Isabelle Mercier

An Absence of Stromal Caveolin-1 Expression Predicts Early Tumor Recurrence and Poor Clinical Outcome in Human Breast Cancers

Previously, we showed that caveolin-1 (Cav-1) expression is down-regulated in human breast cancer-associated fibroblasts. However, it remains unknown whether loss of Cav-1 occurs in the breast tumor stroma in vivo. Here, we immunostained a well-annotated breast cancer tissue microarray with antibodies against Cav-1 and scored its stromal expression. An absence of stromal Cav-1 was associated with early disease recurrence, advanced tumor stage, and lymph node metastasis, resulting in a 3.6-fold reduction in progression-free survival. When tamoxifen-treated patients were selected, an absence of stromal Cav-1 was a strong predictor of poor clinical outcome, suggestive of tamoxifen resistance. Interestingly, in lymph node-positive patients, an absence of stromal Cav-1 predicted an 11.5-fold reduction in 5-year progression-free survival. Clinical outcomes among patients positive for HER2, and patients triple-negative for estrogen receptor, progesterone receptor and HER2, were also strictly dependent on stromal Cav-1 levels. When our results were adjusted for tumor and nodal staging, an absence of stromal Cav-1 remained an independent predictor of poor outcome. Thus, stromal Cav-1 expression can be used to stratify human breast cancer patients into low-risk and high-risk groups, and to predict their risk of early disease recurrence at diagnosis. Based on related mechanistic studies, we suggest that breast cancer patients lacking stromal Cav-1 might benefit from anti-angiogenic therapy in addition to standard regimens. We conclude that Cav-1 functions as a tumor suppressor in the stromal microenvironment.

The autophagic tumor stroma model of cancer or "battery-operated tumor growth": A simple solution to the autophagy paradox.

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”. We have recently reported a simple solution to this paradox. We demonstrated that epithelial cancer cells use oxidative stress to induce autophagy in the tumor microenvironment. As a consequence, the autophagic tumor stroma generates recycled nutrients that can then be used as chemical building blocks by anabolic epithelial cancer cells. This model results in a net energy transfer from the tumor stroma to epithelial cancer cells (an energy imbalance), thereby promoting tumor growth. This net energy transfer is both unilateral and vectorial, from the tumor stroma to the epithelial cancer cells, representing a true host-parasite relationship. We have termed this new paradigm “The Autophagic Tumor Stroma Model of Cancer Cell Metabolism” or “Battery-Operated Tumor Growth”. In this sense, autophagy in the tumor stroma serves as a “battery” to fuel tumor growth, progression, and metastasis, independently of angiogenesis. Using this model, the systemic induction of autophagy will prevent epithelial cancer cells from using recycled nutrients, while the systemic inhibiton of autophagy will prevent stromal cells from producing recycled nutrients—both effectively “starving” cancer cells. We discuss the idea that tumor cells could become resistant to the systemic induction of autophagy, by the up-regulation of natural endogenous autophagy inhibitors in cancer cells. Alternatively, tumor cells could also become resistant to the systemic induction of autophagy, by the genetic silencing/deletion of pro-autophagic molecules, such as Beclin1. If autophagy resistance develops in cancer cells, then the systemic inhibition of autophagy would provide a therapeutic solution to this type of drug resistance, as it would still target autophagy in the tumor stroma. As such, an anti-cancer therapy that combines the alternating use of both autophagy promoters and autophagy inhibitors would be expected to prevent the onset of drug resistance. We also discuss why anti-angiogenic therapy has been found to promote tumor recurrence, progression, and metastasis. More specifically, anti-angiogenic therapy would induce autophagy in the tumor stroma via the induction of stromal hypoxia, thereby converting a non-aggressive tumor type to a “lethal” aggressive tumor phenotype. Thus, uncoupling the metabolic parasitic relationship between cancer cells and an autophagic tumor stroma may hold great promise for anti-cancer therapy. Finally, we believe that autophagy in the tumor stroma is the local microscopic counterpart of systemic wasting (cancer-associated cachexia), which is associated with advanced and metastatic cancers. Cachexia in cancer patients is not due to decreased energy intake, but instead involves an increased basal metabolic rate and increased energy expenditures, resulting in a negative energy balance. Importantly, when tumors were surgically excised, this increased metabolic rate returned to normal levels. This view of cachexia, resulting in energy transfer to the tumor, is consistent with our hypothesis. So, cancer-associated cachexia may start locally as stromal autophagy, and then spread systemically. As such, stromal autophagy may be the requisite precursor of systemic cancer-associated cachexia.

Role of Cholesterol in the Development and Progression of Breast Cancer

Diet and obesity are important risk factors for cancer development. Many studies have suggested an important role for several dietary nutrients in the progression and development of breast cancer. However, few studies have specifically addressed the role of components of a Western diet as important factors involved in breast cancer initiation and progression. The present study examined the role of cholesterol in the regulation of tumor progression in a mouse model of mammary tumor formation. The results suggest that cholesterol accelerates and enhances tumor formation. In addition, tumors were more aggressive, and tumor angiogenesis was enhanced. Metabolism of cholesterol was also examined in this mouse model. It was observed that plasma cholesterol levels were reduced during tumor development but not prior to its initiation. These data provide new evidence for an increased utilization of cholesterol by tumors and for its role in tumor formation. Taken together, these results imply that an increase in plasma cholesterol levels accelerates the development of tumors and exacerbates their aggressiveness.

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.

Short-Term Administration of a Cell-Permeable Caveolin-1 Peptide Prevents the Development of Monocrotaline-Induced Pulmonary Hypertension and Right Ventricular Hypertrophy

Background— Caveolins (Cavs), the principal structural proteins of caveolar microdomains, have been implicated in the development of pulmonary hypertension (PH). Mice with homozygous deletion of the Cav-1 gene develop PH and right ventricular hypertrophy (RVH). Reductions in pulmonary Cav-1 expression have been shown in several animal models of PH and in patients with severe PH. Whether in vivo modulation of Cav-1 expression could affect the development of PH and RVH remains unknown. Therefore, we investigated the effect of in vivo administration of a Cav-1 mimetic peptide on the development of monocrotaline (MCT)-induced PH. Methods and Results— Thirty minutes after injection of saline or 60 mg/kg MCT, rats were assigned to receive a daily injection of saline, a peptide corresponding to the homeodomain of the Drosophila transcription factor antennapedia (AP; 2.5 mg · kg−1 · d−1), or a peptide consisting of the Cav-1–scaffolding domain coupled to AP (AP-Cav; 2.5 mg · kg−1 · d−1) for 2 weeks. MCT and MCT+AP rats developed PH with respective right ventricular systolic pressures of 40.2±1.5 and 39.6±1.5 mm Hg. Administration of AP-Cav to MCT rats significantly reduced the right ventricular systolic pressure to 30.1±1.3 mm Hg. MCT and MCT+AP rats also developed pulmonary artery medial hypertrophy and RVH, which was normalized by administration of AP-Cav. Mechanistically, the development of PH was associated with reduced expression of pulmonary Cav-1 and Cav-2, hyperactivation of the STAT3 signaling cascade, and upregulation of cyclin D1 and D3 protein levels, all of which were prevented by administration of AP-Cav. Conclusions— Short-term administration of a Cav-based cell-permeable peptide to MCT rats prevents the development of pulmonary artery medial hypertrophy, PH, and RVH.

Caveolin-1 deficiency increases cerebral ischemic injury

Caveolins (Cav), the principal structural proteins of the caveolar domains, have been implicated in the pathogenesis of ischemic injury. Indeed, changes in caveolin expression and localization have been reported in renal and myocardial ischemia. Genetic ablation of the Cav-1 gene in mice was further shown to increase the extent of ischemic injury in a model of hindlimb ischemia. However, the role of Cav-1 in the pathogenesis of cerebral ischemia remains unknown. Immunoblot and immunofluorescence analyses of rat brains subjected to middle cerebral artery occlusion revealed marked increases in endothelial Cav-1 and Cav-2 protein levels. To directly assess the functional role of caveolins in the pathogenesis of cerebral ischemic injury, we next investigated the effects of cerebral ischemia in caveolin knockout (KO) mice. Interestingly, Cav-1 KO mice showed a marked increase of cerebral volume of infarction, as compared with wild-type and Cav-2 KO mice. Immunofluorescence analyses showed an increased number of proliferating endothelial cells in wild-type ischemic brains, as compared with Cav-1 KO ischemic brains. Immunoblot analyses of wild-type ischemic brains showed an increase in endothelial nitric oxide synthase protein levels. Conversely, the protein levels of endothelial nitric oxide synthase remained unchanged in Cav-1 KO ischemic brains. TUNEL analysis also showed increased apoptotic cell death in Cav-1 KO ischemic brains, as compared with wild-type ischemic brains. Our findings indicate cerebral ischemia induces a marked increase in endothelial Cav-1 and Cav-2 protein levels. Importantly, genetic ablation of the Cav-1 gene in mice results in increased cerebral volume of infarction. Mechanistically, Cav-1 KO ischemic brains showed impaired angiogenesis and increased apoptotic cell death.

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).

Different Classes of Coactivators Recognize Distinct but Overlapping Binding Sites on the Estrogen Receptor Ligand Binding Domain

We have analyzed interaction of coactivators with the wild-type estrogen receptor α (ER), HEG0, and a mutant, L536P-HEG0, which is constitutively active in several transiently transfected cells and a HeLa line that stably propagates an estrogen-sensitive reporter gene. Different classes of coactivators do not recognize the ER ligand binding domain (LBD) in the same manner. Steroid receptor coactivator-1 (SRC-1), amplified in breast cancer-1 (AIB-1), transcriptional intermediary factor-1 (TIF-1), transcriptional intermediary factor-2 (TIF-2), and receptor interacting protein 140 (RIP140) interacted with HEG0 and L536P-HEG0 in the presence of estradiol, but generally not in the presence of anti-estrogens. However, ICI164,384 stimulated some interaction of RIP140 with LBDs. SRC-1, AIB-1, and RIP140 interacted constitutively with the L536P ER, whereas TIF-1 and TIF-2 interacted only weakly in the absence of hormone. Reciprocal competition for binding to the ER LBD was observed between different classes of coactivators. Moreover, coexpression of RIP140 blocked enhanced transactivation by HEG0 observed in the presence of TIF-2, suggesting that RIP140 may play a negative role in ER signaling. We conclude that constitutive activity of L536P-HEG0 is manifested to similar degrees in different cell types and likely arises from constitutive coactivator binding; different classes of coactivators recognize distinct but overlapping binding sites on the ER LBD. Finally, the observation that L536P-HEG0 interacted constitutively with AIB-1, a coactivator that has been implicated in ER signaling in breast and ovarian cancer, suggests that similar mutations in the ER may contribute to hormone-independent proliferation of breast and ovarian cells.

Caveolin-1, Mammary Stem Cells, and Estrogen-Dependent Breast Cancers

Estrogen exposure is considered a significant risk factor for breast cancer development. Estrogen receptor (ER) alpha is expressed at low levels in normal epithelia, and its expression is dramatically up-regulated as transformation progresses during mammary hyperplasia and adenocarcinoma development. The mechanism(s) driving ERalpha up-regulation during mammary tumorigenesis remains unclear. Caveolin-1 (Cav-1) is the structural protein of plasmalemmal invaginations, termed caveolae, which functions as a tumor suppressor gene. Interestingly, Cav-1 dominant-negative mutations are exclusively found in ERalpha-positive breast cancer samples. In support of these clinical findings, ERalpha expression is increased in Cav-1 (-/-) null mammary epithelia, and estrogen stimulation further enhances the growth of Cav-1-deficient three-dimensional epithelial structures. These phenotypes correlate with augmented levels of cyclin D1. In addition, Cav-1 gene inactivation induces the accumulation of a cell population with the characteristics of adult mammary stem cells. Primary cultures of Cav-1 (-/-) mammary epithelial cells exhibit premalignant changes, such as abnormal lumen formation, epidermal growth factor-independent growth, defects in cell substrate attachment, and increased cell invasiveness. Thus, Cav-1 gene inactivation promotes premalignant alterations in mammary epithelia and induces increased ERalpha expression levels and the up-regulation of cyclin D1. As tumor formation is a multihit process, Cav-1 mutations that occur during the early stages of mammary transformation may be a critical upstream/initiating event leading to increased ERalpha levels.