Given the importance of CAFs in the modulation of breast cancer growth, metastasis and response to therapy, understanding the molecular mechanisms driving CAFs behavior and finding CAFs markers for breast cancer patient stratification has been the focus of research recently58–61.
Amidst these biomarkers, changes in the expression levels of Caveolin-1 (CAV1) in CAFs have been associated with the metabolic reprogramming of the tumor87,88,294 and the modification of
the ECM architecture101, which in turn modulate breast cancer cell growth and metastasis.
1.4.1 Caveolae: Specialized Membrane Microdomains
CAV1 is a scaffolding protein with multiple binding partners that is a key structural component295–297, along with cavins298,299, of cell surface caveolae. Caveolae are non-planar lipid rafts that appear as 50-100nm plasma membrane invaginations under electron
38 of membrane distribution302 and abundance in different cell types303. While endothelial, smooth-muscle, adipocyte and fibroblast cells display a high abundance of caveolae304, lymphocytes, neurons, and hepatocytes show low, but functionally important levels of caveolae305–307.
Figure 9. Caveolae structural organization. EM micrographs (left, top to bottom) showing ultrastructure of caveolae in fibroblasts and complex arrangements of caveolae in cultured adipocytes and in skeletal muscle. Scale bar: 100 nm. Caveolae schematic representation (right). Reproduced with permission from303.
As lipid rafts, caveolae constitute specialized membrane microdomains that pre-organize,
sequester and compartmentalize signaling molecules for efficient signal transduction308. For
instance, the GTPase HRAS309,310, SRC kinases311 and endothelial nitric oxide synthase
(eNOS)312–314 signaling can be regulated by caveolae compartmentalization.
Besides regulating signaling cascades through compartmentalization, caveolae are involved in: (i) detecting physical stimuli like shear stress and membrane tension (mechanosensing)303, (ii) clathrin-independent endocytosis303, (iii) calcium signaling, through the formation of
junctional complexes coupling the plasma membrane with the endoplasmic reticulum (ER)315,
and (iv) cholesterol transport316,317.
In this regard, caveolar lipid rafts are enriched in cholesterol, along with saturated fatty acids and sphingolipids. Moreover, such is the importance of cholesterol, that not only it is essential for caveolae formation318, but also regulates CAV1 expression (by binding to steroid regulatory elements, SREs, of its promoter)316,319. In addition, CAV1 is a cholesterol-binding protein320 that shuttles cholesterol between Golgi, the plasma membrane, and mitochondria. In fact, CAV1 deficiency leads to cholesterol accumulation in mitochondrial membranes, driving mitochondrial dysfunction and aerobic glycolysis317.
1.4.2 Caveolin-1 (CAV1): Structure Related to Function
In order to allow caveolae formation and carry out the aforementioned functions, CAV1 shows a very particular structure (Fig. 10). CAV1 is a 22 kDa integral membrane protein, with a putative hairpin domain embedded within the membrane and both the amino- and carboxy- terminus facing the cytoplasm. This unique configuration is due to the presence of an intramembrane domain (102–134 aa) that prevents CAV1 from completely spanning the
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Figure 10. Domain organization of CAV1. Graphic representation of CAV1 domain organization. CAV1 palmitoylation (Palm) sites, involved in CAV1 targeting to lipid rafts are indicated in green, a Tyrosine phosphorylation (P) site (Y14) in orange, and the starting Methionine (M32) of CAV1β isoform is shown in black. Adapted from303.
Other important domains include the oligomerization domain (61–102 aa), which mediates the
homo-oligomerization of CAV1 necessary for caveolae formation322. The oligomerization
domain also includes the Caveolin scaffolding domain (CSD), not only key for the interaction of CAV1 with caveolae-associated proteins such as eNOS, tyrosine kinases (TKs) and G- protein subunits323, but also important for cholesterol binding324.
Regarding its synthesis, CAV1 is produced in the rough endoplasmic reticulum (RER) as an integral membrane protein, then it traffics through the Golgi complex, where it associates with cholesterol and forms higher-order oligomers, to the cell surface via discrete carriers303.
1.4.3 Caveolae and Caveolin-1 in Disease
Although CAV1 and caveolae are dispensable for life and mice deficient for them are viable, lack of caveolae, either as a result of mutations or gene expression changes in CAV1, results in a variety of diseases, including muscular dystrophy, lipodystrophy, cardiovascular disease and cancer303,308,325.
Even if CAV1 is involved in cancer progression, its role is still unclear. CAV1 deficiency leads to increased proliferation, tumorigenesis326–328 and metastasis90. However, a tumor-promoting role has been reported in prostate cancer329 and melanoma330. These contrasting observations could be explained by differences in which specific tumor compartment is affected by the CAV1 loss (either cancer or stromal cells) and the diverse tumor stages reported.
Regarding CAV1 levels in the tumor stroma, although multiple mechanisms have been proposed to explain the deregulation of CAV1 levels in CAFs, the reason behind these changes still remains a puzzle128,323. While some studies have associated loss of Caveolin-1 (CAV1) in CAFs with poor clinical outcomes in breast cancer in terms of decreased survival, early tumor recurrence, lymph node metastasis and resistance to tamoxifen89,90,331–334, exceptions have
been reported101. CAV1 thus appears to have a complex role in tumor stroma, and further work
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1B. AIMS & OBJECTIVES
The general aim of the research embodied in this thesis report was to explore and contribute further knowledge regarding the impact of the expression levels of stromal CAV1, on breast cancer progression, and assess potential translational opportunities for future studies.
In this regard, previous data had been obtained using mice models where either tumor cells or the stromal component were of murine origin. Precedent studies had shown how data derived from these systems offered mixed results when translated into a clinical setting.
In an attempt to overcome these problems and define with greater precision cancer cell - stromal CAV1 interactions, both the stromal compartment (CAFs) and cancer cells of the presented tumor xenograft are of human origin.
Our specific objectives were:
1. To develop and validate strategies to functionally and mechanistically dissect the specific contribution of stromal CAV1 levels to tumor biology, both in vitro and in vivo. In order to do so, different tailored strategies for the perturbation of CAV1 protein expression in a selective manner were to be deployed and tested.
2. To assess the functional impact on tumor growth and progression in vivo of such selective intervention of stromal CAV1, using state-of-the-art xenograft-based mouse models, which allow for the full characterization of established human breast cancer models, including the recapitulation of the metastatic process.
3. To gain unbiased mechanistic insight on the contribution of stromal CAV1 protein levels, both to basic stromal cell biology as well as at systems-level tumor behavior. A core component of these studies was to be founded on detailed transcriptome profiling across different contexts (in vitro versus in vivo).
4. To explore the therapeutic potential of pharmacological interventions leveraging the novel knowledge generated by our functional and molecular studies.
5. To contribute functional screens for small molecules potentially capable of intervening CAV1 protein levels on cancer-associated fibroblasts (CAFs).
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