Jean-Francois Jasmin

Decreased expression of caveolin 1 in patients with systemic sclerosis: Crucial role in the pathogenesis of tissue fibrosis

OBJECTIVE Recent studies have implicated caveolin 1 in the regulation of transforming growth factor beta (TGFbeta) downstream signaling. Given the crucial role of TGFbeta in the pathogenesis of systemic sclerosis (SSc), we sought to determine whether caveolin 1 is also involved in the pathogenesis of tissue fibrosis in SSc. We analyzed the expression of CAV1 in affected SSc tissues, studied the effects of lack of expression of CAV1 in vitro and in vivo, and analyzed the effects of restoration of caveolin 1 function on the fibrotic phenotype of SSc fibroblasts in vitro. METHODS CAV1 expression in tissues was analyzed by immunofluorescence and confocal microscopy. The extent of tissue fibrosis in Cav1-knockout mice was assessed by histologic/histochemical analyses and quantified by hydroxyproline assays. Cav1-null and SSc fibroblast phenotypes and protein production were analyzed by real-time polymerase chain reaction, immunofluorescence, Western blot, and multiplexed enzyme-linked immunosorbent assay techniques. The effects of restoration of caveolin 1 function in SSc fibroblasts in vitro were also examined using a cell-permeable recombinant CAV1 peptide. RESULTS CAV1 was markedly decreased in the affected lungs and skin of SSc patients. Cav1-knockout mice developed pulmonary and skin fibrosis. Down-regulation of caveolin 1 was maintained in cultured SSc fibroblasts, and restoration of caveolin 1 function in vitro normalized their phenotype and abrogated TGFbeta stimulation through inhibition of Smad3 activation. CONCLUSION Caveolin 1 appears to participate in the pathogenesis of tissue fibrosis in SSc. Restoration of caveolin 1 function by treatment with a cell-permeable peptide corresponding to the CAV1 scaffolding domain may be a novel therapeutic approach in SSc.

Human breast cancer-associated fibroblasts (CAFs) show caveolin-1 downregulation and RB tumor suppressor functional inactivation: Implications for the response to hormonal therapy

It is becoming increasingly apparent that the tumor micro-environment plays a critical role in human breast cancer onset and progression. Therefore, we isolated cancer-associated fibroblasts (CAFs) from human breast cancer lesions and studied their properties, as compared with normal mammary fibroblasts (NFs) isolated from the same patient. Here, we demonstrate that 8 out of 11 CAFs show dramatic down-regulation of caveolin-1 (Cav-1) protein expression; Cav-1 is a well-established marker that is normally decreased during the oncogenic transformation of fibroblasts. Next, we performed gene expression profiling studies (DNA mircoarray) and established a CAF gene expression signature. Interestingly, the expression signature associated with CAFs encompasses a large number of genes that are regulated via the RB-pathway. The CAF gene signature is also predictive of poor clinical outcome in breast cancer patients that were treated with tamoxifen mono-therapy, indicating that CAFs may be useful for predicting the response to hormonal therapy. Finally, we show that replacement of Cav-1 expression in CAFs (using a cell-permeable peptide approach) is sufficient to revert their hyper-proliferative phenotype and prevent RB hyper-phosphorylation. Taken together, these studies highlight the critical role of Cav-1 down-regulation in maintaining the abnormal phenotype of human breast cancer-associated fibroblasts.

Clinical and translational implications of the caveolin gene family: Lessons from mouse models and human genetic disorders

Here we review the clinical and translational implications of the caveolin gene family for understanding the pathogenesis of human diseases, including breast and prostate cancers, pulmonary hypertension, cardiomyopathy, diabetes, and muscular dystrophy. Detailed phenotypic analysis of caveolin knockout mice has served to highlight the crucial role of a caveolin deficiency in the pathogenesis of many human disease processes. Mutations in the human caveolin genes are associated with a number of established genetic disorders (such as breast cancer, lipodystrophy, muscular dystrophy, and cardiomyopathy), making the caveolins important and novel targets for drug development. The implementation of new strategies for caveolin replacement therapy—including caveolin mimetic peptides—is ongoing.

Caveolin-1 and accelerated host aging in the breast tumor microenvironment: chemoprevention with rapamycin, an mTOR inhibitor and anti-aging drug.

Increasing chronological age is the most significant risk factor for human cancer development. To examine the effects of host aging on mammary tumor growth, we used caveolin (Cav)-1 knockout mice as a bona fide model of accelerated host aging. Mammary tumor cells were orthotopically implanted into these distinct microenvironments (Cav-1(+/+) versus Cav-1(-/-) age-matched young female mice). Mammary tumors grown in a Cav-1-deficient tumor microenvironment have an increased stromal content, with vimentin-positive myofibroblasts (a marker associated with oxidative stress) that are also positive for S6-kinase activation (a marker associated with aging). Mammary tumors grown in a Cav-1-deficient tumor microenvironment were more than fivefold larger than tumors grown in a wild-type microenvironment. Thus, a Cav-1-deficient tumor microenvironment provides a fertile soil for breast cancer tumor growth. Interestingly, the mammary tumor-promoting effects of a Cav-1-deficient microenvironment were estrogen and progesterone independent. In this context, chemoprevention was achieved by using the mammalian target of rapamycin (mTOR) inhibitor and anti-aging drug, rapamycin. Systemic rapamycin treatment of mammary tumors grown in a Cav-1-deficient microenvironment significantly inhibited their tumor growth, decreased their stromal content, and reduced the levels of both vimentin and phospho-S6 in Cav-1-deficient cancer-associated fibroblasts. Since stromal loss of Cav-1 is a marker of a lethal tumor microenvironment in breast tumors, these high-risk patients might benefit from treatment with mTOR inhibitors, such as rapamycin or other rapamycin-related compounds (rapalogues).

Towards a new "stromal-based" classification system for human breast cancer prognosis and therapy

Here, we discuss recent evidence that an absence of stromal Cav-1 expression in human breast cancers is a powerful single independent predictor of early disease recurrence, metastasis, and poor clinical outcome. These findings have now been validated in two independent patient populations. Importantly, the predictive value of stromal Cav-1 is independent of epithelial marker status, making stromal Cav-1 a new “universal” or “widely-applicable” breast cancer prognostic marker. We propose based on the expression of stromal Cav-1, that breast cancer patients could be stratified into high-risk and low-risk groups. High-risk patients showing an absence of stromal Cav-1 should be offered more aggressive therapies, such as anti-angiogenic approaches, in addition to the standard therapy regimens. Mechanistically, loss of stromal Cav-1 is a surrogate biomarker for increased cell cycle progression, growth factor secretion, “stemness”, and angiogenic potential in the tumor microenvironment. Since almost all cancers develop within the context of a stromal microenvironment, this new stromal classification system may be broadly applicable to other epithelial and non-epithelial cancer subtypes, as well as “pre-malignant” lesions (carcinoma in situ).

Pressure-Overload–Induced Subcellular Relocalization/Oxidation of Soluble Guanylyl Cyclase in the Heart Modulates Enzyme Stimulation

Rationale: Soluble guanylyl cyclase (sGC) generates cyclic guanosine monophophate (cGMP) upon activation by nitric oxide (NO). Cardiac NO–sGC-cGMP signaling blunts cardiac stress responses, including pressure-overload–induced hypertrophy. The latter itself depresses signaling through this pathway by reducing NO generation and enhancing cGMP hydrolysis. Objective: We tested the hypothesis that the sGC response to NO also declines with pressure-overload stress and assessed the role of heme-oxidation and altered intracellular compartmentation of sGC as potential mechanisms. Methods and Results: C57BL/6 mice subjected to transverse aortic constriction (TAC) developed cardiac hypertrophy and dysfunction. NO-stimulated sGC activity was markedly depressed, whereas NO- and heme-independent sGC activation by BAY 60–2770 was preserved. Total sGC&agr;1 and &bgr;1 expression were unchanged by TAC; however, sGC&bgr;1 subunits shifted out of caveolin-enriched microdomains. NO-stimulated sGC activity was 2- to 3-fold greater in Cav3-containing lipid raft versus nonlipid raft domains in control and 6-fold greater after TAC. In contrast, BAY 60–2770 responses were >10 fold higher in non-Cav3 domains with and without TAC, declining about 60% after TAC within each compartment. Mice genetically lacking Cav3 had reduced NO- and BAY-stimulated sGC activity in microdomains containing Cav3 for controls but no change within non–Cav3-enriched domains. Conclusions: Pressure overload depresses NO/heme-dependent sGC activation in the heart, consistent with enhanced oxidation. The data reveal a novel additional mechanism for reduced NO-coupled sGC activity related to dynamic shifts in membrane microdomain localization, with Cav3-microdomains protecting sGC from heme-oxidation and facilitating NO responsiveness. Translocation of sGC out of this domain favors sGC oxidation and contributes to depressed NO-stimulated sGC activity.

Genetic Ablation of Caveolin-1 Drives Estrogen-Hypersensitivity and the Development of DCIS-Like Mammary Lesions

Caveolin-1 (Cav-1) loss-of-function mutations are exclusively associated with estrogen receptor-positive (ER(+)) human breast cancers. To dissect the role of Cav-1 loss-of-function in the pathogenesis of human breast cancers, we used Cav-1(-/-) null mice as a model system. First, we demonstrated that Cav-1(-/-) mammary epithelia overexpress two well-established ER co-activator genes, CAPER and Foxa1, in addition to ER-alpha. Thus, the functional loss of Cav-1 may be sufficient to confer estrogen-hypersensitivity in the mammary gland. To test this hypothesis directly, we subjected Cav-1(-/-) mice to ovariectomy and estrogen supplementation. As predicted, Cav-1(-/-) mammary glands were hyper-responsive to estrogen and developed dysplastic mammary lesions with adjacent stromal angiogenesis that resemble human ductal carcinoma in situ. Based on an extensive biomarker analysis, these Cav-1(-/-) mammary lesions contain cells that are hyperproliferative and stain positively with nucleolar (B23/nucleophosmin) and stem/progenitor cell markers (SPRR1A and beta-catenin). Genome-wide transcriptional profiling identified many estrogen-related genes that were over-expressed in Cav-1(-/-) mammary glands, including CAPER--an ER co-activator gene and putative stem/progenitor cell marker. Analysis of human breast cancer samples revealed that CAPER is overexpressed and undergoes a cytoplasmic-to-nuclear shift during the transition from pre-malignancy to ductal carcinoma in situ. Thus, Cav-1(-/-) null mice are a new preclinical model for studying the molecular paradigm of estrogen hypersensitivity and the development of estrogen-dependent ductal carcinoma in situ lesions.

ARC (apoptosis repressor with caspase recruitment domain) is a novel marker of human colon cancer.

The ability of cells to escape apoptosis is critical for carcinogenesis as well as resistance to radiation and chemotherapy. ARC (Apoptosis Repressor with CARD (caspase recruitment domain)) is an unusual inhibitor of apoptosis in that it antagonizes both the extrinsic (death receptor) and intrinsic (mitochondrial/ER) apoptosis pathways. ARC is expressed predominantly in terminally differentiated cells such as cardiac and skeletal myocytes and neurons. Recently, however, the abundance of ARC was noted to be markedly increased in the epithelium of primary human breast cancers compared with benign breast tissue and to confer chemo- and radiation-resistance. Whether the induction of ARC is specific to breast cancer or a more general feature of neoplasia remains unknown. In this study, we assessed the abundance and subcellular localization of ARC in 21 human colon cancer cell lines and in 44 primary human colon adenocarcinomas and adjacent benign colonic tissue. ARC was present at high levels in most colon cancer cell lines and in almost all primary colon cancers compared with corresponding controls. Levels of ARC in the cytoplasm were increased in well, moderately, and poorly differentiated cancers compared with benign tissue, while levels of nuclear ARC were increased only in moderately differentiated tumors. Moreover, epithelial cancers of the ovary and cervix exhibited increased ARC abundance compared with controls. These results demonstrate that ARC is a novel marker of human colon cancer and suggest that it may be a feature of epithelial cancers.

Essential Role of Caveolin-3 in Adiponectin Signalsome Formation and Adiponectin Cardioprotection

Objective—Adiponectin (APN) system malfunction is causatively related to increased cardiovascular morbidity/mortality in diabetic patients. The aim of the current study was to investigate molecular mechanisms responsible for APN transmembrane signaling and cardioprotection. Methods and Results—Compared with wild-type mice, caveolin-3 knockout (Cav-3KO) mice exhibited modestly increased myocardial ischemia/reperfusion injury (increased infarct size, apoptosis, and poorer cardiac function recovery; P<0.05). Although the expression level of key APN signaling molecules was normal in Cav-3KO, the cardioprotective effects of APN observed in wild-type were either markedly reduced or completely lost in Cav-3KO. Molecular and cellular experiments revealed that APN receptor 1 (AdipoR1) colocalized with Cav-3, forming AdipoR1/Cav-3 complex via specific Cav-3 scaffolding domain binding motifs. AdipoR1/Cav-3 interaction was required for APN-initiated AMP-activated protein kinase (AMPK)–dependent and AMPK-independent intracellular cardioprotective signalings. More importantly, APPL1 and adenylate cyclase, 2 immediately downstream molecules required for AMPK-dependent and AMPK-independent signaling, respectively, formed a protein complex with AdipoR1 in a Cav-3 dependent fashion. Finally, pharmacological activation of both AMPK plus protein kinase A significantly reduced myocardial infarct size and improved cardiac function in Cav-3KO animals. Conclusion—Taken together, these results demonstrated for the first time that Cav-3 plays an essential role in APN transmembrane signaling and APN anti-ischemic/cardioprotective actions.

Caveolin-1 deficiency exacerbates cardiac dysfunction and reduces survival in mice with myocardial infarction.

Caveolin (Cav)-1 has been involved in the pathogenesis of ischemic injuries. For instance, modulations of Cav-1 expression have been reported in animal models of myocardial infarction and cerebral ischemia-reperfusion. Furthermore, ablation of the Cav-1 gene in mice has been shown to increase the extent of ischemic injury in models of cerebral and hindlimb ischemia. Cav-1 has also been suggested to play a role in myocardial ischemic preconditioning. However, the role of Cav-1 in myocardial ischemia (MI)-induced cardiac dysfunction still remains to be determined. We determined the outcome of a permanent left anterior descending coronary artery (LAD) ligation in Cav-1 knockout (KO) mice. Wild-type (WT) and Cav-1 KO mice were subjected to permanent LAD ligation for 24 h. The progression of ischemic injury was monitored by echocardiography, hemodynamic measurements, 2,3,5-triphenyltetrazolium chloride staining, β-binding analysis, cAMP level measurements, and Western blot analyses. Cav-1 KO mice subjected to LAD ligation display reduced survival compared with WT mice. Despite similar infarct sizes, Cav-1 KO mice subjected to MI showed reduced left ventricular (LV) ejection fraction and fractional shortening as well as increased LV end-diastolic pressures compared with their WT counterparts. Mechanistically, Cav-1 KO mice subjected to MI exhibit reduced β-adrenergic receptor density at the plasma membrane as well as decreased cAMP levels and PKA phosphorylation. In conclusion, ablation of the Cav-1 gene exacerbates cardiac dysfunction and reduces survival in mice subjected to MI. Mechanistically, Cav-1 KO mice subjected to LAD ligation display abnormalities in β-adrenergic signaling.