Caveolin-1 mutants P132L and Y14F are dominant negative regulators of invasion, migration and aggregation in H1299 lung cancer cells.

Caveolin-1 is an essential protein constituent of caveolae. Accumulating evidence indicates that caveolin-1 may act as a positive regulator of cancer progression. In this study, we investigated the function of caveolin-1 in human lung cancer cells. Caveolin-1 knockdown inhibited cell proliferation and reduced focal adhesion kinase (Fak) phosphorylation. Matrix invasion and cell migration as well as expression and activity of matrix metalloproteases were attenuated following caveolin-1 RNAi-mediated knockdown or overexpression of Y14F and P132L mutants, demonstrating dominant-negative activity of these mutants. Time-lapse fluorescence microscopy revealed that caveolin-1 and its mutants P132L and Y14F are localized to the trailing edge of migrating cells during both random and directed cell movement, implying an active role of caveolin-1 in the migration process. Suppression of caveolin-1 function greatly elevated the percentage of H1299 cells exhibiting focal adhesions. In addition, cell aggregation was increased by wild type caveolin-1 and attenuated by both P132L and Y14F mutants. Overexpression of wild type caveolin-1 increased caveolae density, however, P132L and Y14F mutants did not affect caveolae formation, suggesting that in this respect that the mutants do not act in a dominant negative manner, and that effects of caveolin-1 on caveolae and cell invasion, migration, focal adhesion and aggregation, are separable. Our data provide novel mechanistic insights into the role of caveolin-1 in cell motility, invasiveness and aggregation, therefore, expanding our understanding of the tumor-promoting activities of caveolin-1 in advanced-stage cancer.

Ligand interaction scan (LIScan) in the study of ERK8.

ERK8 is the most recent addition for the MAPK family, and its mechanism of activation and function are not yet known, mainly due to the lack of any known physiological stimulator. In this report, we describe the preparation of reagents for the use of a novel method, the ligand interaction scan (LIScan), to study the function of this protein kinase. We generated a set of mutants of ERK8, and identified inhibited as well as stimulated forms. By specifically inhibiting or stimulating the mutants of ERK8, we show that the ERK8-induced inhibition of proliferation is altered. Moreover, we used the developed mutants to show for the first time that ERK8 translocates to the nucleus upon activation. The use of methods such as the ligand interaction scan may thus promote the analyses of the functions of uncharacterized proteins such as ERK8, and possibly help in controlling the activity of target proteins in various experimental systems and applications.

Interaction with MEK causes nuclear export and downregulation of peroxisome proliferator-activated receptor gamma.

The mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) cascade plays a central role in intracellular signaling by many extracellular stimuli. One target of the ERK cascade is peroxisome proliferator-activated receptor gamma (PPARgamma), a nuclear receptor that promotes differentiation and apoptosis. It was previously demonstrated that PPARgamma activity is attenuated upon mitogenic stimulation due to phosphorylation of its Ser84 by ERKs. Here we show that stimulation by tetradecanoyl phorbol acetate (TPA) attenuates PPARgamma's activity in a MEK-dependent manner, even when Ser84 is mutated to Ala. To elucidate the mechanism of attenuation, we found that PPARgamma directly interacts with MEKs, which are the activators of ERKs, but not with ERKs themselves, both in vivo and in vitro. This interaction is facilitated by MEKs' phosphorylation and is mediated by the basic D domain of MEK1 and the AF2 domain of PPARgamma. Immunofluorescence microscopy and subcellular fractionation revealed that MEK1 exports PPARgamma from the nucleus, and this finding was supported by small interfering RNA knockdown of MEK1 and use of a cell-permeable interaction-blocking peptide, which prevented TPA-induced export of PPARgamma from the nucleus. Thus, we show here a novel mode of downregulation of PPARgamma by its MEK-dependent redistribution from the nucleus to the cytosol. This unanticipated role for the stimulation-induced nuclear shuttling of MEKs shows that MEKs can regulate additional signaling components besides the ERK cascade.

Filamin A is a novel caveolin-1-dependent target in IGF-I-stimulated cancer cell migration.

Caveolin-1 is an essential structural constituent of caveolae which is involved in regulation of mitogenic signaling and oncogenesis. Caveolin-1 has been implicated in cell migration but its exact role and mechanism of action in this process remained obscure. We have previously reported that expression of caveolin-1 in stably transfected MCF-7 human breast cancer (MCF-7/Cav1) cells up-regulates phosphorylation of a putative Akt substrate protein, designated pp340 [D. Ravid, S. Maor, H. Werner, M. Liscovitch, Caveolin-1 inhibits cell detachment-induced p53 activation and anoikis by upregulation of insulin-like growth factor-I receptors and signaling, Oncogene 24 (2005) 1338-1347.]. We now show, using differential detergent extraction, SDS-PAGE and mass spectrometry, that the major protein in the pp340 band is the actin filament cross-linking protein filamin A. The identity of pp340 as filamin A was confirmed by immunoprecipitation of pp340 with specific filamin A antibodies. RT-PCR, flow cytometry and Western blot analyses show that filamin A mRNA and protein levels are respectively 3.5- and 2.5-fold higher in MCF-7/Cav1 cells than in MCF-7 cells. Basal filamin A phosphorylation on Ser-2152, normalized to total filamin A levels, is 7.8-fold higher in MCF-7/Cav1 than in MCF-7 cells. Insulin-like growth factor-I (IGF-I) stimulates phosphorylation of filamin A on Ser-2152 in MCF-7 cells and further enhances Ser-2152 phosphorylation over its already high basal level in MCF-7/Cav1 cells. The effect of IGF-I is inhibited by the PI3K inhibitor wortmannin, indicating that IGF-I-stimulated phosphorylation of filamin A occurs via the PI3K/Akt pathway. Co-immunoprecipitation experiments have confirmed a previous report showing that filamin A and caveolin-1 co-exist in a complex and have revealed the presence of active phospho-Akt in this complex. Ser-2152 phosphorylation of filamin A has been implicated in cancer cell migration. Accordingly, caveolin-1 expression dramatically enhances IGF-I-dependent MCF-7 cell migration. These data indicate that caveolin-1 specifies filamin A as a novel target for Akt-mediated filamin A Ser-2152 phosphorylation thus mediating the effects of caveolin-1 on IGF-I-induced cancer cell migration.

Rosiglitazone induces caveolin-1 by PPARgamma-dependent and PPRE-independent mechanisms: the role of EGF receptor signaling and its effect on cancer cell drug resistance.

BACKGROUND: Caveolin-1, a key component of plasma membrane caveolae, has been implicated in the regulation of cancer cell growth and survival. Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a ligand-activated nuclear receptor which plays a pivotal role in many cellular processes. Activation of PPARgamma by its ligand rosiglitazone upregulates caveolin-1 mRNA and protein in human carcinoma cells. MATERIALS AND METHODS: We have used specific signaling inhibitors to dissect the mechanisms of caveolin-1 mRNA and protein induction by rosiglitazone, determined by RT-PCR and Western blotting, respectively. ROS generation was measured by flow cytometry and cell survival was determined by the MTT assay. RESULTS: We show that in HT-29 human colon cancer cells the induction ofcaveolin-1 by rosiglitazone is inhibited by the EGF receptor (EGFR) blocker AG1478. Moreover, rosiglitazone stimulates EGFR phosphorylation, while direct activation of EGFR by EGF up-regulates caveolin-1 mRNA. Inhibitors of Src and the Mek1-Erk1/2 and p38 MAP kinase pathways also inhibit up-regulation of caveolin-1 by rosiglitazone. Furthermore, rosiglitazone stimulates formation of superoxide anions, whereas induction of caveolin-1 expression by rosiglitazone is attenuated by the antioxidant N-acetyl-cysteine. Finally, rosiglitazone increases the resistance of HT-29 cells to doxorubicin and to hydrogen peroxide. The caveolin-1 gene promoter lacks a canonical PPARgamma response element (PPRE) and a PPRE-reporter construct is not sensitive to EGF or EGFR inhibition. CONCLUSION: Our findings indicate that up-regulation of caveolin-1 by rosiglitazone requires superoxide formation and the activation of Src, EGFR, and the Mek1-Erk1/2 and p38 MAP kinase pathways. We suggest a novel mode of action of PPARgamma ligands in the regulation of caveolin-1, and possibly other genes devoid of a PPRE in their promoters, which involves the coordinate activation of PPARgamma and intracellular signaling pathways.

Caveolin-1: a tumor-promoting role in human cancer.

PURPOSE: Caveolae are non-clathrin, flask-shaped invaginations of the plasma membrane. Caveolin-1 is an essential constituent of caveolae and as such acts as a regulator of caveolae-dependent lipid trafficking and endocytosis. Caveolin-1 interacts with a variety of cellular proteins and regulates cell-signaling events. Caveolin-1 appears to act as a tumor suppressor protein at early stages of cancer progression. However, a growing body of evidence indicates that caveolin-1 is up-regulated in several multidrug-resistant and metastatic cancer cell lines and human tumor specimens. Furthermore, caveolin-1 levels are positively correlated with tumor stage and grade in numerous cancer types. CONCLUSION: The available experimental data support the tumor-promoting role of caveolin-1 in advanced-stage cancer.

A case study in misidentification of cancer cell lines: MCF-7/AdrR cells (re-designated NCI/ADR-RES) are derived from OVCAR-8 human ovarian carcinoma cells.

Multidrug-resistant MCF-7 breast adenocarcinoma cells (originally named MCF-7/AdrR cells and later re-designated NCI/ADR-RES) have served as an important and widely used research tool during the last two decades. However, the real identity of these cells has been in doubt since 1998 and has since been debated. The origin of NCI/ADR-RES cells has now been revealed by SNP and karyotypic analyses, carried out at the Sanger Institute and the NCI, respectively. The results of these analyses, recently posted on the Web, show that NCI/ADR-RES cells are derived from OVCAR-8 ovarian adenocarcinoma cells. The case of NCI/ADR-RES cells highlights a wide-spread problem of cell line cross-contamination and misidentification. Fortunately, this is a tractable problem that can be avoided by scrupulous genotyping of cell stocks and adoption of a few simple rules in cell culture practice.

Caveolin-1 inhibits cell detachment-induced p53 activation and anoikis by upregulation of insulin-like growth factor-I receptors and signaling.

Caveolin-1 is an essential structural constituent of caveolae that has been implicated in mitogenic signaling and oncogenesis. Utilizing MCF-7 human breast cancer cells, stably transfected with caveolin-1 (MCF-7/Cav1), we previously demonstrated that caveolin-1 expression decreases MCF-7 cell proliferation and colony formation in soft agar. However, the loss of anchorage-independent growth is associated with inhibition of anoikis, as MCF-7/Cav1 cells exhibit increased survival after detachment. Herein we show that this phenotype is associated with suppression of detachment-induced activation of p53 and of the consequent induction of cyclin-dependent kinase inhibitor p21(WAF1/Cip1). In contrast, activation of p53 and p21(WAF1/Cip1) induced by doxorubicin in MCF-7/Cav1 cells remains largely unaffected. The phenotypic changes observed in MCF-7/Cav1 cells are not accompanied by changes in caspase-6, -7, -8 and -9 and cannot be explained by changes in Bid and Bcl-2 expression. However, MCF-7/Cav1 cells exhibit a constitutively phosphorylated Akt kinase and at least one phosphorylated high molecular weight putative Akt substrate which we designated pp340. In addition, MCF-7/Cav1 cells exhibit elevated expression of insulin-like growth factor-I (IGF-I) receptor expression and increased IGF-I signaling to Erk1/2 and to Akt, as well as IGF-I-induced stimulation of pp340 phosphorylation. The addition of IGF-I to the medium rescues the parental MCF-7 cells from anoikis, indicating that IGF-1 can act as a survival factor for suspended MCF-7 cells. Finally, the levels of caveolin-1 are dramatically elevated in a time-dependent manner upon detachment of anoikis-resistant MCF-7/Cav1 cells and HT-29-MDR human multidrug resistant colon cancer cells. We conclude that expression of caveolin-1 in human breast cancer cells enhances matrix-independent cell survival that is mediated by upregulation of IGF-I receptor expression and signaling.

Caveolin-1 controls BRCA1 gene expression and cellular localization in human breast cancer cells.

The role of caveolae and the caveolin proteins in cancer has been the subject of extensive research. BRCA1 participates in multiple biological pathways including DNA damage repair, transcriptional control, cell growth, apoptosis, and others. Little information, however, is available regarding the cellular mechanisms that control BRCA1 gene expression. The present study examined the potential regulation of BRCA1 gene expression and subcellular localization by Cav-1. Results of Western blots, RT-PCR, and transfection experiments showed that Cav-1 enhances BRCA1 protein and mRNA levels via a mechanism that involves transactivation of the BRCA1 promoter and which is p53-dependent. In addition, immunostaining experiments demonstrate that Cav-1 induced the cytoplasmic sequestration of BRCA1.

Peroxisome proliferator-activated receptor-gamma upregulates caveolin-1 and caveolin-2 expression in human carcinoma cells.

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is a nuclear receptor for eicosanoids that promotes differentiation of human epithelial and mesenchymal cells in vitro and in vivo. PPARgamma was proposed as a target for drug-induced differentiation therapy of cancer. Caveolin-1 is a constituent of plasma membrane caveolae in epithelial cells that is often downregulated upon oncogenic transformation. Caveolin-1 has growth-inhibitory activities and its disruption is sufficient to induce transformation in fibroblasts. Herein we have tested the hypothesis that caveolins are transcriptional target genes for PPARgamma. In human HT-29 colon carcinoma cells, thiazolidinedione PPARgamma ligands increased the levels of caveolin-1 and caveolin-2 proteins two to fivefold in a concentration-dependent manner within 24 h. In human MCF-7 breast adenocarcinoma cells, nonthiazolidinedione PPARgamma ligands elevated caveolin-2 protein three to fourfold, while the thiazoli-dinediones were less effective. Caveolin-1 mRNA levels were found to be upregulated by PPARgamma ligands already after 3 h in both the cell lines. Ectopic expression of a dominant-negative PPARgamma construct attenuated ligand-induced upregulation of caveolins in both HT-29 and HEK-293T cells, indicating that ligand action is mediated by PPARgamma. Ligand-treated MCF-7 cells exhibited a differentiated phenotype, as evinced by analysis of cell-specific differentiation markers: protein levels of maspin were elevated and perinuclear lipid droplets accumulated. In contrast, in HT-29 cells, caveolin expression was not correlated with differentiation. Interestingly, PPARgamma partially cofractionated in lipid rafts and could be coimmunoprecipitated from cell lysates with caveolin-1, indicating that PPARgamma and caveolin-1 may coexist in a complex. Our data indicate that PPARgamma participates in the regulation of caveolin gene expression in human carcinoma cells and suggest that caveolin-1 may mediate some of the phenotypic changes induced by this nuclear receptor in cancer cells. These findings may have potentially important functional implications in the context of cancer differentiation therapy and multidrug resistance.

Caveolin-1 inhibits anchorage-independent growth, anoikis and invasiveness in MCF-7 human breast cancer cells.

Caveolin-1 is an essential structural constituent of caveolae that has been implicated in mitogenic signaling and oncogenesis. Caveolin-1 is down-regulated in oncogene-transformed and tumor-derived cells. Antisense suppression of caveolin-1 or expression of a dominant negative form are sufficient for inducing cellular transformation. Expression of recombinant caveolin-1 inhibits anchorage-independent growth in cancer cells. The present study was designed to determine whether this is caused by inhibition of cancer cell survival or cell proliferation, and to test if another important property of cancer cells, i.e. matrix invasion, is modulated by expression of caveolin. Utilizing MCF-7 human breast adenocarcinoma cells stably transfected with caveolin-1 (MCF-7/Cav1), we demonstrate that caveolin-1 expression decreases MCF-7 cell proliferation rate and markedly reduces their capacity to form colonies in soft agar. The loss of anchorage-independent growth is not associated with stimulation of anoikis; in fact, MCF-7/Cav1 cells exhibit increased survival after detachment as compared with MCF-7 cells, indicating that in these cells caveolin-1 inhibits anoikis. Analysis of matrix metalloprotease release and matrix invasion revealed that expression of caveolin-1 inhibits also these important metastasis-related phenomena. Plating MCF-7 cells on a laminin matrix resulted in activation of ERK1/2, which was dramatically inhibited in MCF-7/Cav1 cells. We conclude that high expression level of caveolin-1 in human breast cancer cells exerts a negative modulatory effect on anchorage-independent growth by inhibiting cell proliferation even though matrix-independent cell survival is enhanced. Caveolin-1 expression inhibits also matrix invasion and blocks laminin-dependent activation of ERK1/2. The inhibitory effect of caveolin-1 on these transformation-dependent processes supports the hypothesis that caveolin-1 acts as a tumor suppressor protein which may impose major phenotypic changes when expressed in human cancer cells.

Bypass of tumor drug resistance by antivascular therapy.

Multidrug resistance (MDR) presents a major obstacle for the successful chemotherapy of cancer. Its emergence during chemotherapy is attributed to a selective process, which gives a growth advantage to MDR cells within the genetically unstable neoplastic cell population. The pleiotropic nature of clinical MDR poses a great difficulty for the development of treatment strategies that aim at blocking MDR at the tumor cell level. Targeting treatment to the nonmalignant vascular network-the lifeline of the tumor-is a promising alternative for the treatment of drug-resistant tumors. The present study demonstrates that MDR in cancer can be successfully circumvented by photodynamic therapy (PDT) using an antivascular treatment protocol. We show that, although P-glycoprotein-expressing human HT29/MDR colon carcinoma cells in culture are resistant to PDT with Pd-bacteriopheophorbide (TOOKAD), the same treatment induces tumor necrosis with equal efficacy (88% vs 82%) in HT29/MDR-derived xenografts and their wild type counterparts, respectively. These results are ascribed to the rapid antivascular effects of the treatment, supporting the hypothesis that MDR tumors can be successfully eradicated by indirect approaches that bypass their inherent drug resistance. We suggest that with progress in ongoing clinical trials, TOOKAD-PDT may offer a novel option for local treatment of MDR tumors.

Cancer multidrug resistance: a review of recent drug discovery research.

Conventional cancer chemotherapy is seriously limited by tumor cells exhibiting multidrug resistance (MDR), caused by changes in the level or activity of membrane transporters that mediate energy-dependent drug efflux and of other proteins that affect drug metabolism and/or drug action. Many inhibitors of MDR transporters have been identified and some are undergoing clinical trials, but currently none are in clinical use. Here we briefly review the status of MDR drugs, outline novel approaches designed to suppress or circumvent MDR mechanisms and discuss the future of MDR therapy in oncology.

Caveats of caveolin-1 in cancer progression.

Caveolin-1, an essential scaffold protein of caveolae and cellular transport processes, lately gained recognition as a stage- and tissue-specific tumor modulator in vivo. Patient studies and rodent models corroborated its janus-faced role as a tumor suppressor in non-neoplastic tissue, its down-regulation (loss of function) upon transformation and its re-expression (regain of function) in advanced-stage metastatic and multidrug resistant tumors. This review is focussed on the role of caveolin-1 in metastasis and angiogenesis and its clinical implications as a prognostic marker in cancer progression.

Ligand interaction scan: a general method for engineering ligand-sensitive protein alleles.

The ligand interaction scan (LIScan) method is a general procedure for engineering small molecule ligand-regulated forms of a protein that is complementary to other 'reverse' genetic and chemical-genetic methods for drug-target validation. It involves insertional mutagenesis by a chemical-genetic 'switch', comprising a genetically encoded peptide module that binds with high affinity to a small-molecule ligand. We demonstrated the method with TEM-1 beta-lactamase, using a tetracysteine hexapeptide insert and a biarsenical fluorescein ligand (FlAsH).

Caveolin-1 up-regulates IGF-I receptor gene transcription in breast cancer cells via Sp1- and p53-dependent pathways.

The insulin-like growth factor (IGF) system plays an important role in the biology of breast cancer. Most of the biological actions of IGF-I and IGF-II are mediated by the IGF-I receptor (IGF-IR), a membrane-bound heterotetramer with potent antiapoptotic and cell survival activities. Caveolin-1 (Cav-1) is one of the main components of caveolae, and it has been shown to interact with multiple signaling molecules. In view of the important roles of IGF-IR and Cav-1 in oncogenically transformed mammary gland cells, in the present study we addressed the potential regulation of IGF-IR gene expression by Cav-1. The results obtained showed that MCF7/Cav-1 cells, expressing the Cav-1 gene in a stable manner, contain significantly higher levels of IGF-IR protein and mRNA than native MCF7 cells. These elevated levels of expression are mediated at the level of transcription, as shown by the results of experiments showing that the activity of the proximal IGF-IR promoter was higher in Cav-1-expressing MCF7 cells than in untransfected MCF7 cells. Furthermore, in subcellular localization studies, intensive IGF-IR staining in membrane ruffles and projections in MCF7/Cav-1 cells were noted, in contrast to typical membrane staining in MCF7 cells. In addition, we demonstrated that transcriptional activation of the IGF-IR gene by Cav-1 requires an intact p53 signaling pathway, since Cav-1 was unable to elevate IGF-IR levels in p53-null cells. Finally, the effect of Cav-1 was associated with an elevation in the levels of Sp1, a zinc-finger protein with important roles in IGF-IR gene transactivation. In summary, we identified the IGF-IR gene as a downstream target for Cav-1 action in breast cancer cells.

Characterization and regulation of yeast Ca2+-dependent phosphatidylethanolamine-phospholipase D activity.

An unconventional phospholipase D (PLD) activity was identified recently in Saccharomyces cerevisiae which is Ca2+-dependent, preferentially hydrolyses phosphatidylethanolamine (PtdEtn) and phosphatidylserine and does not catalyse a transphosphatidylation with primary short-chain alcohols. We have characterized the cytosolic and membrane-bound forms of the yeast PtdEtn-PLD and examined the regulation of its activity under certain growth, nutritional and stress conditions. Both forms of PtdEtn-PLD activity were similarly activated by Ca2+ ions in a biphasic manner. Likewise, other divalent cations affected both cytosolic and membrane-bound forms to the same extent. The yeast PtdEtn-PLD activity was found to interact with immobilized PtdEtn in a Ca2+-dependent manner. The partially purified cytosolic form and the salt-extracted membrane-bound form of yeast PtdEtn-PLD exhibited a similar elution pattern on size-exclusion chromatography, coeluting as low apparent molecular weight peaks. PtdEtn-PLD activity was stimulated, along with Spo14p/Pld1p activity, upon dilution of stationary phase cultures in glucose, acetate and galactose media, but PtdEtn-PLD activation was less pronounced. Interestingly, PtdEtn-PLD activity was found to be elevated by approximately 40% in sec14ts mutants at the restrictive temperature, whereas in other sec mutants it remained unaffected. The activity of PtdEtn-PLD was reduced by 30-40% upon addition to the medium of inositol (75 micro m) in either wild-type yeast or spo14Delta mutants and this effect was seen regardless of the presence of choline, suggesting that transcription of the PtdEtn-PLD gene is down-regulated by inositol. Finally, exposure of yeast cells to H2O2 resulted in a transient increase in PtdEtn-PLD activity followed by a profound, nearly 90% decrease in activity. In conclusion, our results indicate that yeast PtdEtn-PLD activity is highly regulated: the enzyme is acutely activated upon entry into the cell cycle and following inactivation of sec14ts, and is inhibited under oxidative stress conditions. The implications of these findings are discussed.