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Background: Elevated expression of CAV1 in breast cancer increases tumor progression. Extracellular vesicles (EVs) from CAV1-expressing MDA-MB-231 breast cancer cells contain Tenascin C (TNC), but the relevance of TNC remained to be defined. Methods: EVs were characterized by nanotracking analysis, microscopy and western blotting. The uptake of EVs by cells was studied using flow cytometry. The effects of EVs on breast cancer cells were tested in migration, invasion, colony formation and in vivo assays. Results: EVs were taken up by cells; however, only those containing TNC promoted invasiveness. In vivo , EVs lacking TNC ceased to promote tumor growth. Conclusion: CAV1 and TNC contained in breast cancer cell-derived EVs were identified as proteins that favor progression of breast cancer.

Breast cancer is one of the most frequently diagnosed cancers and the leading cause of cancer-related deaths in women worldwide, whereby mortality is largely attributable to the development of distant metastasis. Caveolin-1 (CAV1) is a multifunctional membrane protein that is typically upregulated in the final stages of cancer and promotes migration and invasion of tumor cells. Elevated levels of CAV1 have been detected in extracellular vesicles (EVs) from advanced cancer patients. EVs are lipid enclosed vesicular structures that contain bioactive proteins, DNA and RNAs, which can be transferred to other cells and promote metastasis. Therefore, we hypothesized that CAV1 containing EVs released from breast cancer cells may enhance migration and invasion of recipient cells. EVs were purified from conditioned media of MDA-MB-231 wild-type (WT), MDA-MB-231 (shCAV1; possessing the plasmid pLKO.1 encoding a 'small hairpin' directed against CAV1) and MDA-MB-231 (shC) short hairpin control cells. Nanoparticle tracking analysis revealed an average particle size of 40-350 nm for all preparations. As anticipated, CAV1 was detected in MDA-MB-231 WT and shC EVs, but not in MDA-MB-231 (shCAV1) EVs. Mass spectrometry analysis revealed the presence of specific cell adhesion-related proteins, such as Cyr61, tenascin (TNC) and S100A9 only in WT and shC, but not in shCAV1 EVs. Importantly, EVs containing CAV1 promoted migration and invasion of cells lacking CAV1. We conclude that the presence of CAV1 in EVs from metastatic breast cancer cells is associated with enhanced migration and invasiveness of recipient cells in vitro, suggesting that intercellular communication promoted by EVs containing CAV1 will likely favor metastasis in vivo.

Aim: To track early events during lung metastasis, we labeled cells expressing (B16F10 CAV1 ) or lacking CAV1 (B16F10 mock ) with gold nanoparticles conjugated to the peptide TAT (AuNPs-PEG-TAT)., Methods: B16F10 expressing or lacking CAV1 were labeled with AuNPs-PEG-TAT. The physicochemical properties and cytotoxicity of these nanoparticles, as well as their effects on migration and invasiveness of B16F10 cells in vitro were evaluated. Ex vivo lung distribution of the labeled cells after tail vein injection into C57BL/6 mice was examined., Results: AuNPs-PEG-TAT did not affect B16F10 viability, migration and invasiveness. The metastatic and tumorigenic capability of the labeled B16F10 was also not modified in comparison to unlabeled B16F10 cells. CAV1 expression favored the retention of B16F10 cells in the lungs of mice 2 h post injection, suggesting CAV1 promoted adherence to endothelial cells and transendothelial migration., Conclusions: We developed a protocol to label B16F10 cells with AuNPs-PEG-TAT that permits subsequent tracking of cells in mice. CAV1 overexpression was found to increase retention and transendothelial migration of B16F10 cells in the lung.

Caveolin-1 (CAV1) is a scaffolding protein that plays a dual role in cancer. In advanced stages of this disease, CAV1 expression in tumor cells is associated with enhanced metastatic potential, while, at earlier stages, CAV1 functions as a tumor suppressor. We recently implicated CAV1 phosphorylation on tyrosine 14 (Y14) in CAV1-enhanced cell migration. However, the contribution of this modification to the dual role of CAV1 in cancer remained unexplored. Here, we used in vitro [2D and transendothelial cell migration (TEM), invasion] and in vivo (metastasis) assays, as well as genetic and biochemical approaches to address this question in B16F10 murine melanoma cells. CAV1 promoted directional migration on fibronectin or laminin, two abundant lung extracellular matrix (ECM) components, which correlated with enhanced Y14 phosphorylation during spreading. Moreover, CAV1-driven migration, invasion, TEM and metastasis were ablated by expression of the phosphorylation null CAV1(Y14F), but not the phosphorylation mimicking CAV1(Y14E) mutation. Finally, CAV1-enhanced focal adhesion dynamics and surface expression of beta1 integrin were required for CAV1-driven TEM. Importantly, CAV1 function as a tumor suppressor in tumor formation assays was not altered by the Y14F mutation. In conclusion, our results provide critical insight to the mechanisms of CAV1 action during cancer development. Specific ECM-integrin interactions and Y14 phosphorylation are required for CAV1-enhanced melanoma cell migration, invasion and metastasis to the lung. Because Y14F mutation diminishes metastasis without inhibiting the tumor suppressor function of CAV1, Y14 phosphorylation emerges as an attractive therapeutic target to prevent metastasis without altering beneficial traits of CAV1., Competing Interests: The authors declare no conflicts of interest.

Background: Caveolin-1 (CAV1) has been implicated both in tumor suppression and progression, whereby the specific role appears to be context dependent. Endometrial cancer is one of the most common malignancies of the female genital tract; however, little is known about the role of CAV1 in this disease., Methods: Here, we first determined by immunohistochemistry CAV1 protein levels in normal proliferative human endometrium and endometrial tumor samples. Then using two endometrial cancer cell lines (ECC: Ishikawa and Hec-1A) we evaluated mRNA and protein levels of CAV1 by real time qPCR and Western blot analysis, respectively. The role of CAV1 expression in ECC malignancy was further studied by either inducing its expression in endometrial cancer cells with the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate (4β-TPA) or decreasing expression using short-hairpin RNA constructs, and then evaluating the effects of these changes on ECC proliferation, transmigration, matrigel invasion, and colony formation in soft agar., Results: Immunohistochemical analysis of endometrial epithelia revealed that substantially higher levels of CAV1 were present in endometrial tumors than the normal proliferative epithelium. Also, in Ishikawa and Hec-1A endometrial cancer cells CAV1 expression was readily detectable. Upon treatment with 4β-TPA CAV1 levels increased and coincided with augmented cell transmigration, matrigel invasion, as well as colony formation in soft agar. Reduction of CAV1 expression using short-hairpin RNA constructs ablated these effects in both cell types whether treated or not with 4β-TPA. Alternatively, CAV1 expression appeared not to modulate significantly proliferation of these cells., Conclusion: Our study shows that elevated CAV1, observed in patients with endometrial cancer, is linked to enhanced malignancy of endometrial cancer cells, as evidenced by increased migration, invasion and anchorage-independent growth.

A considerable body of evidence exists implicating high levels of free saturated fatty acids in beta pancreatic cell death, although the molecular mechanisms and the signaling pathways involved have not been clearly defined. The membrane protein caveolin-1 has long been implicated in cell death, either by sensitizing to or directly inducing apoptosis and it is normally expressed in beta cells. Here, we tested whether the presence of caveolin-1 modulates free fatty acid-induced beta cell death by reexpressing this protein in MIN6 murine beta cells lacking caveolin-1. Incubation of MIN6 with palmitate, but not oleate, induced apoptotic cell death that was enhanced by the presence of caveolin-1. Moreover, palmitate induced de novo ceramide synthesis, loss of mitochondrial transmembrane potential and reactive oxygen species (ROS) formation in MIN6 cells. ROS generation promoted caveolin-1 phosphorylation on tyrosine-14 that was abrogated by the anti-oxidant N-acetylcysteine or the incubation with the Src-family kinase inhibitor, PP2 (4-amino-5-(4-chlorophenyl)-7(dimethylethyl)pyrazolo[3,4-d]pyrimidine). The expression of a non-phosphorylatable caveolin-1 tyrosine-14 to phenylalanine mutant failed to enhance palmitate-induced apoptosis while for MIN6 cells expressing the phospho-mimetic tyrosine-14 to glutamic acid mutant caveolin-1 palmitate sensitivity was comparable to that observed for MIN6 cells expressing wild type caveolin-1. Thus, caveolin-1 expression promotes palmitate-induced ROS-dependent apoptosis in MIN6 cells in a manner requiring Src family kinase mediated tyrosine-14 phosphorylation., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Rab5 is a small GTPase that regulates early endosome trafficking and other cellular processes, including cell adhesion and migration. Specifically, Rab5 promotes Rac1 activation and cancer cell migration, but little is known about the upstream regulators of Rab5. We have previously shown that the scaffolding protein Caveolin-1 (CAV1) promotes Rac1 activation and migration of cancer cells. Here, we hypothesized that CAV1 stimulates Rab5 activation, leading to increased Rac1 activity and cell migration. Expression of CAV1 in B16-F10 mouse melanoma and HT-29(US) human colon adenocarcinoma cells increased the GTP loading of Rab5, whereas shRNA-mediated targeting of endogenous CAV1 in MDA-MB-231 breast cancer cells decreased Rab5-GTP levels. Accordingly, shRNA-mediated downregulation of Rab5 decreased CAV1-mediated Rac1 activation, cell migration and invasion in B16-F10 and HT-29(US) cells. Expression of CAV1 was accompanied by increased recruitment of Tiam1, a Rac1 guanine nucleotide exchange factor (GEF), to Rab5-positive early endosomes. Using the inhibitor NSC23766, Tiam1 was shown to be required for Rac1 activation and cell migration induced by CAV1 and Rab5. Mechanistically, we provide evidence implicating p85α (also known as PIK3R1), a Rab5 GTPase-activating protein (GAP), in CAV1-dependent effects, by showing that CAV1 recruits p85α, precluding p85α-mediated Rab5 inactivation and increasing cell migration. In summary, these studies identify a novel CAV1-Rab5-Rac1 signaling axis, whereby CAV1 prevents Rab5 inactivation, leading to increased Rac1 activity and enhanced tumor cell migration and invasion., (© 2014. Published by The Company of Biologists Ltd.)

Melanomas are highly lethal skin tumours that are frequently treated by surgical resection. However, the efficacy of such procedures is often limited by tumour recurrence and metastasis. Caveolin-1 (CAV1) has been attributed roles as a tumour suppressor, although in late-stage tumours, its presence is associated with enhanced metastasis. The expression of this protein in human melanoma development and particularly how the presence of CAV1 affects metastasis after surgery has not been defined. CAV1 expression in human melanocytes and melanomas increases with disease progression and is highest in metastatic melanomas. The effect of increased CAV1 expression can then be evaluated using B16F10 murine melanoma cells injected into syngenic immunocompetent C57BL/6 mice or human A375 melanoma cells injected into immunodeficient B6Rag1-/- mice. Augmented CAV1 expression suppresses tumour formation upon a subcutaneous injection, but enhances lung metastasis of cells injected into the tail vein in both models. A procedure was initially developed using B16F10 melanoma cells in C57BL/6 mice to mimic better the situation in patients undergoing surgery. Subcutaneous tumours of a defined size were removed surgically and local tumour recurrence and lung metastasis were evaluated after another 14 days. In this postsurgery setting, CAV1 presence in B16F10 melanomas favoured metastasis to the lung, although tumour suppression at the initial site was still evident. Similar results were obtained when evaluating A375 cells in B6Rag1-/- mice. These results implicate CAV1 expression in melanomas as a marker of poor prognosis for patients undergoing surgery as CAV1 expression promotes experimental lung metastasis in two different preclinical models.

Caveolin-1 is a member of the caveolin family that has been ascribed a dual role in cancer. In early stages of disease the protein functions predominantly as a tumor suppressor, whereas at later stages, caveolin-1 expression is associated with tumor progression and metastasis. Here, some mechanisms associated with caveolin-1-dependent tumor suppression will be briefly discussed before focusing on the role of this protein and particularly phosphorylation of tyrosine-14 in promoting cell migration, invasion and metastasis. Models are provided summarizing possible explanations for these dramatic changes in function, as well as mechanisms by which this may be achieved.

The role of caveolin-1 (CAV1) in cancer is highly controversial. CAV1 suppresses genes that favor tumor development, yet also promotes focal adhesion turnover and migration of metastatic cells. How these contrasting observations relate to CAV1 function in vivo is unclear. Our previous studies implicate E-cadherin in CAV1-dependent tumor suppression. Here, we use murine melanoma B16F10 cells, with low levels of endogenous CAV1 and E-cadherin, to unravel how CAV1 affects tumor growth and metastasis and to assess how co-expression of E-cadherin modulates CAV1 function in vivo in C57BL/6 mice. We find that overexpression of CAV1 in B16F10 (cav-1) cells reduces subcutaneous tumor formation, but enhances metastasis relative to control cells. Furthermore, E-cadherin expression in B16F10 (E-cad) cells reduces subcutaneous tumor formation and lung metastasis when intravenously injected. Importantly, co-expression of CAV1 and E-cadherin in B16F10 (cav-1/E-cad) cells abolishes tumor formation, lung metastasis, increased Rac-1 activity, and cell migration observed with B16F10 (cav-1) cells. Finally, consistent with the notion that CAV1 participates in switching human melanomas to a more malignant phenotype, elevated levels of CAV1 expression correlated with enhanced migration and Rac-1 activation in these cells., (© 2013 John Wiley & Sons A/S.)

Endometrial tissue of patients with polycystic ovary syndrome (PCOS) shows an impaired expression of insulin signaling molecules. Tyrosine phosphorylation of the insulin receptor (IR) by insulin promotes glucose uptake by activating the PI3K/Akt pathway. IR stability and function depend on the presence of the protein caveolin-1. Activation of IR increases phosphorylation of Y¹⁴caveolin-1. Since the endometrium of PCOS patients is proposed to be insulin resistant, we evaluated the phosphorylation of IR and caveolin-1 in endometria of patients with insulin resistance (PCOSE-IR) compared to controls (CE). To explore the mechanism associated with this condition, cultured endometrial cells (T-HESC) were exposed to high glucose (25 mM, 24 h), an experimental condition that leads to insulin resistance in other cell types. Endometrial protein levels of phospho-Y⁹⁷²IR, phospho-Y¹⁴caveolin-1 and caveolin-1 were determined by Western blotting. In cultured cells, protein levels of caveolin-1, IR, and Akt were evaluated by Western blotting. After acute insulin stimulation, phospho-S⁴⁷³Akt, phospho-Y¹⁴caveolin-1, and 2-deoxyglucose (2-DOG) uptake were determined. PCOSE-IR samples showed high protein levels of caveolin-1, but reduced phospho-Y¹⁴caveolin-1 compared to CE. No differences were observed for phospho-Y⁹⁷²IR between both groups. Cells pretreated with glucose showed a reduction in protein levels of IR and caveolin-1 and were unable to increase 2-DOG uptake, phospho-S⁴⁷³Akt and phospho-Y¹⁴caveolin-1 after insulin stimulation. In conclusion, in PCOSE-IR the impaired phosphorylation of IR downstream molecules such as phospho-Y¹⁴caveolin-1 suggests a diminished insulin sensitivity in endometria, condition that could be supported in vitro by the ability of T-HESCs to become insulin resistant when they are exposed to high glucose., (© Georg Thieme Verlag KG Stuttgart · New York.)

Caveolins are a family of membrane proteins required for the formation of small plasma membrane invaginations called caveolae that are implicated in cellular trafficking processes. In addition to this structural role, these scaffolding proteins modulate numerous intracellular signaling pathways; often via direct interaction with specific binding partners. Caveolin-1 is particularly well-studied in this respect and has been attributed a large variety of functions. Thus, Caveolin-1 also represents the best-characterized isoform of this family with respect to its participation in cancer. Rather strikingly, available evidence indicates that Caveolin-1 belongs to a select group of proteins that function, depending on the cellular settings, both as tumor suppressor and promoter of cellular traits commonly associated with enhanced malignant behavior, such as metastasis and multi-drug resistance. The mechanisms underlying such ambiguity in Caveolin-1 function constitute an area of great interest. Here, we will focus on discussing how Caveolin-1 modulates cell death and survival pathways and how this may contribute to a better understanding of the ambiguous role this protein plays in cancer.

Caveolin-1 is known to promote cell migration, and increased caveolin-1 expression is associated with tumor progression and metastasis. In fibroblasts, caveolin-1 polarization and phosphorylation of tyrosine-14 are essential to promote migration. However, the role of caveolin-1 in migration of metastatic cells remains poorly defined. Here, caveolin-1 participation in metastatic cell migration was evaluated by shRNA targeting of endogenous caveolin-1 in MDA-MB-231 human breast cancer cells and ectopic expression in B16-F10 mouse melanoma cells. Depletion of caveolin-1 in MDA-MB-231 cells reduced, while expression in B16-F10 cells promoted migration, polarization and focal adhesion turnover in a sequence of events that involved phosphorylation of tyrosine-14 and Rac-1 activation. In B16-F10 cells, expression of a non-phosphorylatable tyrosine-14 to phenylalanine mutant failed to recapitulate the effects observed with wild-type caveolin-1. Alternatively, treatment of MDA-MB-231 cells with the Src family kinase inhibitor PP2 reduced caveolin-1 phosphorylation on tyrosine-14 and cell migration. Surprisingly, unlike for fibroblasts, caveolin-1 polarization and re-localization to the trailing edge were not observed in migrating metastatic cells. Thus, expression and phosphorylation, but not polarization of caveolin-1 favor the highly mobile phenotype of metastatic cells.

Augmented expression of cyclooxygenase-2 (COX-2) and enhanced production of prostaglandin E(2) (PGE(2)) are associated with increased tumor cell survival and malignancy. Caveolin-1 is a scaffold protein that has been proposed to function as a tumor suppressor in human cancer cells, although mechanisms underlying this ability remain controversial. Intriguingly, the possibility that caveolin-1 regulates the expression of COX-2 has not been explored. Here we show that augmented caveolin-1 expression in cells with low basal levels of this protein, such as human colon cancer (HT29, DLD-1), breast cancer (ZR75), and embryonic kidney (HEK293T) cells reduced COX-2 mRNA and protein levels and beta-catenin-Tcf/Lef and COX-2 gene reporter activity, as well as the production of PGE(2) and cell proliferation. Moreover, COX-2 overexpression or PGE(2) supplementation increased levels of the inhibitor of apoptosis protein survivin by a transcriptional mechanism, as determined by PCR analysis, survivin gene reporter assays and Western blotting. Furthermore, addition of PGE(2) to the medium prevented effects attributed to caveolin-1-mediated inhibition of beta-catenin-Tcf/Lef-dependent transcription. Finally, PGE(2) reduced the coimmunoprecipitation of caveolin-1 with beta-catenin and their colocalization at the plasma membrane. Thus, by reducing COX-2 expression, caveolin-1 interrupts a feedback amplification loop involving PGE(2)-induced signaling events linked to beta-catenin/Tcf/Lef-dependent transcription of tumor survival genes including cox-2 itself and survivin.

Caveolae are small plasma membrane invaginations that have been implicated in a variety of functions including transcytosis, potocytosis and cholesterol transport and signal transduction. The major protein component of this compartment is a family of proteins called caveolins. Experimental data obtained in knockout mice have provided unequivocal evidence for a requirement of caveolins to generate morphologically detectable caveolae structures. However, expression of caveolins is not sufficient per seto assure the presence of these structures. With respect to other roles attributed to caveolins in the regulation of cellular function, insights are even less clear. Here we will consider, more specifically, the data concerning the ambiguous roles ascribed to caveolin-1 in signal transduction and cancer. In particular, evidence indicating that caveolin-1 function is cell context dependent will be discussed.

Caveolin-1 reportedly acts as a tumor suppressor and promotes events associated with tumor progression, including metastasis. The molecular mechanisms underlying such radical differences in function are not understood. Recently, we showed that caveolin-1 inhibits expression of the inhibitor of apoptosis protein survivin via a transcriptional mechanism involving the beta-catenin-Tcf/Lef pathway. Surprisingly, while caveolin-1 expression decreased survivin mRNA and protein levels in HT29(ATCC) human colon cancer cells, this was not the case in metastatic HT29(US) cells. Survivin down-regulation was paralleled by coimmunoprecipitation and colocalization of caveolin-1 with beta-catenin in HT29(ATCC) but not HT29(US) cells. Unlike HT29(ATCC) cells, HT29(US) cells expressed small amounts of E-cadherin that accumulated in intracellular patches rather than at the cell surface. Re-expression of E-cadherin in HT29(US) cells restored the ability of caveolin-1 to down-regulate beta-catenin-Tcf/Lef-dependent transcription and survivin expression, as seen in HT29(ATCC) cells. In addition, coimmunoprecipitation and colocalization between caveolin-1 and beta-catenin increased upon E-cadherin expression in HT29(US) cells. In human embryonic kidney HEK293T and HT29(US) cells, caveolin-1 and E-cadherin cooperated in suppressing beta-catenin-Tcf/Lef-dependent transcription as well as survivin expression. Finally, mouse melanoma B16-F10 cells, another metastatic cell model with low endogenous caveolin-1 and E-cadherin levels, were characterized. In these cells, caveolin-1-mediated down-regulation of survivin in the presence of E-cadherin coincided with increased apoptosis. Thus, the absence of E-cadherin severely compromises the ability of caveolin-1 to develop activities potentially relevant to its role as a tumor suppressor.

Caveolin-1 is suggested to act as a tumor suppressor. We tested the hypothesis that caveolin-1 does so by repression of survivin, an Inhibitor of apoptosis protein that regulates cell-cycle progression as well as apoptosis and is commonly overexpressed in human cancers. Ectopic expression of caveolin-1 in HEK293T and ZR75 cells or siRNA-mediated silencing of caveolin-1 in NIH3T3 cells caused downregulation or upregulation of survivin mRNA and protein, respectively. Survivin downregulation in HEK293T cells was paralleled by reduced cell proliferation, increases in G0-G1 and decreases in G2-M phase of the cell cycle. In addition, apoptosis was evident, as judged by several criteria. Importantly, expression of green fluorescent protein-survivin in caveolin-1-transfected HEK293T cells restored cell proliferation and viability. In addition, expression of caveolin-1 inhibited transcriptional activity of a survivin promoter construct in a beta-catenin-Tcf/Lef-dependent manner. Furthermore, in HEK293T cells caveolin-1 associated with beta-catenin and inhibited Tcf/Lef-dependent transcription. Similar results were obtained upon caveolin-1 expression in DLD1 cells, where APC mutation leads to constitutive activation of beta-catenin-Tcf/Lef-mediated transcription of survivin. Taken together, these results suggest that anti-proliferative and pro-apoptotic properties of caveolin-1 may be attributed to reduced survivin expression via a mechanism involving diminished beta-catenin-Tcf/Lef-dependent transcription.