR is to contribute to the cellular uptake of triglyceride-rich lipoproteins. We have now tested the effect of apoC-III on the binding of triglyceride-rich lipoproteins to LSR. Supplementation of 125I-very low density lipoprotein (VLDL) with apoC-III inhibited the LSR-mediated binding, internalization, and degradation of 125I-VLDL in primary cultures of rat hepatocytes. Studies using isolated rat liver plasma membranes showed that enrichment of human VLDL and chylomicrons with synthetic or purified human apoC-III decreased their binding to the LSR by about 40%. Supplementation of triglyceride-rich lipoproteins under the same conditions with human apoC-II had no such inhibitory effect, despite the fact that this apoprotein bound as efficiently as apoC-III to these particles. Preincubation of LDL with apoC-III did not modify its binding to LSR. Partitioning studies using 125I-apoC-III showed that this lack of effect was due to apoC-III's inability to efficiently associate with LDL. Purified human apoC-III1 was as efficient as the synthetic nonsialylated form of apoC-III in inhibiting binding of VLDL to LSR. However, despite a 2-fold greater binding of apoC-III2 to VLDL, this isoform was a less efficient inhibitor of the binding of VLDL to LSR than apoC-III1 or nonsialylated apoC-III. Desialylation of apoC-III2 by treatment with neuraminidase increased the inhibition of VLDL binding to LSR to a level similar to that observed with apoC-III1 and nonsialylated apoC-III. We propose that apoC-III regulates in part the rate of removal of triglyceride-rich particles by inhibiting their binding to the LSR, and that the level of inhibition is determined by the degree of apoC-III sialylation.

Mechanism of activation and functional significance of the lipolysis-stimulated receptor. Evidence for a role as chylomicron remnant receptor.

In cultured human and rat cells, the lipolysis-stimulated receptor (LSR), when activated by free fatty acids (FFA), mediates the binding of apoprotein B- and apoprotein E-containing lipoproteins and their subsequent internalization and degradation. To better understand the physiological role of LSR, we developed a biochemical assay that optimizes both the activation and binding steps and, thus, allows the estimation of the number of LSR binding sites expressed in the livers of living animals. With this technique, a strong inverse correlation was found in rats between the apparent number of LSR binding sites in liver and the postprandial plasma triglyceride concentration (r = -0.828, p < 0.001, n = 12). No correlation existed between the number of LSR and plasma triglycerides measured in the same animals after 24 h of fasting. The same membrane binding assay was used to elucidate the mechanism by which FFA induce lipoprotein binding to LSR. The LSR activation step was mediated by direct interaction of FFA with LSR candidate proteins of apparent molecular masses of 115 and 90 kDa and occurred independently of the membrane lipid environment. The FFA-induced conformational shift that revealed the lipoprotein binding site remained fully reversible upon removal of the FFA. However, occupancy of the site by the apoprotein ligand stabilized the active form of LSR. Comparison of the effect of different FFA alone or in combination indicated that the same binding site is revealed by different FFA and that the length and saturation of the FFA monomeric carbon chain are critical in determining the potency of the FFA activating effect. We propose that the LSR pathway represents a limiting step for the cellular uptake of intestinally derived triglyceride-rich lipoproteins and speculate that FFA liberated by lipolysis initiate this process by altering the conformation of LSR to reveal the lipoprotein binding site.

Inhibitory effect on the lipolysis-stimulated receptor of the 39-kDa receptor-associated protein.

Adenovirus vector-mediated transfer of the receptor-associated protein (RAP) gene into low density lipoprotein (LDL) receptor-deficient mice was shown to achieve plasma concentrations ranging between 20 and 200 micrograms/ml and to result in the accumulation of remnant lipoproteins (Willnow, T. E., Sheng, Z., Ishibashi, S., and Herz, J. (1994) Science 264, 1471-1474). Both this finding and the observation that in addition to various other members of the LDL receptor gene family, RAP binds to a yet unidentified protein of apparent molecular mass of 105 kDa prompted us to examine the effect of high concentrations of RAP on the lipolysis-stimulated receptor (LSR). LSR is a receptor distinct from the LDL receptor and the LDL receptor-related protein and is capable of binding apoB and apoE when activated by free fatty acids. Data reported here show that in fibroblasts isolated from a subject homozygous for familial hypercholesterolemia, RAP fusion protein inhibited LSR-mediated binding of 125I-LDL and the subsequent internalization and degradation of the particles. Studies on the interaction of RAP with LSR in isolated rat liver membranes revealed that at concentrations > or = 10 micrograms/ml, RAP inhibited in a dose-dependent manner the binding of LDL to LSR; half-maximum inhibition was obtained with 20 micrograms/ml RAP. Ligand blotting studies revealed that RAP bound directly to two rat liver membrane proteins of apparent molecular masses identical to those that bind 125I-LDL after preincubation with oleate. However, unlike LDL, binding of 125I-RAP to LSR did not require preincubation with oleate. Preincubation of nitrocellulose membranes with an excess of unlabeled RAP fusion protein decreased oleate-induced binding of 125I-LDL to LSR candidate proteins, whereas preincubation with excess unlabeled LDL was unable to prevent the subsequent binding of 125I-RAP to the LSR proteins. Both the latter data and analysis of the mechanism of inhibition were consistent with the RAP inhibitory effect on LSR being achieved by interference with a site distinct from the oleate-induced LDL binding site. In conclusion, this study shows that at concentrations reported to delay chylomicron remnant removal in LDL receptor-deficient mice, RAP exerted a significant inhibitory effect on LSR.

Lipolysis-stimulated receptor: a newcomer on the lipoprotein research scene.

It has been widely accepted that the remnants of the intestinally-derived lipoprotein chylomicrons, i.e., chylomicron remnants (CMR), are cleared from the circulation by a receptor genetically distinct from the well-known LDL-receptor. This second receptor was initially considered as a receptor specific for apo E, in contrast to the LDL-receptor, which binds both apo B and apoE. This article critically examines the current dogma of the putative CMR receptor, as well as both supporting and conflicting evidence for the recently-proposed identity of this receptor with the LDL-receptor related protein (LRP). Next, we introduce the lipolysis-stimulated receptor, LSR, which bears all the biochemical characteristics of the CMR receptor. In addition, the apparent number of LSR expressed in the liver is inversely correlated with nonfasting levels of plasma triglycerides. A change in LSR expression and parallel inverse change in plasma triglycerides is observed in rats treated with hyperlipidemic (retinoic acid) or hypolipidemic (fish oil in MaxEPA) agents, indicating that LSR represents a definite target for pharmacological management of hyperlipidemia. In support of this notion is the observation that MaxEPA, which causes an increase in LSR expression, also reduces both plasma triglyceride and cholesterol levels in the thus far intractable homozygous Watanabe heritable hyperlipidemic rabbit.

Furosemide-sensitive K+ transport in transformed and nontransformed rat liver epithelial cells: regulation by protein kinase C and involvement in cell growth.

Using Rb+ as a K+ tracer and atomic absorption spectrophotometry for measuring the Rb+ stable isotope, we studied K+ transport systems and their regulation by protein kinase C in nontransformed and spontaneously transformed rat liver epithelial cells. Ouabain-sensitive Na+/K(+)-ATPase and the furosemide-sensitive Na+/K+/Cl- cotransport had comparable activity ratios in both cell types (0.92 and 1 in nontransformed and transformed rat liver epithelial cells, respectively). The protein kinase C activators, dioctanoylglycerol and phorbol myristate acetate, partly inhibited the Na+/K+/Cl- cotransport in both cell types but their effect was stronger in nontransformed cells, suggesting that, in transformed cells, the Na+/K+/Cl- cotransport had partly lost the ability to be inhibited by protein kinase C. In both cell types, phorbol myristate acetate had little and dioctanoylglycerol had no inhibitory effect on Na+/K(+)-ATPase. Furosemide (1 mM) partly inhibited the [3H]thymidine incorporation in both cell types, suggesting an involvement of the Na+/K+/Cl- cotransport in rat liver epithelial cell growth.

[Liver mechanisms for the elimination of lipoproteins of intestinal origin]. / Mécanismes hépatiques d'élimination des lipoprotéines d'origine intestinale.

This article critically examines the concept of the putative chylomicron remnant receptor (CMR). The molecular nature of this second lipoprotein receptor remains disputed. Indeed, two proteins, the low density lipoprotein receptor-related protein (LRP) and the lipolysis stimulated receptor (LSR) have been proposed as candidates for this function. The LRP bears significant structural homology with the LDL receptor and mediates the internalisation of beta-VLDL enriched with apo E. In addition, LRP binds several ligands not related to the lipoprotein system. Thus, LRP's contribution to the clearance of CMR has been questioned. The precise biochemical structure of LSR remains unclear. However, a series of observations support the hypothesis that LSR is the CMR receptor. LSR, which is activated by free fatty acis (FFA), the products of lipolysis, is present in primary cultures of rat hepatocytes. It displays the highest affinity for triglyceride-rich lipoproteins and is inhibited by lactoferrin. The existence of a strong inverse correlation in rats between the apparent number of hepatic LSR and the plasma triglyceride concentration measured in the post-prandial state, indicate that LSR represents a rate-limiting step for the removal of triglyceride-rich lipoproteins. Moreover, the ability of MAXEPA to enhance the expression of LSR in parallel with its well documented hypotriglyceridemic effect indicates that, contrary to popular belief, the putative CMR receptor represents a target for pharmacological management of hyperlipidemia.

Characterization of specific binding sites for [3H]2-MeS-ADP on megakaryocytoblastic cell lines in culture.

Binding of [3H]2-methyl thio-adenosine 5' diphosphate ([3H]2-MeS-ADP), a stable analogue of adenosine 5' diphosphate (ADP) to DAMI and Meg-01, two megakaryocytoblastic cell lines, was time-dependent, reversible and saturable. Scatchard analysis of the saturation binding data indicated that [3H]2-MeS-ADP bound to one class of specific binding sites with high affinity (dissociation constants = 45.3 +/- 13.4 and 48.2 +/- 17.7 nM, and maximum binding capacities = 341.2 +/- 31.1 and 903 +/- 98 fmole/10(6) cells for DAMI and Meg-01, respectively) (N = 3). Unlabelled 2-MeS-ADP competitively and selectively inhibited the specific binding of [3H]2-MeS-ADP on DAMI and Meg-01 with inhibitory constant values of 118 +/- 11 and 38 +/- 11 nM, respectively (N = 3). ADP was 3 to 10 times less potent than 2-Mes-ADP in displacing [3H]2-MeS-ADP from its binding sites on DAMI and Meg-01, whereas other ADP analogues, such as AMP, GDP, UDP, adenosine or FSBA, did not interfere with the binding of [3H]2-MeS-ADP, suggesting that DAMI and Meg-01 contain ADP-specific receptors.