Early in cancer development, tumour cells express vascular endothelial growth factor (VEGF), a secreted molecule that is important in all stages of angiogenesis, an essential process that provides nutrients and oxygen to the nascent tumor and thereby enhances tumor-cell survival and facilitates growth. Survivin, another protein involved in angiogenesis, is strongly expressed in most human cancers, where it promotes tumor survival by reducing apoptosis as well as favoring endothelial cell proliferation and migration. The mechanisms by which cancer cells induce VEGF expression and angiogenesis upon survivin up-regulation remain to be fully established. Since the PI3K/Akt signalling and β-catenin-Tcf/Lef dependent transcription have been implicated in the expression of many cancer-related genes, including survivin and VEGF, we evaluated whether survivin may favor VEGF expression, release from tumor cells and induction of angiogenesis in a PI3K/Akt-β-catenin-Tcf/Lef-dependent manner. Here, we provide evidence linking survivin expression in tumor cells to increased β-catenin protein levels, β-catenin-Tcf/Lef transcriptional activity and expression of several target genes of this pathway, including survivin and VEGF, which accumulates in the culture medium. Alternatively, survivin downregulation reduced β-catenin protein levels and β-catenin-Tcf/Lef transcriptional activity. Also, using inhibitors of PI3K and the expression of dominant negative Akt, we show that survivin acts upstream in an amplification loop to promote VEGF expression. Moreover, survivin knock-down in B16F10 murine melanoma cells diminished the number of blood vessels and reduced VEGF expression in tumors formed in C57BL/6 mice. Finally, in the chick chorioallantoid membrane assay, survivin expression in tumor cells enhanced VEGF liberation and blood vessel formation. Importantly, the presence of neutralizing anti-VEGF antibodies precluded survivin-enhanced angiogenesis in this assay. These findings provide evidence for the existance of a posititve feedback loop connecting survivin expression in tumor cells to PI3K/Akt enhanced β-catenin-Tcf/Lef-dependent transcription followed by secretion of VEGF and angiogenesis.

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 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 Rab5-GTP loading, whereas shRNA-mediated targeting of endogenous CAV1 in MDA-MB-231 breast cancer cells decreased Rab5-GTP levels. Accordingly, shRNA-mediated down-regulation of Rab5 decreased Rac1 activation, cell migration and invasion due to CAV1 in B16-F10 and HT-29(US) cells. Expression of CAV1 was accompanied by increased recruitment of the Rac1-GEF Tiam1 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 the Rab5-GAP p85α in CAV1-dependent effects, by showing that CAV1 recruits p85α, precluding 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.

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.

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.

Close contacts between endoplasmic reticulum and mitochondria enable reciprocal Ca²⁺ exchange, a key mechanism in the regulation of mitochondrial bioenergetics. During the early phase of endoplasmic reticulum stress, this inter-organellar communication increases as an adaptive mechanism to ensure cell survival. The signalling pathways governing this response, however, have not been characterized. Here we show that caveolin-1 localizes to the endoplasmic reticulum–mitochondria interface, where it impairs the remodelling of endoplasmic reticulum–mitochondria contacts, quenching Ca²⁺ transfer and rendering mitochondrial bioenergetics unresponsive to endoplasmic reticulum stress. Protein kinase A, in contrast, promotes endoplasmic reticulum and mitochondria remodelling and communication during endoplasmic reticulum stress to promote organelle dynamics and Ca²⁺ transfer as well as enhance mitochondrial bioenergetics during the adaptive response. Importantly, caveolin-1 expression reduces protein kinase A signalling, as evidenced by impaired phosphorylation and alterations in organelle distribution of the GTPase dynamin-related protein 1, thereby enhancing cell death in response to endoplasmic reticulum stress. In conclusion, caveolin-1 precludes stress-induced protein kinase A-dependent remodelling of endoplasmic reticulum–mitochondria communication.