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INVITED REVIEW
Departments of Molecular Pharmacology and Medicine, and The Albert Einstein Cancer Center, Albert Einstein College of Medicine, Bronx, New York
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ABSTRACT |
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 TOP ABSTRACT CAV-1, CELL-CYCLE PROGRESSION,...ROLE OF CAV-1 IN...Loss of Cav-1 During...CAVEOLIN-1, TUMOR SUPPRESSORS,...CAV-1 IN HUMAN CANCERS:...CAV-1: IS IT A...OTHER CAVEOLIN GENES: ROLE...FUTURE DIRECTIONS: POTENTIAL...REFERENCES |
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caveolae; cellular signaling; tumor formation; cell invasiveness
In 1992, the first caveolar marker protein was identified and cloned, and was termed caveolin/VIP21 (now known as caveolin-1 or Cav-1) (92), thus paving the way for the molecular analysis of caveolar function. Caveolin was first isolated with the use of a screen to identify proteins that are tyrosine phosphorylated upon v-Src-mediated cell transformation (30–32). In contrast, VIP21 was cloned simultaneously as an integral membrane protein of trans-Golgi-derived transport vesicles (50).
In 2001, the development of Cav-1(–/–) null mice demonstrated an absolute requirement for Cav-1 in the formation of caveolae because virtually all tissues in Cav-1 null mice completely lack these organelles (16, 89). Currently, the functional roles attributed to caveolae and Cav-1 are quite diverse, ranging from vesicular transport (transcytosis, endocytosis, and potocytosis) and cholesterol homeostasis, to the suppression of cell transformation and the regulation of signal transduction (for a review of caveolae and vesicular trafficking/cholesterol, see Ref. 90).
The establishment of novel biochemical techniques to purify caveolae (by virtue of their unique biophysical properties) enabled the large-scale preparation of these organelles and the determination of their associated protein components (62, 97). With the use of Cav-1 as a marker protein, caveolae have now been demonstrated to concentrate a wide variety of signaling molecules, including glycosyl phosphatidylinositol-linked proteins, Src-family tyrosine kinases, H-Ras, heterotrimeric G protein subunits, PKC isoforms, and endothelial nitric oxide synthase (eNOS) (for a more complete list, see Ref. 90). These findings led to the proposal of the "Caveolae Signaling Hypothesis": that caveolae function to compartmentalize signaling molecules and to regulate signal transduction (61).
Interestingly, many signaling molecules have now been shown to directly interact with Cav-1. These interactions occur through a defined modular protein domain, known as the caveolin-scaffolding domain (CSD; residues 82–101 in Cav-1) (13). For example, in 1995, a Cav-1-derived mimetic peptide containing the CSD was shown to directly inhibit the activation of heterotrimeric G proteins (60). Since then, Cav-1 and its scaffolding domain have been shown to negatively regulate the signaling of many other proteins, including eNOS (29, 44, 71), epidermal growth factor receptor (EGFR) (14), Src tyrosine kinases (59), H-Ras (19), and c-Neu (18). Undoubtedly, the ability of Cav-1 and caveolae to modulate signaling has important implications for the process of cell transformation and tumor formation.
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CAV-1, CELL-CYCLE PROGRESSION, AND ERK-1/2 ACTIVATION |
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TOPABSTRACTCAV-1, CELL-CYCLE PROGRESSION,...ROLE OF CAV-1 IN...Loss of Cav-1 During...CAVEOLIN-1, TUMOR SUPPRESSORS,...CAV-1 IN HUMAN CANCERS:...CAV-1: IS IT A...OTHER CAVEOLIN GENES: ROLE...FUTURE DIRECTIONS: POTENTIAL...REFERENCES |
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For example, Galbiati and colleagues (27) have shown that Cav-1 expression levels are negatively regulated by growth factor stimuli and that recombinant overexpression of Cav-1 can inhibit cellular proliferation, by mediating cell cycle arrest in G0/G1. In addition, Cav-1(–/–) null mouse embryonic fibroblasts (MEFs) show increased proliferation rates, concomitant with increased S-phase fractions and decreased G0/G1 fractions, as well as altered expression of several cell cycle-related proteins (89, 121). More specifically, the loss of Cav-1 results in decreased expression of p21Cip1, cyclin D1, and PCNA overexpression, as well as hyperactivation of the Ras-p42/44 MAP kinase cascade.
Elucidation of the mechanisms by which Cav-1 exerts its negative regulatory control of cell proliferation has uncovered some novel molecular pathways. For example, Cav-1 normally functions as a transcriptional repressor of Cyclin D1, an important regulatory component of the cyclin-cdk complex that phosphorylates Rb, thereby controlling entry into S-phase (40). Furthermore, the induction of G0/G1 arrest by Cav-1 overexpression appears to operate through a p53/p21Cip1-dependent pathway (27).
Cav-1 also controls signaling along the Ras-p42/44 MAP kinase cascade. Many components of Ras-p42/44 MAP kinase signaling appear to be compartmentalized within caveolae, including growth factor receptors (such as Neu/Erb-B2 and EGFR), Ras, Raf-1, MEK-1/2, and ERK-1/2 (64, 67, 68, 72, 93, 105, 106). In addition, Cav-1 directly inhibits ERK-1/2 activation, both in vitro and in vivo (17, 131). In NIH-3T3 cells, antisense mediated downregulation of Cav-1 is sufficient to cause the constitutive hyperactivation of ERK-1/2 signaling (26). Furthermore, hyperactivation of ERK-1/2 also occurs in Cav-1(–/–) null mouse cells and tissues under various conditions (9, 12, 121). Interestingly, there appears to be a reciprocal relationship between Cav-1 and ERK-1/2 because activation of the Ras-p42/44 MAP kinase cascade causes the downregulation of Cav-1 expression in NIH-3T3 cells and in the mammary gland (21). Thus it appears that Cav-1 functions as a natural endogenous inhibitor of the p42/44 MAP kinase cascade.
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ROLE OF CAV-1 IN PROGRAMMED CELL DEATH: PRO- OR ANTI-APOPTOTIC? |
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TOPABSTRACTCAV-1, CELL-CYCLE PROGRESSION,...ROLE OF CAV-1 IN...Loss of Cav-1 During...CAVEOLIN-1, TUMOR SUPPRESSORS,...CAV-1 IN HUMAN CANCERS:...CAV-1: IS IT A...OTHER CAVEOLIN GENES: ROLE...FUTURE DIRECTIONS: POTENTIAL...REFERENCES |
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Currently, the role of Cav-1 in apoptosis remains controversial. On one hand, caveolae are highly enriched in both sphingomyelin (a precursor of ceramide) and sphingomyelinase (the enzyme responsible for generating ceramide) (65). Ceramide induces cell death by inhibiting the well-characterized phosphatidylinositol 3-kinase (PI3-kinase)/Akt survival pathway (133). Interestingly, Cav-1 has been shown to interact with PI3-kinase and Cav-1 overexpression sensitizes fibroblasts to ceramide-induced death, through a PI3-kinase-dependent mechanism (134). Furthermore, we have demonstrated that Cav-1 expression sensitizes both NIH-3T3 fibroblasts and T24 bladder carcinoma cells to cell death initiated by staurosporine, a chemical inducer of apoptosis (63).
In contrast, disruption of caveolae using cholesterol-sequestering agents has been shown to block IL-6 and IGF-1-induced activation of the PI3-kinase/Akt signaling pathway (83). Therefore, caveolae and Cav-1 are required to mediate proper survival signals through the PI3-K/Akt pathway. Moreover, caveolin-1 overexpression in Rat1A cells and human prostate cancer cells (LNCaP), or Cav-1 upregulation in androgen-insensitive LNCaP clones renders these cells more resistant to apoptosis (113, 114). In addition, antisense mediated downregulation of Cav-1 results in prostate cancer cells that are more sensitive to apoptosis (57, 58, 74). Finally, Li and colleagues (57) have demonstrated that caveolin-1 overexpression mediates cell survival by sustaining Akt activation, through the binding and inhibition of the serine/threonine protein phosphatases, namely PP1 and PP2A. Taken together, these results argue that Cav-1 also has anti-apoptotic activities.
The apparent incongruity of the pro-apoptotic or anti-apoptotic functions of Cav-1 may be explained by cell-type specific effects: anti-apoptotic activity in prostate cancer cells and proapoptotic activity in other cell types. Alternatively, the disparate effects of Cav-1 may be due to the use of different apoptotic inducers. Additional studies will be necessary to distinguish between these two possibilities.
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Loss of Cav-1 During Cell Transformation |
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TOPABSTRACTCAV-1, CELL-CYCLE PROGRESSION,...ROLE OF CAV-1 IN...Loss of Cav-1 During...CAVEOLIN-1, TUMOR SUPPRESSORS,...CAV-1 IN HUMAN CANCERS:...CAV-1: IS IT A...OTHER CAVEOLIN GENES: ROLE...FUTURE DIRECTIONS: POTENTIAL...REFERENCES |
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CAVEOLIN-1, TUMOR SUPPRESSORS, AND ONCOGENES |
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TOPABSTRACTCAV-1, CELL-CYCLE PROGRESSION,...ROLE OF CAV-1 IN...Loss of Cav-1 During...CAVEOLIN-1, TUMOR SUPPRESSORS,...CAV-1 IN HUMAN CANCERS:...CAV-1: IS IT A...OTHER CAVEOLIN GENES: ROLE...FUTURE DIRECTIONS: POTENTIAL...REFERENCES |
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Two important properties of neoplastic cells are immortalization and transformation. In some sense, all cells within a microenvironment undergo the process of natural selection, and those cells that are able to more quickly attain immortalization and transformation are more likely to survive and propagate. Therefore, it is important to determine the relative contribution of different genes in promoting immortalization and/or transformation.
While Cav-1 does not appear to have a direct role in immortalization, Cav-1 does synergize with other immortalizing genes. Loss of the INK4a locus, encoding both p16INK4a and p19ARF cell cycle regulators, is sufficient to allow cells to become immortalized. We have demonstrated that concomitant loss of Cav-1 and INK4a results in cells with a striking proliferative advantage, demonstrating that the loss of Cav-1 expression cooperates or synergizes with genetic mutations that abolish INK4a function (121). Furthermore, transformation of INK4a (–/–)/Cav-1(–/–) fibroblasts with various oncogenes [H-Ras (G12V) or v-Src] renders these cells more neoplastic, generating up to 40-fold larger tumors in nude mice (Fig. 3). These results suggest that mutation or downregulation of caveolin-1 expression, in combination with an INK4a mutation, would impart cells with a profound neoplastic advantage over those cells with a mutation in either gene alone.
Another well-characterized tumor suppressor, p53, appears to be directly involved in regulating Cav-1 expression. Razani et al. (88) have demonstrated that p53 is a positive transcriptional and translational regulator of Cav-1, and that inactivation of p53 through viral oncoproteins results in reduced Cav-1 expression. Cav-1 levels are also dramatically reduced in p53-deficient fibroblasts (56). In support of these cellular findings, we have demonstrated that p53 (–/–) null mice show dramatically reduced levels of Cav-1 in vivo (121). However, a molecular dissection of the transcriptional or translational regulatory aspects of this relationship awaits further studies.
Preliminary evidence also suggests that Cav-1 may have a role in regulating PTEN activity as a substantial amount of PTEN (a phosphatase possessing tumor suppressor functions) localizes to caveolae and specifically forms molecular complexes with Cav-1 (11). Aldred et al. (1) have suggested that this relationship has implications for follicular thyroid neoplasms because Cav-1 and PTEN levels are simultaneously downregulated, suggesting perhaps that the downregulation of Cav-1 may contribute to dysregulation of PTEN function.
A whole host of cellular oncogenes have been shown to reduce Cav-1 expression in cells (Table 1). These include, but are not limited to, c-Myc, HPV E6, v-Abl, Bcr-Abl, H-RasG12V, v-Src, and Neu/ErbB2 (18, 48, 79, 113). Virtually all of these oncoproteins appear to downregulate Cav-1 expression through transcriptional mechanisms. In addition, Ras- and Raf-mediated downregulation of Cav-1 relies upon ERK-1/2 activation, as ERK-1/2 inhibition restores Cav-1 expression in Ras- and Raf-transformed cells, as well as in human fibrosarcoma cells (21, 117). Activation of other signaling molecules has also been demonstrated to downregulate Cav-1 expression, including PKA and PKC-
(21, 123).
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CAV-1 IN HUMAN CANCERS: CHROMOSOMAL LOCALIZATION, GENE MUTATIONS, AND COMPLEX EXPRESSION PATTERNS |
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TOPABSTRACTCAV-1, CELL-CYCLE PROGRESSION,...ROLE OF CAV-1 IN...Loss of Cav-1 During...CAVEOLIN-1, TUMOR SUPPRESSORS,...CAV-1 IN HUMAN CANCERS:...CAV-1: IS IT A...OTHER CAVEOLIN GENES: ROLE...FUTURE DIRECTIONS: POTENTIAL...REFERENCES |
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Importantly, sequence analysis of Cav-1 in human tumors has also revealed sporadic mutations. In a cohort of patients with primary breast cancer, Hayashi and colleagues (36) detected a sporadic P132L mutation in up to 16% of the cases examined. Furthermore, this mutation induces cellular transformation, acts in a dominant negative fashion by causing the mislocalization and intracellular retention of wild-type Cav-1, and causes ERK-1/2 hyperactivation (Fig. 4D) (36, 52). Interestingly, this mutation is analogous to the CAV-3 (P104L) mutation that is associated with several skeletal muscle disorders, including autosomal dominant limb-girdle muscular dystrophy (LGMD-1C) (73). Independently, another group has recently identified novel Cav-1 mutations in human oral squamous cell cancers (34).
Cav-1 expression has now been assessed in a wide range of human tumors (Table 2). On the basis of the transformation suppressor activity of Cav-1 in cultured cells, we would predict that human tumors would show reductions in Cav-1 expression. However, Cav-1 expression levels are reduced, unchanged, or upregulated, depending on the tumor cell type. Interestingly, within tumor types derived from the same cell type or tissue, Cav-1 expression levels are consistently upregulated or downregulated in the majority of cases. For instance, Cav-1 downregulation is typical of ovarian, lung, and mammary carcinomas, as well as mesenchymal sarcomas. On the other hand, Cav-1 is consistently upregulated in bladder, esophagus, thyroid (papillary subtype), and prostate carcinomas, with some exceptions (82). Further research will assist in identifying the modifying factors that determine whether it is the upregulation or downregulation of Cav-1 in these latter tumor types that facilitates tumorigenesis. Such Cav-1 upregulation may be due to the observation that Cav-1 levels are increased during multidrug resistance (4, 51). However, multidrug resistant cells with higher levels of Cav-1 show significant reductions in anchorage-independent growth.
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In other reports (37, 43, 46, 75), Cav-1 overexpression has been correlated with metastasis in esophageal squamous cell carcinoma, clear cell renal cell carcinoma, mammary adenocarcinoma, and metastatic cell lines derived from lung adenocarcinoma. These results have implications for those human cancers where Cav-1 is consistently downregulated and suggest that the upregulation of Cav-1 may represent an acquired feature that contributes to a metastatic phenotype. This concept of a "biphasic" expression pattern for Cav-1 was elegantly demonstrated in colon carcinoma cells after selection for metastatic variants (5).
This notion of two opposing roles for a protein, depending on the stage of tumor progression, is not unique. In early stages of transformation and carcinogenesis, transforming growth factor-
(TGF-) demonstrates tumor suppressive activity, by inducing cell cycle arrest and apoptosis, thereby inhibiting primary tumor growth (6, 91). However, at later stages of tumor progression, increased secretion of TGF- by tumor and stromal cells promotes tumor invasion, angiogenesis, and metastasis, as well as immunosuppression of host surveillance mechanisms. Therefore, the role of TGF- in tumorigenesis can be either suppressive or promoting, depending on the tumor developmental stage. Siegel et al. (103) definitively demonstrated these contrasting roles for TGF- in breast cancer by interbreeding transgenic mice expressing either constitutively activated or dominant negative forms of TGF- to MMTV-Neu/ErbB2 transgenic mice.
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CAV-1: IS IT A TUMOR SUPPRESSOR, AN ONCOGENE, OR BOTH? |
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TOPABSTRACTCAV-1, CELL-CYCLE PROGRESSION,...ROLE OF CAV-1 IN...Loss of Cav-1 During...CAVEOLIN-1, TUMOR SUPPRESSORS,...CAV-1 IN HUMAN CANCERS:...CAV-1: IS IT A...OTHER CAVEOLIN GENES: ROLE...FUTURE DIRECTIONS: POTENTIAL...REFERENCES |
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Tyrosine Phosphorylation
The dually contrasting roles for Cav-1 in tumor progression may be partly explained by the observation that Cav-1 has several peptide domains with opposing functions. A molecular dissection of the Cav-1 protein has revealed distinct regions that may counteract the effects of the growth-inhibitory CSD (Fig. 5). First, Tyr14 at the extreme NH2 terminus is important for the binding and recruitment of a c-Src/Grb7 signaling complex (55). This residue is constitutively phosphorylated in v-Src- and v-Abl-transformed cells, transiently phosphorylated during growth factor stimulation in other cells, and localizes to focal adhesions, which are the predominant sites of tyrosine kinase signaling (Fig. 6A). Functionally, tyrosine 14-phosphorylated Cav-1 binds Grb7 and enhances both anchorage-independent growth (Fig. 6B) and EGF-stimulated cell migration (55). Thus tyrosine phosphorylated Cav-1 may function like a growth factor receptor that recruits SH2 domain-containing proteins to the plasma membrane. Cav-2 also undergoes phosphorylation (at Tyr19 and Tyr27) and similarly recruits SH2 domain-containing proteins, such as c-Src, Nck, and Ras-GAP (53).
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A second region of Cav-1 also appears to have growth-stimulatory properties. Serine phosphorylation of Cav-1 changes its topology, thereby converting Cav-1 from an integral membrane protein to a secreted protein product. Normally, the majority of Cav-1 is associated with the plasma membrane. However, Cav-1 also appears to be secreted, especially in exocrine cell types, where it is packaged into secretory vesicles (66, 101). Phosphorylation of Cav-1 at Ser80 directly regulates its conversion to a secreted protein. For example, mutation of Ser80 to glutamate (S80E, which mimics chronic phosphorylation) results in preferential targeting of Cav-1 to the ER membrane and directs its subsequent packaging for secretion (Fig. 7) (101). Furthermore, phosphorylation at Ser80 is required for proper secretion because its mutation to alanine (S80A; which abrogates phosphorylation) results in no detectable secretion of Cav-1 (101), concomitant with its intracellular accumulation (Fig. 7A, bottom). Mechanistically, in terms of cellular transformation, shunting Cav-1 for secretion to the extracellular environment would subvert its normal intracellular tumor suppressive functions. Essentially, these changes in Cav-1 membrane topology have the same consequences as a loss of Cav-1 expression.
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Dominant-Negative Point Mutations
Finally, the previously mentioned identification of Cav-1 (P132L) mutations in up to 16% of human breast cancers provides a third distinct mechanism to inactivate the tumor suppressor function of caveolin-1. This mutation drives cellular transformation in NIH 3T3 cells. Briefly, NIH 3T3 cells expressing Cav-1 (P132L) show augmented growth in soft agar, as well as increased invasiveness, and increased chemotaxis (36).
Thus these three defined mechanisms provide a clear explanation for how the growth-inhibitory functions of the CSD may be subverted.
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OTHER CAVEOLIN GENES: ROLE OF CAVEOLIN-2 AND CAVEOLIN-3 |
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TOPABSTRACTCAV-1, CELL-CYCLE PROGRESSION,...ROLE OF CAV-1 IN...Loss of Cav-1 During...CAVEOLIN-1, TUMOR SUPPRESSORS,...CAV-1 IN HUMAN CANCERS:...CAV-1: IS IT A...OTHER CAVEOLIN GENES: ROLE...FUTURE DIRECTIONS: POTENTIAL...REFERENCES |
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Despite the fact that the CAV-2 gene is co-localized with CAV-1 at the 7q31.1/D7S522 locus (20), the bulk of caveolae-related cancer research has focused on Cav-1. This is primarily due to the early observation that Cav-2 levels are not downregulated in response to oncogenic transformation, in contrast to Cav-1 (18, 100).
Recent research, however, is beginning to implicate the other caveolin family members in human cancers. Cav-2 expression has been detected in various lung cancers using a cDNA microarray approach, and its expression correlates with shorter survival in stage I adenocarcinomas (119). In addition, Cav-2 upregulation is also observed in esophageal and bladder carcinomas (24, 39). In another study on germ cell tumors of the testis, Cav-3 overexpression was detected in seminomas (45). Future research will undoubtedly uncover altered Cav-2 and Cav-3 expression patterns within other human tumors, therefore necessitating studies to address their functional importance in tumor growth and metastasis.
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FUTURE DIRECTIONS: POTENTIAL CAVEOLIN-BASED ANTI-CANCER THERAPIES |
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TOPABSTRACTCAV-1, CELL-CYCLE PROGRESSION,...ROLE OF CAV-1 IN...Loss of Cav-1 During...CAVEOLIN-1, TUMOR SUPPRESSORS,...CAV-1 IN HUMAN CANCERS:...CAV-1: IS IT A...OTHER CAVEOLIN GENES: ROLE...FUTURE DIRECTIONS: POTENTIAL...REFERENCES |
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-catenin transcriptional activation, and results in increased invasiness in neoplastic cells. Because invasiveness and the loss of E-cadherin expression are common features of metastatic cells, these results have important implications for metastasis. Likewise, a novel association among caveolae, caveolin-1, and matrix metalloproteinases (MMPs), a family of matrix-degrading enzymes that facilitate invasion/metastasis, is being established. Several groups have shown that MT1-MMP and MMP-2 are localized to caveolae and that caveolae are required for proper MT1-MMP localization and function (2, 28, 85). Furthermore, we (122) have recently shown that recombinant expression of Cav-1 in mammary epithelial cells suppresses their metastatic capacity, inhibits their invasiveness, and prevents their ability to secrete MMPs (MMP-2/9). Similar results were obtained using a cell-permeable peptide containing the CSD (Cav-1; residues 82–101). Thus it may be fruitful to develop novel anticancer therapies that mimic the activity of the CSD.
Because of the heterogeneity of Cav-1 expression in different tumors, therapies targeting Cav-1 will need to consider the specific roles or functions of this protein within that particular type of tumor cell. In one interesting gene therapy strategy, the Cav-1 promoter was used to specifically target prostate cancer cells in vitro and in vivo with minimal toxicity (84).
Another type of anti-tumor therapy is based on the observation that Cav-1 is highly expressed in endothelium. Therefore, targeting a tumor's blood supply provides an interesting opportunity for Cav-1-directed therapies. In exciting confirmation of this hypothesis, Gratton et al. (33) have demonstrated that a cell-permeable peptide derived from the CSD markedly reduced tumor progression in mice by regulating microvascular permeability.
In summary, current research has clearly established a role for Cav-1 as a "tumor and metastasis-modifying gene." Future studies will undoubtedly reveal novel relationships between Cav-1 and a variety of signaling pathways, and offer exciting opportunities to develop anti-cancer therapies that target Cav-1 and caveolae, both in primary tumors and in metastatic disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTCAV-1, CELL-CYCLE PROGRESSION,...ROLE OF CAV-1 IN...Loss of Cav-1 During...CAVEOLIN-1, TUMOR SUPPRESSORS,...CAV-1 IN HUMAN CANCERS:...CAV-1: IS IT A...OTHER CAVEOLIN GENES: ROLE...FUTURE DIRECTIONS: POTENTIAL...REFERENCES |
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8-fold increase for H-Ras-transfected cells and 
