KEGG pathways endocytosis

KEGG pathways Focal adhesions

KEGG pathways CAV-1 in viral myocarditis

Post-transcriptional modifications

Tyr14 phosphorylation by Src, Fyn or Abl tyrosine kinases, has been linked to various cellular phenomena including mechano-transduction, signal transduction, endocytosis, cell migration and focal adhesion dynamics. PY14Cav1 has been localized to focal adhesions using a monoclonal antibody, suggesting a specific function in cell migration processes. Similarly, pY14Cav1 was shown to stabilize FAK, focal adhesion kinase and other components within focal adhesions, and lead to enhanced focal adhesions turnover and tumor cell migration. PY14cav1 is essential for cav1 binding to intermediate filaments, a step required for anterior polarization of cav1 in transmigrating cells. Strong evidence suggests that pY14cav1 plays both a structural and signalling role in focal adhesion function. Recent work shows that pYCav1 in cooperation with the Mgat5/galectin-3 lattice, regulates FAK dynamics in focal adhesions and favours focal adhesion disassembly ad tumor cell migration.
Studies suggest that pY14Cav1-mediated control of raft internalization plays an important role in integrin-mediated control of cell proliferation that, when de-regulated, can lead to anchorage-independent growth and transformation and thereby the acquisition of tumorigenic potential.
Ser80 phosphorylation serves to convert Cav1 to a soluble secreted protein.
Palmytolation on Cys133, Cys143 and Cys156; while this modification is not essential for Cav1 localization to caveolae it is required for Cav1 oligomerization.

Signalling through the CSD

The Cav1 CSD of 20 amino-acids (residues 82–101) binds to numerous signalling molecules including Src family tyrosine kinases, growth factor receptors, endothelial nitric oxide synthase (eNOS), G proteins and G-protein-coupled receptors (GPCRs).
The ability of Cav1 to sequester and compartmentalize the spatio-temporal pairing of activators and effectors that regulate cellular signalling may play an important role in its regulation of tumor progression.

In particular, the CSD was shown to interact with the following pathways:
# eNos
# EGFR
# c-myc
# PI3K
# ERalpha
# PDGFR
# Cyclin-D1
# Insulin receptor
# TGFbeta
# WNT and beta-catenin
# ELK1
# TNFalpha and ceramide-mediated apoptosis
# beta-integrin
# SRC
# RhoA
# p53
# MAP kinase cascade
# VEGF
# Filamin-A
# Mgat5/galectin-3
# mTor and autophagy

Caveolin-1 in tumor progression: the good, the bad and the ugly, 2008

The role of CAV-1 in different tumors

Breast cancer
92 human breast cancers, the majority of which were invasive carcinomas, showed a sporadic mutation in Cav1 at codon 132 (P132L) in 16% of cases. In vitro, this mutant form of Cav1 behaves in a dominant-negative manner, explaining why only a single mutated Cav1 allele is found in patients with breast cancer, and leads to formation of misfolded Cav1 oligomers that are retained within the Golgi complex and are not targeted to caveolae. Recombinant expression of the Cav1 P132L mutant induced cellular transformation and invasion in NIH 3T3 fibroblasts. A different group reported that the P132L mutation was present in 19% of Eraplha positive breast cancers but not in Eralha negative ones. High levels of oxidative-stress are associated with aggressiveness in breast cancers. Several findings indicate that BRCA1 may protect against oxidative stress, while metastatic breast cancers show low BCRA1 protein expression due to promoter methylation. Elevated ROS levels induce glycolytic metabolism in the tumor stroma, with increased expression of the L-lactate exporter MCT4, which is downregulated in cancer associated fibroblasts by use of antioxidant. In fact, loss of BRCA1 function in epithelial cancer cells leads to hydrogen peroxide generation in both epithelial breast cancer and CAFs, with increased MCT4 and decreased Cav-1 expression in the stroma.
BRCA1 mutations drive oxidative stress and glycolysis in the tumor micro-environment: implications for breast cancer with antioxidant therapies., 2012

prostate cancer
the cav1 promoter region was found to be hypermetilated in prostate cancer, therefore both expression levels and functional mutations contribute to the role of cav1 in cancer. Recently stromal production of cav1 was found involved in a paracrine antiapoptotic loop in perineural invasion suggesting that it acts as an autocrine/paracrine factor to increase the proliferative activity and decrease apoptosis of prostate cancer cells. Cav1 secreted by prostate cancer cells has been shown to enable angiogenesis and tumor growth during prostate cancer progression. Thompson et al. showed that secreted Cav1 could stimulate prostate tumor cell growth and survival.
Metastasis-related genes in prostate cancer: the role of caveolin-1, 2009

Lung adenocarcinoma
Caveolin-1 immunoreactivity was either totally absent or just barely detectable in a few lung adenocarcinoma cells from cases diagnosed as lung adenocarcinoma without regional lymph node metastasis. In contrast, increased caveolin-1 immunoreactivity both in number and intensity was detected in primary lung adenocarcinoma cells as well as in cancer cells that metastasized to regional lymph nodes from the cases diagnosed as advanced lung adenocarcinoma with nodal metastases. Multivariate analysis considering caveolin-1 immunoreactivity in addition to the established prognostic parameters such as pT stage, pN in these patients confirmed that caveolin-1 is an independent functional predictor of poor survival. It has been further revealed that up-regulated caveolin-1 in cell lines is necessary for mediating filopodia formation, which may enhance the invasive ability of lung adenocarcinoma cells.
Up-Regulated Caveolin-1 Accentuates the Metastasis Capability of Lung Adenocarcinoma by Inducing Filopodia Formation, 2002

Malignant pleural mesothelioma
a review of transcriptome studies has shown that Cav-1 can be considered a "mesothelioma gene", since it is reproducibly downregulated among independent studies (it is unspecified if mesothelioma cell lines and tissue samples used for these studies were epithelioid or sarcomatoid type). Moreover, it can be partially responsible for cisplatin resistance. Since many viruses use caveolin-mediated endocytosis to enter human cells, Cav-1 can also be the target SV40 uses to infect mesothelial cells. In 2009 gene expression profiling has been suggested to allow the differential diagnosis between MPM and lung adenocarcinoma. Cell-adhesion pathways are deregulated in MPM, including osteopontin, mesothelin, integrin-beta1 and their downstream proteins, such as FAK, Akt, rpS6 and Stat3. These findings underline the importance of the interaction between MPM and its extracellular micro-environment, in which growth factors like VEGF, FGF and PDGF play a role. In this context, normal fibroblasts, adequately induced, help to promote the tumor progression and can infiltrate within the tumor mass.
A review of transcriptome studies combined with data mining reveals novel potential markers of malignant pleural mesothelioma, 2011

Multiple Myeloma
Caveolin-1 is required for vascular endothelial growth factor-triggered multiple myeloma cell migration and is targeted by bortezomib.vascular endothelial growth factor (VEGF) triggers Src-dependent phosphorylation of caveolin-1, which is required for p130(Cas) phosphorylation and multiple myeloma cell migration. Conversely, depletion of caveolin-1 by antisense methodology abrogates p130(Cas) phosphorylation and VEGF-triggered multiple myeloma cell migration. The proteasome inhibitor bortezomib both inhibited VEGF-triggered caveolin-1 phosphorylation and markedly decreased caveolin-1 expression. Consequently, bortezomib inhibited VEGF-induced multiple myeloma cell migration. Bortezomib also decreased VEGF secretion in the bone marrow microenvironment and inhibited VEGF-triggered tyrosine phosphorylation of caveolin-1, migration, and survival in human umbilical vascular endothelial cells.

Tumor stage and CAV-1 expression

One way to reconcile the conflicting data about cav1 expression levels in different cancer cells is to consider that the role of cav1 may depend on tumor stage. In vivo, Cav1 is highly expressed in normal and differentiated tissues that, through various mechanisms, can undergo oncogenic transformation in which decreased Cav1 expression confers a growth advantage to these cancer cells. This cellular expansion is then followed in later stages by re-induction of Cav1 expression that enables the cancer cells to survive by suppressing apoptosis and by acquiring multi-drug resistance abilities (MDR). These abilities favour growth and survival of highly metastatic cells that will invade the blood circulation and spread to distant locations to form secondary tumors. Therefore, targeting Cav1 expression may represent a novel method for preventing metastasis development. Cav1 has also been involved in tumor vasculature maturation by promoting smooth muscle cell migration. Particularly, Cav1 secreted by prostate cancer cells has been shown to enable angiogenesis and tumor growth during prostate cancer progression. Tumor blood vessels are known to be hyperpermeable to macromolecules when compared to normal vasculature. Cav1 is involved in this phenomenon as Cav1−/− mice displayed increased tumor microvascular permeability, angiogenesis, and growth.

Cav-1: tumor-suppressor or pro-oncogenic protein?

Cav1 plays varied, and at times seemingly conflicting, roles in tumor progression. The role of Cav1 is dependent on expression of various other molecular effectors that interact with or influence Cav1 function either directly or indirectly. For instance, recruitment of EGFR and other receptors to positive regulatory Mgat5/galectin-dependent macromolecular complexes competes with receptor recruitment to negative regulatory Cav1-containing domains.The ability to override the negative regulatory scaffolding function of Cav1, by either mutation or recruitment to the Mgat5/galectin lattice, demonstrates the critical role of this function in Cav1 suppressor activity.

Cav1 can therefore be considered a conditional tumor suppressor whose suppressor function is dependent on expression of Mgat5 and the galectin lattice.

Expression of the Mgat5/galectin-3 lattice will therefore enable high Cav1 levels associated with poor prognosis in some tumor types. Galectin-3 can stimulate Cav1 phosphorylation and elevated Cav1 levels in a cell expressing the Mgat5/galectin lattice should lead to Cav1 tyrosine phosphorylation and associated increased focal adhesion turnover. This suggests that while the Mgat5/galectin lattice will compete with negative regulatory Cav1 microdomains to promote EGFR signalling, the lattice will work together with pY14Cav1 to promote focal adhesion domain stabilization and consequent turnover. As such, in advanced tumors expressing Mgat5 and galectin-3, not only will the suppressor function of Cav1 be overridden, but the role of pY14Cav1 as a promoter of tumor cell migration will be enabled.
The dependence of Cav1 function in tumor progression on expression of Mgat5 and galectin-3, and potentially other functional interactors, can explain the discordant results observed for Cav1 expression in tumor progression. For example, Cav1 inhibition of EGF-stimulated lamellipodial protrusion and cell migration in a metastatic rat mammary adenocarcinoma cell line may reflect its ability to dampen EGFR signalling while its promotion of migration in other systems may reflect its ability to impact on focal adhesion dynamics.

Competitive interaction between Cav1 and the Mgat5/galectin lattice domains to negatively regulate cytokine receptor signalling, and concerted action between the two to promote focal adhesion turnover and tumor cell migration could potentially be manipulated as a strategy for controlling Cav1 function in tumor progression. Targeting galectin-3 in tumors expressing elevated levels of both Cav1 and galectin-3 could conceivably switch Cav1 from being a promoter of tumor progression and metastasis to a suppressor of growth signalling. Galectin-3 targeting agents have been described, notably oral ingestion of pectin, and utilization of such agents for treatment of Cav1/galectin-3 positive tumors may prove a worthy avenue of future research.

CAV-1 and tumor stroma

Activated myo-fibroblasts are critical for normal wound healing and are generated via the TGFb mediated differentiation of normal fibroblasts. As such, the tumor microenvironment has been postulated to play a key role in tumor initiation, progression, and metastasis. In this regard, cancer-associated fibroblasts may be thought of as activated myo-fibroblasts that cannot regress to the unactivated state. A loss of Cav-1 expression may be sufficient to induce a constitutive myo-fibroblastic phenotype; in fact, a loss of Cav-1 in mammary stromal fibroblasts appears to drive the onset of the myo-fibroblastic differentiation program, upregulating muscle-related genes and TGFbeta ligands. An absence of stromal Cav-1 was specifically associated with a high rate of mammary tumor recurrence, metastasis and tamoxifen-resistance, resulting in poor clinical outcome. More than 25 candidate stromal biomarkers have been identified, all upregulated by a loss of Cav-1 in stromal cells. Interestingly, these proteins include five myo-fibroblast markers, three signaling molecules, one oncogene, eight metabolic and glycolytic enzymes, as well as three extra-cellular matrix proteins—known to be associated with fibrosis and tumorigenesis. The eight glycolytic enzymes include the M2-isoform of pyruvate kinase and lactate dehydrogenase, which are key regulators known to mediate the Warburg effect. Two markers of oxidative stress were also upregulated, suggesting the over-production of reactive oxygen species (ROS) in Cav-1 deficient stromal cells.

The "reversed Warburg effect" in the tumor stroma

The “Warburg Hypothesis” was first formulated by Otto Warburg in the early 1920s. He hypothesized that tumor metabolism is different from normal metabolism, and relies on glycolysis for the production of energy in the form of ATP, despite
the presence of oxygen. Thus, aerobic glycolysis has come to be known as the “Warburg Effect,” and was originally attributed to mitochondrial mal-functioning.
Importantly, aerobic glycolysis results in the production of two metabolic end-products, pyruvate and lactate, which can then be secreted by cancer cells. Secreted lactate and pyruvate can be taken up by adjacent cancer cells and provides a feed-forward mechanism for tumor growth, as these metabolites can then enter into the TCA cycle in cancer cells which are using oxidative metabolism. Lactate dehyrogenase (LDH) is essential for this process, as it is a bi-directional enzyme that coverts lactate to pyruvate and vice-versa. So LDH converts lactate to pyruvate, which enters the TCA cycle. Bi-directional transport of lactate and pyruvate (into and out of cancer cells) is accomplished by a family of mono-carboxylate transporters (such as MCT1 and MCT4).
Not all tumors are associated with increased aerobic glycolysis, and in fact it is now clear that cancer cells utilize both glycolysis and oxidative phosphorylation to satisfy their metabolic needs. Experimental assessments of ATP production in cancer cells have demonstrated that oxidative pathways play a significant role in energy generation, and may be responsible for about 50 to 80% of the ATP generated. Also, it should be considered that most studies were performed using isolated cancer cells, which may behave very differently from cancer cells in vivo, surrounded by their natural microenvironment. Consistent with this new stromal hypothesis, lactate by itself is sufficient to promote aerobic glycolysis, fibrosis and angiogenesis. So, secreted lactate and pyruvate derived from cancer-associated fibroblasts may also be used by endothelial progenitor cells, pericytes, and endothelial cells to drive angiogenesis. Similarly, a high tumor content of lactate is a powerful predictive biomarker for recurrence, metastasis and poor clinical outcome.
We should therefore consider that the Warburg effect may be a stromal phenomenon: epithelial cancer cells induce the Warburg effect (aerobic glycolysis) in neighboring stromal fibroblasts. These cancer-associated fibroblasts, then undergo myo-fibroblastic differentiation, and secrete lactate and pyruvate (energy metabolites resulting from aerobic glycolysis). Epithelial cancer cells could then take up these energy-rich metabolites and use them in the mitochondrial TCA cycle, thereby promoting efficient energy production (ATP generation via oxidative phosphorylation), resulting in a higher proliferative capacity. In this alternative model of tumorigenesis, the epithelial cancer cells instruct the normal stroma to transform into a wound-healing stroma, providing the necessary energy-rich micro-environment for facilitating tumor growth and angiogenesis. In essence, the fibroblastic tumor stroma would directly feed the epithelial cancer cells, in a type of host-parasite relationship.This new idea has been termed the “Reverse Warburg Effect.”, suggesting that aerobic glycolysis may take place in the “fibroblastic” tumor stromal compartment, rather than in the epithelial cancer cells themselves. In this scenario, the epithelial tumor cells “corrupt” the normal stroma, turning it into a factory for the production of energy-rich metabolites. This alternative model is still consistent with Warburg’s original observation that tumors show a metabolic shift towards aerobic glycolysis. In support of this idea, unbiased proteomic analysis and transcriptional profiling of a new model of cancer-associated fibroblasts (caveolin-1 deficient stromal cells), shows the upregulation of both myo-fibroblast markers and glycolytic enzymes, under normoxic conditions. The expression of these proteins has been validated in the fibroblastic stroma of human breast cancer tissues that lack stromal Cav-1. Importantly, a loss of stromal Cav-1 in human breast cancers is associated with tumor recurrence, metastasis, and poor clinical outcome. Thus, an absence of stromal Cav-1 may be a biomarker for the “Reverse Warburg Effect,” explaining its powerful predictive value. Genetic ablation of Cav-1 in murine fibroblasts is indeed sufficient to functionally induce the onset of aerobic glycolysis via mitochondrial dysfunction. These new data indicate that cancer cells and CAFs develop a 'symbiotic' or 'parasitic' relationship, with the vectorial and unilateral transfer of energy from glycolytic stromal cells to oxidative cancer cells.
A simple prediction of the “Reverse Warburg Effect” is that tumors with an increased percentage of stroma would have a worse prognosis, because they would be expected to have increased lactate production/secretion. If the “Reverse Warburg Effect” is correct, then lactate transport inhibitors would be a promising therapeutic strategy.

The reverse Warburg effect: aerobic glycolysis in cancer associated fibroblasts and the tumor stroma, 2009

CAV-1 as a marker of hypoxia and oxydative stress

Further studies are needed to determine how loss of Cav-1 induces the expression of myo-fibroblast markers and glycolytic enzymes: however it was shown that these genes are normally upregulated by hypoxia and/or are targets of the HIF genes.
A loss of Cav-1 may induce oxydative stress, which is sufficient to upregulate HIF.
These results indicate that a loss of stromal Cav-1 is a marker of hypoxia and oxidative stress. Mitophagy, or mitochondrial-autophagy, is particularly important to remove damaged ROS-generating mitochondria. An autophagy/mitophagy program is also triggered by hypoxia. Hypoxia is a common feature of solid tumors, and promotes cancer progression, invasion and metastasis. Interestingly, via induction of autophagy, hypoxia is sufficient to induce a dramatic loss of Cav-1 in fibroblasts. The hypoxia-induced loss of Cav-1 can be inhibited by the autophagy inhibitor chloroquine, or by pharmacological inhibition of HIF1α. Conversely, small interfering RNA-mediated Cav-1 knock-down is sufficient to induce pseudo-hypoxia, with HIF1α and NFκB activation, and to promote autophagy/mitophagy, as well as a loss of mitochondrial membrane potential in stromal cells
In a co-culture model, autophagy in cancer-associated fibroblasts was shown to promote tumor cell survival via the induction of the pro-autophagic HIF1α and NFκB pathways in the tumor stromal microenvironment. Finally, the mitophagy marker Bnip3L is selectively up-regulated in the stroma of human breast cancers lacking Cav-1, but is notably absent from the adjacent breast cancer epithelial cells

It is indeed well documented that, as a consequence of intra-tumoral hypoxia, the hypoxia-inducible factor (HIF)1α pathway is activated in many tumors cells, resulting in the direct up-regulation of lactate dehydrogenase (LDH) and increased glucose consumption.

Via oxidative stress, cancer cells activate two major transcription factors in adjacent stromal fibroblasts (hypoxia-inducible factor (HIF)1α and NFκB). This leads to the onset of both autophagy and mitophagy, as well as aerobic glycolysis, which then produces recycled nutrients (such as lactate, ketones, and glutamine). These high-energy chemical building blocks can then be transferred and used as fuel in the tricarboxylic acid cycle (TCA) in adjacent cancer cells. The outcome is high ATP production in cancer cells, and protection against cell death. Cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts (CAFs). This activates reactive oxygen species (ROS) production and autophagy. ROS production in CAFs, via the bystander effect, serves to induce random mutagenesis in epithelial cancer cells, leading to double-strand DNA breaks and aneuploidy, which is associated with poor clinical outcome. Cancer cells mount an anti-oxidant defense and up-regulate molecules that protect them against ROS and autophagy, preventing them from undergoing apoptosis. So, stromal fibroblasts conveniently feed and mutagenize cancer cells, while protecting them against death.
In further support of the existence of a 'lactate shuttle' in human tumors, it has been now shown that CAFs express MCT4 (for lactate extrusion), while breast cancer cells express MCT1 (for lactate uptake). Interestingly, MCT4 expression in CAFs is induced by oxidative stress, and MCT4 is a known HIF1α target gene.

Autophagy in the tumor stroma and CAV-1 expression

Autophagy is a cellular self-catabolic process in which cytoplasmic constituents are sequestered in double membrane vesicles that fuse with lysosomes where they are degraded. As this catabolic activity generates energy, autophagy is often induced under nutrient limiting conditions providing a mechanism to maintain cell viability and may be exploited by cancer cells for survival under metabolic stress. The role of autophagy in tumorigenesis is controversial. Both autophagy inhibitors (chloroquine) and autophagy promoters (rapamycin) block tumorigenesis by unknown mechanism(s). This is called the "Autophagy Paradox".
As HIF1α triggers autophagy in both fibroblasts and cancer cells, these data demonstrate that the role of autophagy in driving tumor formation is cell-type specific, and that stromal autophagy, and not cancer cell autophagy, favors tumor growth.Several studies have demonstrated that the over-expression of autophagic markers, such as ATG16L and cathepsin K and D, in the stroma and not in tumor cells predicts poor prognosis. Similarly, loss of autophagic markers, such as Beclin 1, in tumor cells correlates with poor clinical outcome, suggesting that activation of an autophagic program in tumor cells reduces tumor aggressiveness
Metabolome profiling of several types of human cancer tissues versus corresponding normal tissues have consistently shown that cancer tissues are highly catabolic, with the significant accumulation of many amino acids and TCA cycle metabolites. The levels of reduced glutathione were decreased in primary and metastatic prostate cancers compared to benign adjacent prostate tissue, suggesting that aggressive disease is associated with increased oxidative stress. Also, these data show that the tumor microenvironment has increased oxidative-stress-induced autophagy and increased catabolism. Taken together, all these findings suggest an integrated model whereby a loss of stromal Cav-1 induces autophagy/mitophagy in the tumor stroma, via oxidative stress. This creates a catabolic micro-environment with the local accumulation of chemical building blocks and recycled nutrients (such as amino acids and nucleotides), directly feeding cancer cells to sustain their survival and growth. Sotgia et. alii have termed this novel idea the 'autophagic tumor stroma model of cancer'. This new paradigm may explain the 'autophagy paradox', Autophagy inhibitors (such as chloroquine) functionally block the catabolic transfer of metabolites from the stroma to the tumor, inducing cancer cell starvation and death. Conversely, autophagy inducers (such as rapamycin) promote autophagy in tumor cells and induce cell death. Thus, both inhibitors and inducers of autophagy will have a similar effect by severing the metabolic coupling of the stroma and tumor cells, resulting in tumor growth inhibition (cutting 'off ' the fuel supply).
Finally, the autophagic tumor stroma model can also provide an explanation for systemic cachexia, which is progressive skeletal muscle and adipose tissue wasting, affecting up to 50% of all cancer patients and resulting in severe weight loss and shortened survival. Cachexia is the result of increased energy consumption and higher metabolic rates. Cancer leads to a generalized catabolic state via an autophagic-mechanism that generates building blocks and starves the rest of the body. Oxidative stress-induced autophagy functions as a driver of muscle wasting. Thus, cachexia may start locally as stromal autophagy, and then spread systemically via cytokine production and inflammation, which also drive autophagy.
In direct support that cancer cells use mitochondrial oxidative metabolism, many investigators have shown that cancer cells are 'addicted' to glutamine . Glutamine is a non-essential amino acid that is metabolized to glutamate and enters the TCA cycle as alpha-ketoglutarate, resulting in high ATP generation via oxidative phosphorylation.
Recent studies also show that ammonia is a by-product of glutaminolysis. In addition, ammonia can act as a diffusible inducer of autophagy. Given these observations, glutamine addiction in cancer cells provides another mechanism for driving and/or maintaining autophagy in the tumor micro-environment. It has been previously shown that a loss of Cav-1 in the stroma is sufficient to drive autophagy, resulting in increased glutamine production in the tumor micro-environment. Thus, this concept defines a new vicious cycle in which autophagy in the tumor stroma transfers glutamine to cancer cells, and the by-product of this metabolism, ammonia, maintains autophagic glutamine production.
Interestingly, there may be a connection between angiogenesis and autophagy; in fact, anti-angiogenic therapy has been found to promote tumor recurrence, progression and metastasis. A possible explanation may be that anti-angiogenic therapy induces autophagy in the tumor stroma via the induction of stromal hypoxia, thereby converting a non-aggressive tumor type to a "lethal" aggressive tumor phenotype.
The autophagic tumor stroma model of cancer or battery-operated tumor growth: A simple solution to the autophagy paradox 2010

Extracellular matrix regulation of autophagy: mTor/Pras40 and cancer dormancy

Integrin-mediated attachment of epithelial cells to extracellular matrix (ECM) is critical for proper growth and survival. Although detachment leads to apoptosis, termed anoikis, recent work demonstrates that ECM detachment also robustly induces autophagy, a tightly regulated lysosomal self-digestion process that actually promotes survival.
Autophagy presumably protects epithelial cells from the stresses of ECM detachment, allowing them to survive provided they reattach in a timely manner
The recent discovery of AuTophaGy-related genes (ATGs) has provided the means to manipulate and monitor the autophagic process in experimental models
Autophagy is induced in response to a variety of stress conditions. In cells starved for nutrients or growth factors, autophagy produces critical nutrients and energy that enhance cell survival through the breakdown of cytosolic components. Recent studies demonstrate that other stresses, including hypoxia, growth factor withdrawal, and ER stress, also induce autophagy to enhance cell survival.
Basal levels of autophagy are required to maintain homeostasis by preventing the build-up of damaged proteins and organelles; although autophagy is vital for maintaining cell survival, excessive autophagy results programmed cell death, termed autophagic or type 2 cell death.
The dual roles of autophagy in cell survival and death likely play important roles in the pathogenesis of numerous human diseases including neurodegeneration, aging, viral and bacterial pathogenesis, and cancer.
Detachment-induced autophagy directly results from the loss of ECM-integrin engagement.
The pathways linking loss of integrin engagement at the cell surface to the autophagy machinery remain elusive; they have been grouped into three categories: growth factor and nutrient-sensing pathways; energy-sensing pathways; and integrated stress response.
Group 1:
Downregulation of growth factor receptors or nutrient sensors on the cell surface leads to the inactivation of multiple growth promoting pathways, notably, the mammalian target of rapamycin (mTOR) pathway, which is the archetypal inhibitor of autophagy. Accordingly, mTOR downregulation is also observed following ECM detachment.
Remarkably, mTOR activity may be directly controlled in response to cell-matrix adhesion through focal adhesion kinase (FAK).
FAK, a critical component of adhesion mediated signaling, can tyrosine phosphorylate TSC2, an upstream mTOR regulator, to suppress its activity and maintain mTOR activation.
Group 2:
During times of bioenergetic stress, autophagy provides nutrients to cell through degradation products released from the lysosome. Presumably, these basic components can be recycled and used to synthesize new proteins and to provide inputs for energy cycles that produce ATP. In fact, in response to diverse stressors, autophagy inhibition profoundly reduces intracellular ATP levels
Decreased energy (ATP) can be monitored in part by AMP-activated protein kinase (AMPK), which is activated due to increased AMP to ATP ratios via the upstream kinase, LKB1. Upon activation, AMPK phosphorylates and activates the tuberous sclerosis complex (TSC1/2 complex) resulting in downstream inhibition of mTOR. mTOR inhibition not only limits pro-growth signals, but also induces autophagy, which in turn, provides ATP through the recycling of degradation products. AMPK is robustly activated during ECM detachment.
Group3:
Phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) on serine 51 is a critical convergent point in the integrated stress response (ISR), a general stress-response program conserved from yeast to mammals.
Mammary epithelial cells grown in suspension or treated with β1 integrin blocking antibodies exhibit increased eIF2α phosphorylation, which depends on the endoplasmic reticulum kinase PERK, a known upstream eIF2α kinase.
ER stress, induced via chemicals or the expression of toxic polyglutamine repeat proteins, elicits autophagosome formation that depends on both PERK activation and eIF2α phosphorylation.
Recently, both in vitro and mouse studies have identified the disruption of β1 integrin function as a unique activator of cellular dormancy. Inhibition of β1 integrin activity prevents tumor cell proliferation, but not cell viability, leading to the induction of a dormant state. Further studies indicate that dormancy is mediated downstream of β1 integrin through decreases in FAK and MAPK signaling and activation of p38 and eIF2α.
Notably, the stress pathways induced during dormancy also activate autophagy in other contexts; accordingly, one can speculate that detachment-induced autophagy in disseminated tumor cells may be vital for maintaining a dormant state or promoting the survival of dormant cancer cells.
Insulin stimulates protein synthesis and cell growth by activation of the protein kinases Akt (also known as protein kinase B, PKB) and mammalian target of rapamycin (mTOR). It was reported that Akt activates mTOR by phosphorylation and inhibition of tuberous sclerosis complex 2 (TSC2). However, in recent studies the physiological requirement of Akt phosphorylation of TSC2 for mTOR activation has been questioned. Vander Haar et alii identified PRAS40 (proline-rich Akt/PKB substrate 40 kDa) as a novel mTOR binding partner that mediates Akt signals to mTOR. PRAS40 binds the mTOR kinase domain and its interaction with mTOR is induced under conditions that inhibit mTOR signalling, such as nutrient or serum deprivation or mitochondrial metabolic inhibition. Binding of PRAS40 inhibits mTOR activity and suppresses constitutive activation of mTOR in cells lacking TSC2. PRAS40 silencing inactivates insulin-receptor substrate-1 (IRS-1) and Akt, and uncouples the response of mTOR to Akt signals. Furthermore, PRAS40 phosphorylation by Akt and association with 14-3-3, a cytosolic anchor protein, are crucial for insulin to stimulate mTOR. These findings identify PRAS40 as an important regulator of insulin sensitivity of the Akt–mTOR pathway and a potential target for the treatment of cancers, insulin resistance and hamartoma syndromes.
Extracellular matrix regulation of autophagy, 2008

Caveolin: pulmonary arterial hypertension and lung fibrosis

Pulmonary arterial hypertension (PAH) is a poorly understood disease that carries a devastating prognosis. Increased pulmonary vascular tone in PAH leads to right heart failure and death.
PAH is characterized by increased and dysfunctional angiogenesis of the pulmonary circulation, associated with fibrosis and inflammation. In fact Caveolin-1 is highly expressed in a variety of cell types in solid tissues including epithelial cells, endothelial cells, fibroblasts, and adipocytes.
A population of true endothelial cells found in the vessel wall called endothelial colony-forming cells (ECFC) is believed to contribute to angiogenesis through cell proliferation. While sprouting angiogenesis can be an appropriate response to vascular injury and hypoxia, increased and dysregulated angiogenesis is a hallmark of many disease states such as cancer, macular degeneration and pulmonary arterial hypertension. ECFC possess significant clonal proliferative potential and are implicated in both physiologic and pathologic angiogenesis. For example, ECFC demonstrate the ability to participate in vascular repair in mouse models of hind limb ischemia. Conversely, circulating ECFC dysfunction has been demonstrated in diseases of dysregulated angiogenesis, such as diabetes mellitus and PAH.
Caveolin-1 is linked to the regulation of fibrosis through its effects on the regulation of ECM production by fibroblasts. High levels of caveolin-1 are found in normal lung fibroblasts (NLF), whereas much lower levels are found in lung fibroblasts isolated from the fibrotic lung tissue of scleroderma patients (SLF) and IPF patients. The loss of caveolin-1 results in the hyperactivation of signaling molecules (MEK, ERK, JNK, Akt) leading to the overexpression of collagen, tenascin-C, and the myofibroblast marker ASMA (α-smooth muscle actin).
Effects of loss of caveolin-1 in fibrosis include activation of TGF-β signaling and upregulation of CXCR4 in monocytes resulting in their enhanced migration into damaged tissue. There are multiple points of intersection between TGF-β and caveolin-1 signaling. First of all, TGF-β inhibits caveolin-1 expression in a variety of cell types including fibroblasts (both lung and dermal) and monocytes. Caveolin-1 also regulates TGF-β signaling via its effects on the endocytosis of TGF-β ligand-receptor complexes. TGF-β receptors are present in both caveolin-1-rich lipid rafts and early endosomes. Caveolin-1-dependent internalization in lipid rafts leads to receptor degradation, thereby inhibiting TGF-β signaling. Therefore, the decreased caveolin-1 expression observed in IPF fibroblasts facilitates their hyperproliferation. In fact PTEN, like caveolin-1, is present at reduced levels in IPF fibroblasts. The lack of PTEN results in the activation of PI3K/Akt signaling.
A variety of studies have suggested that caveolin-1 may be a useful therapeutic target in fibrotic diseases of the lung and other tissues because in these diseases a low level of caveolin-1 expression is associated with a high level of collagen expression and fibrosis. The use of caveolin-1 as a molecular therapeutic target for treating fibrosis may be a good idea because caveolin-1 is a master regulator of several signaling pathways. The pharmaceutical industry has spent billions of dollars to find strong, specific inhibitors of individual signaling pathways and they have succeeded in designing these inhibitors. Yet they have had little or no success in developing a treatment for fibrosis and only limited success in treating cancer.

Caveolin-1 Signaling in Lung Fibrosis,2012

Pulmonary artery endothelium resident endothelial colony-forming cells in pulmonary arterial hypertension, 2011

Caveolins: cardiovascular diseases

Caveolins play a significant role in cardiovascular disease and dysfunction. In particular, caveolin-1 (Cav-1) and caveolin-3 (Cav-3) have been identified as potential regulators of vascular dysfunction and heart disease and might even confer cardiac protection in certain settings. In the cardiovascular endothelium, Cav-1 is a key regulator of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS), calcium, and vascular growth and remodeling.
One of the main ways Cav-1 regulates cellular signaling is through a sequence known as the caveolin scaffold domain (CSD). When bound to the CSD, eNOS is held in an inactive state, thereby limiting NO production. Calmodulin, an activator of eNOS, has been shown to compete with Cav-1 for this binding site. Changes in NO regulation contributing to pathophysiological processes, termed endothelial dysfunction, is clinically characterized by reduced NO bioavailability and is associated with worse cardiovascular outcomes. Beyond endothelial dysfunction, the pathophysiological role NO is also seen in the setting of pulmonary hypertension. Both humans and Cav-1 deficient mice are noted to develop pulmonary hypertension as a result of uncontrolled eNOS activation due to a lack of Cav-1. Similar constitutive hyper-activation of eNOS is also known to drive cardiomyopathy seen in Cav-1 deficiency.

Caveolin-3 in cardiovascular disease and protection
Cav-3 is essential for formation of caveolae in cardiac and skeletal muscle and is the dominant caveolin isoform in cardiomyocytes. Cav-3 is a player in both cardiac disease and protection. Cav-3 dependent preconditioning involves complicated and often controversial signaling cascades. Mice lacking Cav-3 have an inability to be preconditioned to ischemic injury through ischemia preconditioning. Therefore, given the central role of Cav-3 in ischemic preconditioning, Cav-3 presents itself as an interesting therapeutic target for inducing cardioprotection from ischemia. Following the finding that hypoxic cardiomyocytes from rats that were subsequently reoxygenated had oxidative damage and decreased Cav-3 levels, the authors demonstrated that use of a Cav-3 peptide of the scaffold domain was able to eliminate oxidative damage. In this case, protection from oxidative damage was attributed to inhibiting production of O2−, increased superoxide dismutase activity (SOD), and inhibition of the caspase-3.
In summary, both increased eNOS activity and deregulation of Cav-3 within the heart can result in hypertrophic cardiomyopathy.
However, while caveolin targeted therapeutics have great potential, due to complex signaling pathways associated with caveolins they require further development to ensure specificity in order to reach clinical applicability. One method of doing so is to develop compounds to directly target the interaction between eNOS and Cav-1 and thereby prevent or limit inhibition eNOS by Cav-1. In this pursuit, peptides of Cav-1 CSD show promise in being able to regulate NO production. Furthermore, targeting of Cav-1 to regulate cellular signaling outside of eNOS is also a front of intense research.

Mechanical unloading increases caveolin expression in the failing human heart
Loss of caveolin-3 expression results in the activation of a program of progressive hypertrophy in cardiac myocytes, and deletion of both caveolin-1 and -3 results in severe cardiomyopathy. Hypoxia, elevated catecholamines and genetic perturbations in alpha1-adrenergic signaling are associated with suppression of caveolin gene expression.
It is demonstrated that mechanical unloading of patients with end-stage (NYHA Class IV) heart failure induces expression of all three caveolin isoforms. The induction of caveolin-1 is associated with the reciprocal suppression of ANF, suggesting that the changes in the expression of both genes are linked to decreased hemodynamic load. The upregulation of caveolin expression following mechanical unloading of failing human hearts does not represent a simple reversal of the changes associated with advanced heart failure, but rather is consistent with the remodeling of lipid metabolism and adrenergic signaling.
There are, however, substantial differences in these responses depending on the type of the underlying cardiomyopathy. The response of individuals with a non-ischemic cardiomyopathy both in terms of induction of receptor tyrosine kinases and caveolins is very heterogeneous whereas the subset of patients with ischemic cardiomyopathy shows a much more uniform response. In particular, in these patients there is a coordinated upregulation of all three caveolin isoforms. Combined induction of caveolin-1 and caveolin-3 may result in beneficial cardiac effects. While in myocytes reduced nitric oxide levels resulting from increased caveolin-3 expression may cause augmentation of the inotropic properties, it has been shown that in fibroblasts and endothelial cells caveolin-1 attenuates cardiac dysfunction after ischemia–reperfusion by maintaining nitric oxide release from the endothelium through the inhibition of protein kinase C, that is associated with improved cardiac performance.
In cardiac myocytes, several components of the adrenergic signal transduction pathways, including the β2-adrenergic receptor, are sequestered in caveolin-rich membrane microdomains. Some studies have shown that caveolin-dependent sequestration improves the coupling between the β2-adrenergic receptor and adenylate cyclase. Thus, increased expression of caveolins in human myocardium may be a factor in improving β-adrenergic responsiveness.
In conclusion, data suggest that changes linked to reverse remodeling are associated with consistent transcriptional upregulation of caveolin expression and of CD36. The restoration of caveolin expression following mechanical unloading may result not only in normalization and improvements in myocardial fatty acid transport and insulin sensitivity, but also modulation of β-adrenergic responsiveness. CD36 (FAT/fatty acid translocase), the major myocardial fatty acid transport protein, is also sequestered in caveolae. Loss of CD36 results in both insulin resistance and impaired fatty acid transport. CD36 deficiency in humans can lead to the development of hypertrophic cardiomyopathy. The upregulation of CD36 expression following mechanical unloading may contribute to improvements in both myocardial fatty acid transport and insulin sensitivity.

Caveolin as a potential drug target for cardiovascular protection, 2012

Mechanical unloading increases caveolin expression in the failing human heart, 2003

Caveolin: primary open-angle glaucome (POAG)

CAV1 and CAV2 are involved in the formation of caveolae which are specialized invaginations of the plasma membrane that are rich in cholesterol and other lipids, and they take part in transcytosis. Caveolae recruit and compartmentalize various signaling molecules through direct physical interaction mediated by the cave-olin scaffolding domain (CSD) in CAV1. This interaction generally results in inhibition of signaling. Caveolins have been suggested as regulators of adult neural stem cell proliferation, as evidenced by increased proliferation of adult neural stem cells in Cav1, Cav2 and Cav3 knockout mice. The regulation by CAV1 of the endothelial nitric oxide synthase (eNOS), an enzyme that produces nitric oxide, is well documented, but the interaction of CAV1 and eNOS leads to eNOS inactivation and reduced nitric oxide production. Nitric oxide plays an important role in the regulation of many physiological functions in the cardiovascular system and the central and peripheral nervous systems. Nitric oxide produced in excessive amounts causes cytotoxicity, neurodegeneration, apoptotic cell death and circulatory failure.
Interestingly, CAV-1 scaffolding domain (amino acids 81–100) is able to bind α-synuclein – a member of the synuclein family implicated in the process of neuro-degeneration. Both α- and γ-synuclein are expressed in TM cells and participate in glaucomatous alterations. α- synuclein upregulates expression of CAV-1 which might be important in the pathogenesis of neurodegenerative disorders.
Elevated levels of CAV-1 and CAV-2 have been associated with several forms of cancer, Alzheimer disease, and other human diseases. One of the mechanisms underlying their involvement in pathology is linked to alterations of the cholesterol distribution in the cellular plasma membrane leading to the dysregulation of cholesterol homeostasis.

Caveolin SNPs and their possible implication in pathogenesis of POAG
Recently, a genome-wide association study (GWAS) confirmed that the identified SNPs (that interest CAV1/CAV2 genes) are associated with primary open-angle glaucoma(POAG) and that specific haplotypes located in the CAV1/CAV2 intergenic region are associated with the disease. It was also confirmed that associations with several CAV1/CAV2 SNPs are significant mostly in women.
Caveolins are implicated in a wide range of processes including modulation of the endothelial cell membrane, a process that participates in drainage of fluid (aqueous humor) from the eye. Loss of caveolin-1 function can cause increased expression of NOS3, suggesting that caveolin-1 could modulate IOP through a mechanism that includes eNOS. Moreover, recent data suggest that caveolin-1 is required for the activation of endothelial nitric oxide synthase in response to 17-beta-estradiol, providing additional evidence that these interactions could be sex related.
Our previous studies identifying an interaction between NOS3, postmenopausal hormone use and POAG in women suggested that sex may significantly influence specific pathways responsible for POAG development. So there is a possibility that sex and genotype have a synergistic effect on POAG risk. NOS3 SNPs were preferentially associated with women with the high-tension variant of POAG.
Endothelial cell function could broadly influence glaucoma pathogenesis; however, a major pathway for ocular fluid flow related to IOP involves the endothelium of Schlemm's canal, which connects the trabecular meshwork outflow pathways to the episcleral venous system. In response to increased fluid flow and increased IOP, Schlemm's canal inner wall endothelial cells form giant vacuoles. Vacuole formation involves modulation of endothelial cell signaling and vascular tone, processes that could be influenced by both caveolin-1 and eNOS. Interestingly, increased caveolin-1 expression has been observed soon after elevation of IOP in an in vitro glaucoma model. In the study, the CAV1/CAV2 SNPs were significantly associated with POAG overall; however, in the population, these associations were mostly significant in women, a finding that is supported by possible molecular interactions between caveolin, eNOS and 17beta-estradiol. These results, taken together with previous studies on the relation of NOS3, POAG and sex suggest that caveolin proteins, potentially through an interaction with eNOS and estrogen, may regulate IOP through a mechanism involving endothelial cell function.
Overall, the CAV1/CAV2 SNPs conferred modest risk to glaucoma susceptibility accounting for a small percentage of POAG heritability, suggesting that other, as yet unknown, genetic and/or environmental factors also contribute to this condition.

Common variants near CAV1 and CAV2 are associated with primary open-angle glaucoma, 2010

Expression of caveolin in trabecular meshwork cells and its possible implication in pathogenesis of primary open angle glaucoma, 2011

Common variants near CAV1 and CAV2 are associated with primary open-angle glaucoma in Caucasians from the USA, 2011

Caveolin: hypertension and insulinoresistence

Variants of the CAV1 gene are associated with hyperinsulinemia and IR in humans with hypertension. Probably decreased CAV1 levels lead to alterations in glucose metabolism. These findings have important clinical implications. First, they identify a genetic marker that might aid in identifying individuals at risk for metabolic disease. Second, this study identifies a novel pathway that contributes to IR in humans. New therapies targeting this pathway may provide individualized treatment to patients identified to have a defect in the CAV1 gene.
To assess the importance of CAV1 on the metabolic phenotype, it was used a CAV1 KO animal model. CAV1 KO mice have increased systolic blood pressure (SBP) levels (A), an exaggerated response to a glucose tolerance test (B), and greater fasting insulin ©.
CAV1 is a known regulator of insulin signaling and insulin receptor stability. Furthermore, depletion of CAV1 results in a 90% decrease in adipocyte insulin receptor levels in CAV1 KO mice. Although the role of CAV1 in insulin-mediated glucose uptake is less clear, CAV1 has also been shown to be involved in glucose transporter-4 translocation to the plasma membrane in both adipocytes and muscle cells. It is possible that alterations in the CAV1 gene are affecting one or both of these processes, leading to the hyperinsulinemic state.

Variants of the Caveolin-1 Gene: A Translational Investigation Linking Insulin Resistance and Hypertension, 2011