Caveolin-1 function at the plasma membrane and in intracellular compartments in cancer.

Caveolin-1 (CAV1) is commonly considered to function as a cell surface protein, for instance in the genesis of caveolae. Nonetheless, it is also present in many intracellular organelles and compartments. The contributions of these intracellular pools to CAV1 function are generally less well understood, and this is also the case in the context of cancer. This review will summarize literature available on the role of CAV1 in cancer, highlighting particularly our understanding of the canonical (CAV1 in the plasma membrane) and non-canonical pathways (CAV1 in organelles and exosomes) linked to the dual role of the protein as a tumor suppressor and promoter of metastasis. With this in mind, we will focus on recently emerging concepts linking CAV1 function to the regulation of intracellular organelle communication within the same cell where CAV1 is expressed. However, we now know that CAV1 can be released from cells in exosomes and generate systemic effects. Thus, we will also elaborate on how CAV1 participates in intracellular communication between organelles as well as signaling between cells (non-canonical pathways) in cancer.

Hypoxia-Induced Caveolin-1 Expression Promotes Migration and Invasion of Tumor Cells.

BACKGROUND: Exacerbated proliferation of cancer cells in nascent tumors leads to the genesis of a hypoxic microenvironment, which is associated with poor patient prognosis, because these stress conditions enhance migratory, invasive and metastatic capacities of tumor cells. These changes are associated with the induction of the hypoxia-inducible factors (HIFs, mainly HIF1α) and increased expression of target genes, including Caveolin-1 (CAV1). Results from our group have shown that CAV1 expression in metastatic cancer cells promotes cell migration/invasion in vitro and metastasis in vivo in a manner dependent on tyrosine-14 phosphorylation by src family kinases. Here, we evaluated whether hypoxia-induced expression of CAV1 was required for hypoxia-dependent migration and invasion in cancer cells. METHODS: B16-F10 murine melanoma and HT29(US) colon adenocarcinoma cells were exposed to hypoxia (1% O2). CAV1 expression was evaluated by western blotting. Endogenous CAV1 and HIF1α were knocked-down using different shRNA constructs. Cell migration and invasion were evaluated in Boyden Chamber and Matrigel assays, respectively. RESULTS: We observed that hypoxia increased CAV1 protein levels in a HIF1 α- dependent manner, in B16-F10 and HT29(US) cells. Importantly, hypoxia-dependent migration of both tumor cell lines was blocked upon CAV1 knock-down. Likewise, pharmacological inhibition of HIF prevented hypoxia-induced migration and invasion in B16-F10 cells. Finally, hypoxia-induced migration was also blocked by the src-family kinase inhibitor 4-amino-5-(4-chloro-phenyl)-7-(t-butyl) pyrazolo3,4-dpyrimidine (PP2), an inhibitor of CAV1 phosphorylation. CONCLUSION: Hypoxia induced migration and invasion of metastatic cancer cells require HIF1α-dependent induction of CAV1 expression and src family kinase activation.

Astrocyte-to-neuron communication through integrin-engaged Thy-1/CBP/Csk/Src complex triggers neurite retraction via the RhoA/ROCK pathway.

Two key proteins for cellular communication between astrocytes and neurons are αvß3 integrin and the receptor Thy-1. Binding of these molecules in the same (cis) or on adjacent (trans) cellular membranes induces Thy-1 clustering, triggering actin cytoskeleton remodeling. Molecular events that could explain how the Thy-1-αvß3 integrin interaction signals have only been studied separately in different cell types, and the detailed transcellular communication and signal transduction pathways involved in neuronal cytoskeleton remodeling remain unresolved. Using biochemical and genetic approaches, single-molecule tracking, and high-resolution nanoscopy, we provide evidence that upon binding to αvß3 integrin, Thy-1 mobility decreased while Thy-1 nanocluster size increased. This occurred concomitantly with inactivation and exclusion of the non-receptor tyrosine kinase Src from the Thy-1/C-terminal Src kinase (Csk)-binding protein (CBP)/Csk complex. The Src inactivation decreased the p190Rho GTPase activating protein phosphorylation, promoting RhoA activation, cofilin, and myosin light chain II phosphorylation and, consequently, neurite shortening. Finally, silencing the adaptor CBP demonstrated that this protein was a key transducer in the Thy-1 signaling cascade. In conclusion, these data support the hypothesis that the Thy-1-CBP-Csk-Src-RhoA-ROCK axis transmitted signals from astrocytic integrin-engaged Thy-1 (trans) to the neuronal actin cytoskeleton. Importantly, the ß3 integrin in neurons (cis) was not found to be crucial for neurite shortening. This is the first study to detail the signaling pathway triggered by αvß3, the endogenous Thy-1 ligand, highlighting the role of membrane-bound integrins as trans acting ligands in astrocyte-neuron communication.

The Porphyromonas gingivalis O antigen is required for inhibition of apoptosis in gingival epithelial cells following bacterial infection.

BACKGROUND AND OBJECTIVE: Porphyromonas gingivalis infection induces apoptosis inhibition in gingival epithelial cells; however, it is not fully understood which bacterial effectors are involved in this process. The aim of this study is to evaluate whether the P. gingivalis lipopolysaccharide (LPS), specifically the O-antigen region, affects adherence, invasion, viability and apoptosis of gingival epithelial cells. MATERIAL AND METHODS: Gingival epithelial cells (OKF6/TERT2 line) were infected by different freshly prepared P. gingivalis clinical isolates, obtained from subjects with chronic periodontitis (CP3 and CP4) and healthy individuals (H1 and H3). Periodontitis and healthy isolates show differences in O-antigen production, as healthy isolates lack the O-antigen region. In addition, cells were infected by a site-specific mutant lacking the O-antigen portion. After 24 h postinfection, cell proliferation, viability and apoptosis were evaluated by Trypan blue, MTS and annexin V assays, respectively. Bacterial invasion, adhesion and proliferation were measured by gentamicin/metronidazole protection assays. Finally, toll-like receptor (TLR)2 and TLR4 mRNA expression was evaluated by quantitative reverse transcription-polymerase chain reaction. Statistical analysis was performed using ANOVA, Tukey's or Dunnett's tests (p < 0.05). RESULTS: At 24 h postinfection, strains lacking the O-antigen region (healthy isolates and O-antigen ligase-deficient strain) were unable to increase proliferation and viability, or decrease apoptosis as compared with strains producing intact LPS (periodontitis isolates and reference strain). However, the presence of the O-antigen neither contributed to changes in the ability of the bacteria to adhere to or invade cells, nor to intracellular survival. The presence of O-antigen also increased the expression of TLR4 (nearly sixfold), which correlated with inhibition of apoptosis. CONCLUSION: The O-antigen region of P. gingivalis LPS is required to increase gingival epithelial cell viability upon infection by bacteria and this increase is attributable to a reduction in apoptosis. Moreover, although bacterial internalization is required, the effects observed are not due to alterations in P. gingivalis adherence, invasion or intracellular survival. Interestingly, inhibition of apoptosis correlates with increased TLR4 expression, suggesting a role for TLR4 in this process.

The twisted survivin connection to angiogenesis.

Survivin, a member of the inhibitor of apoptosis family of proteins (IAPs) that controls cell division, apoptosis, metastasis and angiogenesis, is overexpressed in essentially all human cancers. As a consequence, the gene/protein is considered an attractive target for cancer treatment. Here, we discuss recent findings related to the regulation of survivin expression and its role in angiogenesis, particularly in the context of hypoxia. We propose a novel role for survivin in cancer, whereby expression of the protein in tumor cells promotes VEGF synthesis, secretion and angiogenesis. Mechanistically, we propose the existence of a positive feed-back loop involving PI3-kinase/Akt activation and enhanced ß-Catenin-TCF/LEF-dependent VEGF expression followed by secretion. Finally, we elaborate on the possibility that this mechanism operating in cancer cells may contribute to enhanced tumor vascularization by vasculogenic mimicry together with conventional angiogenesis.

Caveolin-1 in cell migration and metastasis.

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.

Testosterone modulates the expression of molecules linked to insulin action and glucose uptake in endometrial cells.

Polycystic ovary syndrome (PCOS) is a common hyperandrogenic disorder associated with insulin resistance. Insulin exerts its metabolic function by activating the PI3K/Akt pathway and favoring glucose uptake. Caveolin-1 is a scaffolding protein which increases insulin receptor (IR) stability. Alternatively, activation of IR increases caveolin-1 phosphorylation on tyrosine-14. Furthermore, endometrial tissue from PCOS patients is proposed to be insulin resistant; however, the particular role of testosterone in modulating the metabolic effects of insulin remains unexplored in endometrial stromal cells. To evaluate whether androgens modulate the response to insulin, T-HESCs cells were stimulated with 100 nM testosterone for 24 h and changes in the protein levels of caveolin-1, IR, and Akt were determined by Western blotting (WB). After testosterone treatment, the consequences of acute insulin stimulation were evaluated by WB analysis of phospho-S473Akt and phospho-Y14Caveolin-1, as well as by measuring glucose incorporation analyzing 2-deoxyglucose (2-DOG) uptake. For cells pretreated with testosterone, higher IR, IRS-1, and caveolin-1 protein levels compared with control conditions were detected. However, in testosterone treated cells acute insulin stimulation did not increase phospho-S473Akt and phospho-Y14caveolin-1 levels and reduced 2-DOG uptake was observed compared to control cells. Our results suggest that testosterone may have a detrimental role on the metabolic effects of insulin in endometrial stromal cell cultures. Thus, the high androgen levels in patients with PCOS may favor insulin resistance observed in endometria from these women.

Sjögren's syndrome and the epithelial target: a comprehensive review.

The most difficult component in our understanding of human autoimmunity remains a rigorous dissection of etiological events. Indeed, the vast literature on autoimmune diseases focuses on the inflammatory response, with the hope of developing drugs that reduce inflammation. However, there is increasing recognition that understanding the immunobiology of target tissues will also have direct relevance to disease natural history, including breach of tolerance. Sjögren's syndrome is essentially an epitheliitis and there are major changes to normal architectural salivary organization. We propose that loss of homeostasis is the initial event that precipitates inflammation and that such inflammatory response includes not only the adaptive response, but also an intense innate immune/bystander response. To understand these events this review focuses on the architecture, phenotype, function and epithelial cell organization. We further submit that there are several critical issues that must be defined to fully understand epithelial cell immunobiology in Sjögren's syndrome, including defining epithelial cell polarity, cell-cell and cell to extracellular matrix interactions and a variety of chemical and mechanical signals. We also argue that disruption of tight junctions induces disorganization of the apical pole of salivary acinar cells in Sjögren's syndrome. In addition, there will be a critical role of inflammatory cytokines in the apico-basal relocation of tight junction proteins. Further, the altered disorganization and relocation of proteins that participate in secretory granule formation are also dysregulated in Sjögren's syndrome and will contribute to abnormalities of mucins within the extracellular matrix. Our ability to understand Sjögren's syndrome and develop viable therapeutic options will depend on defining these events of epithelial cell biology.

Oral dryness in Sjögren's syndrome patients. Not just a question of water.

Sjögren's syndrome (SS) is a chronic autoimmune disease of undefined etiology. Patients with this syndrome suffer from severe alterations in both the quality and quantity of saliva and tears, due to impaired function of the relevant exocrine glands. Prevalent symptoms experienced by SS-patients include a persistent dry mouth sensation (xerostomia) and dry eyes (keratoconjunctivitis sicca). Water content of saliva depends of acetylcholine levels, glandular innervation, M3R signaling, calcium tunneling and water release, among other factors. However, unstimulated salivary flow correlates only poorly with symptoms of mouth dryness, raising the question as to which other components of saliva may be involved in mouth dryness experienced by SS-patients? Salivary mucins are glycoproteins characterized by the presence of large oligosaccharide side chains attached to the protein backbone. These molecules are key saliva components that are required to sequester water and thereby moisturize, as well as lubricate the oral mucosa. In the labial salivary glands of SS patients, morphological and functional alterations are detectable that affect the maturation and trafficking of salivary mucins. In this review, we will focus the discussion on these aspects of reduced salivary flow and decreased quality of salivary mucins, since they are likely to be responsible for xerostomia in SS-patients.

The caveolin-1 connection to cell death and survival.

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.

Decreased phosphorylation of Y¹4caveolin-1 in endometrial tissue of polycystic ovary syndrome patients may be related with an insulin resistant state in this tissue.

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.

Reduced sulfation of muc5b is linked to xerostomia in patients with Sjögren syndrome.

OBJECTIVES: MUC5B contains sulfated and sialylated oligosaccharides that sequester water required for moisturising the oral mucosa. Xerostomia, in patients with Sjögren syndrome, is generally associated with reduced quantities, rather than altered properties, of saliva. Here, we determined the amount of MUC5B (mRNA and protein) as well as sulfation levels in salivary glands of patients with normal or altered unstimulated salivary flow. Localisation of MUC5B and sulfated MUC5B, as well as total levels sulfated groups were determined and compared with acini basal lamina disorganisation. PATIENTS AND METHODS: In all, 18 patients with normal or altered unstimulated salivary flow and 16 controls were studied. MUC5B mRNA and protein were evaluated in salivary glands by semiquantitative RT-PCR and Western blot analysis. MUC5B sulfation was determined by Western blotting. MUC5B and sulfo-Lewis(a) antigen localisation were assessed by immunohistochemistry. The total amount of sulfated oligosaccharides was determined microdensitometrically. RESULTS: No significant differences were detected in MUC5B mRNA and protein levels between controls and patients, while sulfo-Lewis(a) antigen levels were lower in patients. The number of sulfo-Lewis(a) positive mucous acini was reduced in patients but no correlation was observed between lower levels of sulfation and unstimulated salivary flow. Microdensitometric data confirmed the presence of reduced sulfated oligosaccharides levels in mucous acini from patients with highly disorganised basal lamina. CONCLUSION: Disorganisation of the basal lamina observed in patients with Sjögren syndrome may lead to dedifferentiation of acinar mucous cells and, as a consequence, alter sulfation of MUC5B. These changes are suggested to represent a novel mechanism that may explain xerostomia in these patients.

Casein kinase 2 (CK2) increases survivin expression via enhanced beta-catenin-T cell factor/lymphoid enhancer binding factor-dependent transcription.

Increased expression of casein kinase 2 (CK2) is associated with hyperproliferation and suppression of apoptosis in cancer. Mutations in the tumor suppressor APC (adenomatous polyposis coli) are frequent in colon cancer and often augment beta-catenin-T cell factor (Tcf)/lymphoid enhancer binding factor (Lef)-dependent transcription of genes such as c-myc and cyclin-D1. CK2 has also been implicated recently in the regulation of beta-catenin stability. To identify mechanisms by which CK2 promotes survival, effects of the specific CK2 inhibitors 4,5,6,7-tetrabromobenzotriazole (TBB) and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole were assessed. TBB and 2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole significantly decreased proliferation and increased apoptosis of HT29(US) colon cancer cells. RT-PCR and immunoblot analysis revealed that both inhibitors decreased survivin mRNA and protein levels in HT29(US) cells. Similar effects were observed with TBB in human DLD-1 and SW-480 colorectal cells as well as ZR-75 breast cancer cells and HEK-293T embryonic kidney cells. Expression of GFP-CK2alpha in HEK-293T cells resulted in beta-catenin-Tcf/Lef-dependent up-regulation of survivin and increased resistance to anticancer drugs. Augmented beta-catenin-Tcf/Lef-dependent transcription and resistance to apoptosis observed upon GFP-CK2alpha expression were abolished by TBB. Alternatively, HEK-293T cells expressing GFP-survivin were resistant to TBB-induced apoptosis. Finally, siRNA-mediated down-regulation of CK2alpha in HEK-293T cells coincided with reduced beta-catenin and survivin levels. Taken together, these results suggest that CK2 kinase activity promotes survival by increasing survivin expression via beta-catenin-Tcf/Lef-mediated transcription. Hence, selective CK2 inhibition or down-regulation in tumors may provide an attractive opportunity for the development of novel cancer therapies.