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Caveolin-1 as a potential new therapeutic target in multiple myeloma
Cancer Letters 233 (2006) 10–15 www.elsevier.com/locate/canlet
Mini-review
Caveolin-1 as a potential new therapeutic target in multiple myeloma* Klaus Podar*, Kenneth C. Anderson* Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Jerome Lipper Multiple Myeloma Center, 44 Binney Street, Boston, MA 02115, USA Received 18 February 2005; accepted 23 February 2005
Abstract Caveolae are specialized flask-shaped lipid rafts enriched in cholesterol, sphingolipids, and structural marker proteins termed caveolins. Caveolins are highly conserved hairpin loop-shaped, oligomeric proteins of 22–24 kDa. Besides the plasma cell membrane, caveolins are also present in mitochondria, the endoplasmatic reticulum, the Golgi/trans-Golgi network, and secretory vesicles. They play a critical role in normal vesicular transport, cholesterol homeostasis, and signal transduction. Conversely, dysregulation of caveolin-1 has been associated with several human diseases including multiple myeloma, an incurable malignancy characterized by excess monoclonal plasma cells within the bone marrow. In this mini-review, we characterize the functional role of caveolin-1 in multiple myeloma, and present the preclinical rationale for novel potential therapeutic approaches targeting caveolin-1 in multiple myeloma. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Caveolin-1; Multiple myeloma; IL-6; IGF-1; VEGF
1. Introduction Multiple myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells in the bone marrow (BM) associated with bone lesions, renal dysfunction, and immunodeficiency [1]. Despite high dose therapy and more recent novel therapeutic approaches, the median survival remains at 4–5 years. * This work was supported by National Institutes of Health Grants IP50 CA100707, PO-1 78378, and RO-1 CA 50945. * Corresponding authors. Tel.: C1 617 632 2144; fax: C1 617 632 2140. E-mail addresses: [email protected] (K. Podar), [email protected] (K.C. Anderson).
The annual incidence of MM is approximately 3.8 per 100,000 population (approximately 14,400 new cases of MM/year), and it is almost twice as common in the black versus Caucasian population [2]. The increased incidence of MM in African-Americans could, at least in part, be related to increased serum cholesterol levels [3] since cholesterol is an essential component of MM cell membranes, it has been implicated in disease pathogenesis [4], and is associated with higher mortality in black versus Caucasian men [5]. In support of this view, a prospectively studied cohort of more than 900,000 US adults showed that increased body weight is associated with increased death rates from cancers, including MM [6].
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.02.035
K. Podar, K.C. Anderson / Cancer Letters 233 (2006) 10–15
Lipid rafts are ‘liquid-ordered’ membranous microdomains with a unique lipid composition (enrichment in cholesterol, sphingolipids, and phosphatidylethanolamine) in the otherwise homogeneous, phospholipid-rich, two-dimensional lipid layer of the plasma cell membrane [7]. Caveolae (‘little caves’) are specialized lipid raft microdomains, which classically form dynamic, flask-shaped, vesicular invaginations (50–100 nm) and are functionally implicated in pinocytosis, transcytosis and endocytosis; cholesterol homeostasis; cell transformation; and signal transduction modulating cell growth, survival, adhesion, and migration [8,9]. Typically, caveolae require the presence of functionally highly conserved, hairpin loop-shaped, 22–24 kDa, oligomeric proteins, termed caveolins, in particular caveolin-1 (Cav-1) [10–13]. Expression of caveolins is very heterogeneous in different cells: in contrast to Cav-3, which is specifically expressed in muscle cells, Cav-1 isoforms (Cav-1a, containing residues 1–178; and Cav-1b, containing residues 32–178) and also Cav-2 isoforms (full-length Cav-2a and two truncated variants, Cav-2b and Cav-2g) are expressed in most cell types including fibroblasts, adipocytes, endothelial cells, and type I pneumocytes, but not in human peripheral blood cells or myeloid, lymphoid, and erythroid cell lines [14–16]. Dysregulation of Cav-1 is associated with several human malignancies, including MM. Initial studies have demonstrated that Cav-1 negatively regulates several signaling molecules, thereby mediating cell growth inhibition. However, more recent studies show that Cav-1 can also function as a tumor-promoter, dependent on the tumor type and the tumor stage; as well as promote acquisition of multidrug resistance [9,17]. The tumor-suppressor function of Cav-1 is mediated via the Cav-1 scaffolding domain (CSD), i.e. by direct inhibition of heterotrimeric G proteins [18]. Conversely, the tumor-promoter function may be mediated via: (1) growth-factor-induced tyrosinephosphorylation of Cav-1 (Tyr14), which recruits SH2-domain-containing proteins to the plasma membrane, thereby enhancing activation of downstream signaling proteins [19,20]; (2) growth-factor-induced serine-phosphorylation of Cav-1 (Ser80), which converts Cav-1 to a secreted protein, which may function as an autocrine or paracrine growth and survival factor [21]; and (3) up-regulation of Cav-1
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expression by direct binding of cholesterol to the Cav-1 promoter [22,23]. Increased levels of cholesterol also contribute to enhanced production of hormones including testosterone, growth factors, and lipid raft/caveolae formation. Finally, a P132L mutation of Cav-1 observed in human breast cancer cells is associated with increased cell growth, invasiveness, and chemotaxis in NIH3T3 cells [24]. Importantly, Cav-1 over-expression is also present in multidrug resistant (MDR) colon and human breast adenocarcinomas [25].
2. Pathophysiologic roles of Cav-1 in MM In contrast to normal B cells, Cav-1 is expressed in MM cell lines and patient MM cells. Moreover, Cav-1 expression is upregulated in MM versus monoclonal gammopathy of undetermined significance (MGUS), suggesting a role for caveolae in the transition of MGUS to MM and MM pathogenesis [20]. Studies are now underway to evaluate whether Cav-1 expression may, as in prostate cancer [26], function as a diagnostic and prognostic marker in MM. Our recent studies suggest a functional model in which caveolae play a pivotal role in interleukin-6 (IL-6)-, insulin-like growth factor-1 (IGF-1)-, and vascular endothelial growth factor (VEGF)-induced signaling, thereby enhancing MM cell survival and conferring resistance to dexamethasone, as well as modulating MM cell migration [20,27]. Specifically, both IL6-receptor signal transducing chain gp130 and IGF-1 receptor constitutively co-localize with Cav-1 in the lipid raft fraction isolated by sucrose density gradient separation. IL-6 induces Src-family kinasedependent tyrosine phosphorylation of Cav-1 (Tyr14) and triggers subsequent formation of gp130$SHPTP2/PI3-K-containing complexes. Conversely, disruption of caveolae inhibits complex formation and subsequent PI3-K/Akt-1, as well as STAT 3, activation. IGF-1 induces Src-family kinase-dependent tyrosine phosphorylation of Cav-1 (Tyr14) and subsequent tyrosine-phosphorylation of IRS-1, which is required for transducing downstream signals via interaction with SH2-domains on other proteins (e.g. the regulatory p85 subunit of PI3-K). Disruption of caveolae also inhibits IGF-1-triggered phosphorylation of IRS-1 and downstream activation
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K. Podar, K.C. Anderson / Cancer Letters 233 (2006) 10–15
of PI3-K/Akt-1. In contrast, ERK signal transduction is not modulated by disruption of caveolae. Taken together, these data demonstrate that caveolae and Cav-1 mediate IL-6- and IGF-1-triggered survival signals through both the PI3-K/Akt-1, as well as the STAT3, pathways [20]. VEGF levels and increased angiogenesis in the bone marrow are correlated with clinical outcome in MM. The multiple functional sequelae of VEGF are due to its broad spectrum of target cells. Besides its regulatory role in physiologic and pathologic angiogenesis, VEGF also triggers growth, survival, and migration of MM cells via paracrine and autocrine pathways; inhibits maturation of dendritic cells, thereby contributing to immunodeficiency; and increases bone-resorbing activity, thereby contributing to bone destruction in MM [28]. Cav-1 has an important role in mediating cell motility: specifically, Cav-1 accumulates at the leading edge of cultured fibroblasts [13], human umbilical vein smooth muscle cells [29], and Cav-1-deficient FRT cells expressing recombinant Cav-1 [30]; as well as at the trailing edge of bovine aortic endothelial cells [31]. In MM cells, VEGF receptor-1 (Flt-1) is associated with Cav-1; and VEGF binding triggers Src-dependent tyrosine phosphorylation of Cav-1, which is required for p130Cas phosphorylation and MM cell migration. Conversely, depletion of Cav-1 by antisense methodology abrogates VEGF-triggered MM cell migration and associated p130Cas phosphorylation, further confirming the essential role of Cav-1 in MM cell migration [27]. Bortezomib, a promising novel anti-MM agent recently approved by the United States Food and Drug Administration (FDA) for therapy of patients with progressive MM after previous treatment, targets proteins related to apoptosis, growth signaling/cell cycle, heat shock proteins, and the proteasome pathway. We recently identified Cav-1 as an additional target: Bortezomib inhibits VEGF-triggered Cav-1 phosphorylation and later downregulation of Cav-1 in MM cells. Furthermore, Bortezomib also inhibits VEGF-triggered Cav-1 phosphorylation in endothelial cells and thereby abrogates endothelial cell migration within the bone marrow environment of MM patients. These findings demonstrate multiple facets of the anti-myeloma activity of Bortezomib and further support novel treatment strategies targeting caveolae in MM [27].
3. Potential new therapeutic approaches to target Cav-1 and caveolae in MM In contrast to plasma cells, Cav-1 is expressed in MM cell lines and patient MM cells and mediates tumor cell survival and VEGF-triggered migration; blocking its function abrogates these sequelae. Moreover, targeting Cav-1 in other cells within the MM BM microenvironment, e.g. endothelial cells, may also inhibit their tumor promoting activity and angiogenesis. Therefore, therapeutics developed to target components of caveolae, e.g. cholesterol, and Cav-1 may decrease MM progression and overcome drug resistance, e.g. to dexamethasone. Potential therapeutic approaches to target caveolae include the following. 3.1. Targeting Cav-1 and caveolae by modulating cholesterol-production and caveolar lipid composition Cav-1 directly binds cholesterol, a cofactor in formation of caveolae, forming homo- and heterooligomers [13,32,33]. The threshold of plasma membrane cholesterol below which caveolae cannot be formed is 40 mol% [33]. Cav-1 mRNA levels are up- and down-regulated by cholesterol and oxysterols, respectively, through two steroid regulatory binding elements in the Cav-1 promoter [22,23]. Therefore, therapeutic approaches to target caveolar lipid composition in general, and cholesterol in particular, represent potential novel treatments for MM patients. Strategies include: First, cholesterol sequestration by filipin, nystatin, and amphotericin; second, poreformation by saponin, digitonin, and streptolysin O; third, cholesterol depletion by b-cyclodextrin; and fourth, inhibition of cholesterol-biosynthesis by statins, e.g. lovastatin. Moreover, perturbation of lipid raft stability using dietary polyunsaturated fatty acids may be of additional therapeutic benefit. Specifically, statins (i.e. lovastatin) are irreversible inhibitors of hydroxymethylglutaryl-CoA (HMGCoA) reductase, which block the production of mevalonate, an intermediate product in cholesteroland isoprenoid-synthesis. Previous studies have reported decreased viability of MM cell lines and patient MM cells in the presence of lovastatin, due to induction of apoptosis and inhibition of
K. Podar, K.C. Anderson / Cancer Letters 233 (2006) 10–15
proliferation [34]. In addition, our own studies show that lovastatin, like b-CD, decreases IL-6- and IGF-Iinduced activation of Akt-1/PKB in the mm cell line MM.1S and patient plasma cell leukemia cells, confirming the requirement for cholesterol and an intact plasma membrane structure in MM cells to mediate PI3-K/Akt-1 signaling. 3.2. Direct targeting of Cav-1 and caveolae Cav-1 antisense or Cav-1 targeting small interfering RNA are used in many current studies to further delineate Cav-1 functions in MM cells and other cells of the MM BM microenvironment. In addition, these methodologies may be the basis for novel genetherapy based strategies. Furthermore, targeting Cav-1 can abrogate VEGFtriggered increased angiogenesis/permeability, thereby blocking blood supply to MM tumor cells. Specifically, Cav-1 is highly expressed in endothelial cells. VEGF-binding to its endothelial receptors increases the production of nitric oxide (NO) via activation of endothelial nitric oxide synthetase (eNOS) and thereby enhances tumor vessel permeability, a hallmark of tumor vasculature. Cavtratin, a recently developed peptide derived from the eNOS inhibitory CSD sequence of Cav-1 protein, selectively inhibits tumor microvascular permeability and evasculature of pro-oncogenic macromolecules, thereby blocking tumor progression [35]. Another promising gene-therapeutic approach is the use of an adenoviral vector, which is regulated by the Cav-1 promoter. Preclinical studies in prostate cancer demonstrated high efficacy with minimal toxicity [36]. Finally, our recent studies show downregulation of Cav-1 expression evidenced by microarray analysis and western blot analysis upon treatment of MM cells with novel anti-MM therapeutics including bortezomib, and the pan-VEGF receptor inhibitor GW786034.
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also be used for delivery of new targeted therapies [37,38]. In contrast to clathrin-dependent endocytosis, the caveolae-associated internalization pathway avoids lysosomes, thereby bypassing acidic and harmful milieu. This may be a major advantage for drug delivery via the caveolar pathway. Specific binding of anticancer drugs or gene vectors to Cav-1 or other caveolar components may therefore be used to directly target MM cells or endothelial cells, thereby blocking tumor cell growth and tumor angiogenesis/permeability; and to modulate expression of tumor-associated genes, i.e. growth factors.
4. Conclusions In conclusion, caveolae and Cav-1 are now recognized as important modulators of tumor growth, survival, and migration. Cav-1 is expressed and caveolae are formed in MM cells, but not in normal human peripheral blood cells or myeloid, lymphoid, and erythroid lines, and modulate MM cell survival, drug resistance and migration. Ongoing studies are determining: the role of subcellular Cav-1 localization (endoplasmatic reticulum, Golgi apparatus); whether, as in prostate cancer [39], Cav-1 is secreted by MM cells or BM stromal cells, thereby stimulating MM cell growth and survival; whether Cav-1 can be used as a prognostic marker in MM; and whether there is cell-type-specific caveola composition (e.g. in MM cells, endothelial cells, fibroblasts) that allows selective therapy strategies targeting Cav-1. Taken together, our recent studies clearly show a role of Cav1 in MM pathogenesis. Therapeutics which directly or indirectly modulate components of caveolae, e.g. cholesterol, and/or Cav-1 within the BM microenvironment have already shown clinical promise, further supporting development of Cav-1/caveolae targeted treatment strategies to improve patient outcome in MM.
3.3. Using Cav-1 and caveolae as a drug-delivery system
Acknowledgements
Caveolae function is associated with endocytotic pathways (i.e. folate, cholesterol; viruses, bacteria, fungi, parasites, toxins; and prions) and may therefore
The authors thank all members of the Jerome Lipper Multiple Myeloma Center and Dr Martin Sattler for many helpful discussions.
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Mini-review
Caveolin-1 as a potential new therapeutic target in multiple myeloma* Klaus Podar*, Kenneth C. Anderson* Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Jerome Lipper Multiple Myeloma Center, 44 Binney Street, Boston, MA 02115, USA Received 18 February 2005; accepted 23 February 2005
Abstract Caveolae are specialized flask-shaped lipid rafts enriched in cholesterol, sphingolipids, and structural marker proteins termed caveolins. Caveolins are highly conserved hairpin loop-shaped, oligomeric proteins of 22–24 kDa. Besides the plasma cell membrane, caveolins are also present in mitochondria, the endoplasmatic reticulum, the Golgi/trans-Golgi network, and secretory vesicles. They play a critical role in normal vesicular transport, cholesterol homeostasis, and signal transduction. Conversely, dysregulation of caveolin-1 has been associated with several human diseases including multiple myeloma, an incurable malignancy characterized by excess monoclonal plasma cells within the bone marrow. In this mini-review, we characterize the functional role of caveolin-1 in multiple myeloma, and present the preclinical rationale for novel potential therapeutic approaches targeting caveolin-1 in multiple myeloma. q 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Caveolin-1; Multiple myeloma; IL-6; IGF-1; VEGF
1. Introduction Multiple myeloma (MM) is characterized by the clonal proliferation of malignant plasma cells in the bone marrow (BM) associated with bone lesions, renal dysfunction, and immunodeficiency [1]. Despite high dose therapy and more recent novel therapeutic approaches, the median survival remains at 4–5 years. * This work was supported by National Institutes of Health Grants IP50 CA100707, PO-1 78378, and RO-1 CA 50945. * Corresponding authors. Tel.: C1 617 632 2144; fax: C1 617 632 2140. E-mail addresses: [email protected] (K. Podar), [email protected] (K.C. Anderson).
The annual incidence of MM is approximately 3.8 per 100,000 population (approximately 14,400 new cases of MM/year), and it is almost twice as common in the black versus Caucasian population [2]. The increased incidence of MM in African-Americans could, at least in part, be related to increased serum cholesterol levels [3] since cholesterol is an essential component of MM cell membranes, it has been implicated in disease pathogenesis [4], and is associated with higher mortality in black versus Caucasian men [5]. In support of this view, a prospectively studied cohort of more than 900,000 US adults showed that increased body weight is associated with increased death rates from cancers, including MM [6].
0304-3835/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2005.02.035
K. Podar, K.C. Anderson / Cancer Letters 233 (2006) 10–15
Lipid rafts are ‘liquid-ordered’ membranous microdomains with a unique lipid composition (enrichment in cholesterol, sphingolipids, and phosphatidylethanolamine) in the otherwise homogeneous, phospholipid-rich, two-dimensional lipid layer of the plasma cell membrane [7]. Caveolae (‘little caves’) are specialized lipid raft microdomains, which classically form dynamic, flask-shaped, vesicular invaginations (50–100 nm) and are functionally implicated in pinocytosis, transcytosis and endocytosis; cholesterol homeostasis; cell transformation; and signal transduction modulating cell growth, survival, adhesion, and migration [8,9]. Typically, caveolae require the presence of functionally highly conserved, hairpin loop-shaped, 22–24 kDa, oligomeric proteins, termed caveolins, in particular caveolin-1 (Cav-1) [10–13]. Expression of caveolins is very heterogeneous in different cells: in contrast to Cav-3, which is specifically expressed in muscle cells, Cav-1 isoforms (Cav-1a, containing residues 1–178; and Cav-1b, containing residues 32–178) and also Cav-2 isoforms (full-length Cav-2a and two truncated variants, Cav-2b and Cav-2g) are expressed in most cell types including fibroblasts, adipocytes, endothelial cells, and type I pneumocytes, but not in human peripheral blood cells or myeloid, lymphoid, and erythroid cell lines [14–16]. Dysregulation of Cav-1 is associated with several human malignancies, including MM. Initial studies have demonstrated that Cav-1 negatively regulates several signaling molecules, thereby mediating cell growth inhibition. However, more recent studies show that Cav-1 can also function as a tumor-promoter, dependent on the tumor type and the tumor stage; as well as promote acquisition of multidrug resistance [9,17]. The tumor-suppressor function of Cav-1 is mediated via the Cav-1 scaffolding domain (CSD), i.e. by direct inhibition of heterotrimeric G proteins [18]. Conversely, the tumor-promoter function may be mediated via: (1) growth-factor-induced tyrosinephosphorylation of Cav-1 (Tyr14), which recruits SH2-domain-containing proteins to the plasma membrane, thereby enhancing activation of downstream signaling proteins [19,20]; (2) growth-factor-induced serine-phosphorylation of Cav-1 (Ser80), which converts Cav-1 to a secreted protein, which may function as an autocrine or paracrine growth and survival factor [21]; and (3) up-regulation of Cav-1
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expression by direct binding of cholesterol to the Cav-1 promoter [22,23]. Increased levels of cholesterol also contribute to enhanced production of hormones including testosterone, growth factors, and lipid raft/caveolae formation. Finally, a P132L mutation of Cav-1 observed in human breast cancer cells is associated with increased cell growth, invasiveness, and chemotaxis in NIH3T3 cells [24]. Importantly, Cav-1 over-expression is also present in multidrug resistant (MDR) colon and human breast adenocarcinomas [25].
2. Pathophysiologic roles of Cav-1 in MM In contrast to normal B cells, Cav-1 is expressed in MM cell lines and patient MM cells. Moreover, Cav-1 expression is upregulated in MM versus monoclonal gammopathy of undetermined significance (MGUS), suggesting a role for caveolae in the transition of MGUS to MM and MM pathogenesis [20]. Studies are now underway to evaluate whether Cav-1 expression may, as in prostate cancer [26], function as a diagnostic and prognostic marker in MM. Our recent studies suggest a functional model in which caveolae play a pivotal role in interleukin-6 (IL-6)-, insulin-like growth factor-1 (IGF-1)-, and vascular endothelial growth factor (VEGF)-induced signaling, thereby enhancing MM cell survival and conferring resistance to dexamethasone, as well as modulating MM cell migration [20,27]. Specifically, both IL6-receptor signal transducing chain gp130 and IGF-1 receptor constitutively co-localize with Cav-1 in the lipid raft fraction isolated by sucrose density gradient separation. IL-6 induces Src-family kinasedependent tyrosine phosphorylation of Cav-1 (Tyr14) and triggers subsequent formation of gp130$SHPTP2/PI3-K-containing complexes. Conversely, disruption of caveolae inhibits complex formation and subsequent PI3-K/Akt-1, as well as STAT 3, activation. IGF-1 induces Src-family kinase-dependent tyrosine phosphorylation of Cav-1 (Tyr14) and subsequent tyrosine-phosphorylation of IRS-1, which is required for transducing downstream signals via interaction with SH2-domains on other proteins (e.g. the regulatory p85 subunit of PI3-K). Disruption of caveolae also inhibits IGF-1-triggered phosphorylation of IRS-1 and downstream activation
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of PI3-K/Akt-1. In contrast, ERK signal transduction is not modulated by disruption of caveolae. Taken together, these data demonstrate that caveolae and Cav-1 mediate IL-6- and IGF-1-triggered survival signals through both the PI3-K/Akt-1, as well as the STAT3, pathways [20]. VEGF levels and increased angiogenesis in the bone marrow are correlated with clinical outcome in MM. The multiple functional sequelae of VEGF are due to its broad spectrum of target cells. Besides its regulatory role in physiologic and pathologic angiogenesis, VEGF also triggers growth, survival, and migration of MM cells via paracrine and autocrine pathways; inhibits maturation of dendritic cells, thereby contributing to immunodeficiency; and increases bone-resorbing activity, thereby contributing to bone destruction in MM [28]. Cav-1 has an important role in mediating cell motility: specifically, Cav-1 accumulates at the leading edge of cultured fibroblasts [13], human umbilical vein smooth muscle cells [29], and Cav-1-deficient FRT cells expressing recombinant Cav-1 [30]; as well as at the trailing edge of bovine aortic endothelial cells [31]. In MM cells, VEGF receptor-1 (Flt-1) is associated with Cav-1; and VEGF binding triggers Src-dependent tyrosine phosphorylation of Cav-1, which is required for p130Cas phosphorylation and MM cell migration. Conversely, depletion of Cav-1 by antisense methodology abrogates VEGF-triggered MM cell migration and associated p130Cas phosphorylation, further confirming the essential role of Cav-1 in MM cell migration [27]. Bortezomib, a promising novel anti-MM agent recently approved by the United States Food and Drug Administration (FDA) for therapy of patients with progressive MM after previous treatment, targets proteins related to apoptosis, growth signaling/cell cycle, heat shock proteins, and the proteasome pathway. We recently identified Cav-1 as an additional target: Bortezomib inhibits VEGF-triggered Cav-1 phosphorylation and later downregulation of Cav-1 in MM cells. Furthermore, Bortezomib also inhibits VEGF-triggered Cav-1 phosphorylation in endothelial cells and thereby abrogates endothelial cell migration within the bone marrow environment of MM patients. These findings demonstrate multiple facets of the anti-myeloma activity of Bortezomib and further support novel treatment strategies targeting caveolae in MM [27].
3. Potential new therapeutic approaches to target Cav-1 and caveolae in MM In contrast to plasma cells, Cav-1 is expressed in MM cell lines and patient MM cells and mediates tumor cell survival and VEGF-triggered migration; blocking its function abrogates these sequelae. Moreover, targeting Cav-1 in other cells within the MM BM microenvironment, e.g. endothelial cells, may also inhibit their tumor promoting activity and angiogenesis. Therefore, therapeutics developed to target components of caveolae, e.g. cholesterol, and Cav-1 may decrease MM progression and overcome drug resistance, e.g. to dexamethasone. Potential therapeutic approaches to target caveolae include the following. 3.1. Targeting Cav-1 and caveolae by modulating cholesterol-production and caveolar lipid composition Cav-1 directly binds cholesterol, a cofactor in formation of caveolae, forming homo- and heterooligomers [13,32,33]. The threshold of plasma membrane cholesterol below which caveolae cannot be formed is 40 mol% [33]. Cav-1 mRNA levels are up- and down-regulated by cholesterol and oxysterols, respectively, through two steroid regulatory binding elements in the Cav-1 promoter [22,23]. Therefore, therapeutic approaches to target caveolar lipid composition in general, and cholesterol in particular, represent potential novel treatments for MM patients. Strategies include: First, cholesterol sequestration by filipin, nystatin, and amphotericin; second, poreformation by saponin, digitonin, and streptolysin O; third, cholesterol depletion by b-cyclodextrin; and fourth, inhibition of cholesterol-biosynthesis by statins, e.g. lovastatin. Moreover, perturbation of lipid raft stability using dietary polyunsaturated fatty acids may be of additional therapeutic benefit. Specifically, statins (i.e. lovastatin) are irreversible inhibitors of hydroxymethylglutaryl-CoA (HMGCoA) reductase, which block the production of mevalonate, an intermediate product in cholesteroland isoprenoid-synthesis. Previous studies have reported decreased viability of MM cell lines and patient MM cells in the presence of lovastatin, due to induction of apoptosis and inhibition of
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proliferation [34]. In addition, our own studies show that lovastatin, like b-CD, decreases IL-6- and IGF-Iinduced activation of Akt-1/PKB in the mm cell line MM.1S and patient plasma cell leukemia cells, confirming the requirement for cholesterol and an intact plasma membrane structure in MM cells to mediate PI3-K/Akt-1 signaling. 3.2. Direct targeting of Cav-1 and caveolae Cav-1 antisense or Cav-1 targeting small interfering RNA are used in many current studies to further delineate Cav-1 functions in MM cells and other cells of the MM BM microenvironment. In addition, these methodologies may be the basis for novel genetherapy based strategies. Furthermore, targeting Cav-1 can abrogate VEGFtriggered increased angiogenesis/permeability, thereby blocking blood supply to MM tumor cells. Specifically, Cav-1 is highly expressed in endothelial cells. VEGF-binding to its endothelial receptors increases the production of nitric oxide (NO) via activation of endothelial nitric oxide synthetase (eNOS) and thereby enhances tumor vessel permeability, a hallmark of tumor vasculature. Cavtratin, a recently developed peptide derived from the eNOS inhibitory CSD sequence of Cav-1 protein, selectively inhibits tumor microvascular permeability and evasculature of pro-oncogenic macromolecules, thereby blocking tumor progression [35]. Another promising gene-therapeutic approach is the use of an adenoviral vector, which is regulated by the Cav-1 promoter. Preclinical studies in prostate cancer demonstrated high efficacy with minimal toxicity [36]. Finally, our recent studies show downregulation of Cav-1 expression evidenced by microarray analysis and western blot analysis upon treatment of MM cells with novel anti-MM therapeutics including bortezomib, and the pan-VEGF receptor inhibitor GW786034.
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also be used for delivery of new targeted therapies [37,38]. In contrast to clathrin-dependent endocytosis, the caveolae-associated internalization pathway avoids lysosomes, thereby bypassing acidic and harmful milieu. This may be a major advantage for drug delivery via the caveolar pathway. Specific binding of anticancer drugs or gene vectors to Cav-1 or other caveolar components may therefore be used to directly target MM cells or endothelial cells, thereby blocking tumor cell growth and tumor angiogenesis/permeability; and to modulate expression of tumor-associated genes, i.e. growth factors.
4. Conclusions In conclusion, caveolae and Cav-1 are now recognized as important modulators of tumor growth, survival, and migration. Cav-1 is expressed and caveolae are formed in MM cells, but not in normal human peripheral blood cells or myeloid, lymphoid, and erythroid lines, and modulate MM cell survival, drug resistance and migration. Ongoing studies are determining: the role of subcellular Cav-1 localization (endoplasmatic reticulum, Golgi apparatus); whether, as in prostate cancer [39], Cav-1 is secreted by MM cells or BM stromal cells, thereby stimulating MM cell growth and survival; whether Cav-1 can be used as a prognostic marker in MM; and whether there is cell-type-specific caveola composition (e.g. in MM cells, endothelial cells, fibroblasts) that allows selective therapy strategies targeting Cav-1. Taken together, our recent studies clearly show a role of Cav1 in MM pathogenesis. Therapeutics which directly or indirectly modulate components of caveolae, e.g. cholesterol, and/or Cav-1 within the BM microenvironment have already shown clinical promise, further supporting development of Cav-1/caveolae targeted treatment strategies to improve patient outcome in MM.
3.3. Using Cav-1 and caveolae as a drug-delivery system
Acknowledgements
Caveolae function is associated with endocytotic pathways (i.e. folate, cholesterol; viruses, bacteria, fungi, parasites, toxins; and prions) and may therefore
The authors thank all members of the Jerome Lipper Multiple Myeloma Center and Dr Martin Sattler for many helpful discussions.
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