Stromal Caveolin-1 Research Articles

Abstract 1486: The contribution of stromal caveolin-1 to breast cancer metastasis

Abstract In a previous study, our group has shown that the expression of caveolin-1 (CAV1) in normal tissue surrounding a primary breast cancer is a powerful prognostic indicator of subsequent metastatic disease. Whilst reports linking expression of CAV1 within breast tumour cells to clinical outcome have not led to any clear conclusions, our finding of a strong positive correlation between loss of CAV1 expression in breast tumour stroma and poor prognosis (p<0.0001) (Sloan et al, 2009, AJP 174: 2035-2043), is novel and exciting as it offers the potential of a reliable prognostic indicator of metastatic disease. Tumours arising from non- or weakly metastatic mammary cells grow at a faster or similar rate when co-injected with CAV1 null mammary fibroblasts. In contrast, co-injection with CAV1 expressing mammary fibroblasts has a suppressive effect on tumour growth. To mimic this phenotype observed in vivo, 3D co-culture assays are being developed, and although they have demonstrated a positive effect of fibroblasts on tumour cell migration and proliferation, no stromal CAV1 specific effect has been observed. To further understand the consequences of the loss of stromal CAV1, profiling of CAV1 expressing and null mouse mammary fibroblasts was conducted using cytokine arrays and cDNA microarrays. Differences in the secretion of known CAV1 associated cytokines such as RANTES, Gas6 and IL-6 were observed, with no significant changes in their expression. Based on these findings, CAV1 may be contributing directly to the progression of breast cancer and thus provide the basis of a stromal targeted therapy. An understanding of the mechanism by which CAV1 expression is lost from host cells, and how this loss leads to progressive disease, could allow a therapy that restores or substitutes for CAV1 activity in stromal cells or that targets the key cytokines involved in this paracrine signalling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1486. doi:1538-7445.AM2012-1486

Expression of Caveolin-1, Caveolin-2 and Caveolin-3 in Thyroid Cancer and Stroma

Objective: To study the expression status of caveolin-1, caveolin-2 and caveolin-3 in the epithelial and stromal compartments of conventional papillary thyroid carcinoma (PTC), diffuse sclerosing variant of papillary carcinoma (DSVPC) and anaplastic carcinoma (AC). Methods: Tissue microarrays were constructed from 70 PTC, 41 DSVPC and 12 AC, and immunohistochemical stains were performed with caveolin-1, caveolin-2, caveolin-3, cytokeratin, vimentin and E-cadherin. The expression status of these markers in the epithelial and stromal cells was evaluated, and the results were correlated with the clinicopathologic variables. Results: Epithelial caveolin-3 expression was absent in the majority of PTC and DSPVC, and was significantly increased in AC (p < 0.001). The stromal expression of caveolin-1, caveolin-2 and caveolin-3 increased in frequency from PTC to DSVPC to AC, and was significantly increased in the stroma of AC (p < 0.001). Cytokeratin and E-cadherin were more frequently negative in AC compared to PTC (p = 0.003) and DSVPC (p < 0.001), while vimentin was more frequently expressed in AC compared to PTC and DSVPC (both p < 0.001). Conclusion: Epithelial caveolin-3 expression is increased in AC compared to PTC and DSVPC, and this may be linked to the epithelial-mesenchymal transition process of AC. In addition, stromal caveolin-1, caveolin-2 and caveolin-3 expression was more frequent in AC compared to PTC and DSVPC, and the specific expression of caveolin-3 in the stroma of AC could suggest a possible role of myofibroblasts.

Pyruvate kinase expression (PKM1 and PKM2) in cancer-associated fibroblasts drives stromal nutrient production and tumor growth

We have previously demonstrated that enhanced aerobic glycolysis and/or autophagy in the tumor stroma supports epithelial cancer cell growth and aggressive behavior, via the secretion of high-energy metabolites. These nutrients include lactate and ketones, as well as chemical building blocks, such as amino acids (glutamine) and nucleotides. Lactate and ketones serve as fuel for cancer cell oxidative metabolism, and building blocks sustain the anabolic needs of rapidly proliferating cancer cells. We have termed these novel concepts the “Reverse Warburg Effect,” and the “Autophagic Tumor Stroma Model of Cancer Metabolism.” We have also identified a loss of stromal caveolin-1 (Cav-1) as a marker of stromal glycolysis and autophagy. The aim of the current study was to provide genetic evidence that enhanced glycolysis in stromal cells favors tumorigenesis. To this end, normal human fibroblasts were genetically-engineered to express the two isoforms of pyruvate kinase M (PKM1 and PKM2), a key enzyme in the glycolytic pathway. In a xenograft model, fibroblasts expressing PKM1 or PKM2 greatly promoted the growth of co-injected MDA-MB-231 breast cancer cells, without an increase in tumor angiogenesis. Interestingly, PKM1 and PKM2 promoted tumorigenesis by different mechanism(s). Expression of PKM1 enhanced the glycolytic power of stromal cells, with increased output of lactate. Analysis of tumor xenografts demonstrated that PKM1 fibroblasts greatly induced tumor inflammation, as judged by CD45 staining. In contrast, PKM2 did not lead to lactate accumulation, but triggered a “pseudo-starvation” response in stromal cells, with induction of an NFκB-dependent autophagic program, and increased output of the ketone body 3-hydroxy-buryrate. Strikingly, in situ evaluation of Complex IV activity in the tumor xenografts demonstrated that stromal PKM2 expression drives mitochondrial respiration specifically in tumor cells. Finally, immuno-histochemistry analysis of human breast cancer samples lacking stromal Cav-1 revealed PKM1 and PKM2 expression in the tumor stroma. Thus, our data indicate that a subset of human breast cancer patients with a loss of stromal Cav-1 show profound metabolic changes in the tumor microenvironment. As such, this subgroup of patients may benefit therapeutically from potent inhibitors targeting glycolysis, autophagy and/or mitochondrial activity (such as metformin).

Stromal Caveolin-1 Expression in Breast Carcinoma. Correlation with Early Tumor Recurrence and Clinical Outcome

Caveolin- (cav-1) has been linked to tumor progression and clinical outcome in breast cancer, but its role as a prognostic marker is still unclear. We evaluated stromal and tumor caveolin-1 expression in 91 breast carcinomas, and assessed the association between their expression and clinicopathologic variables as well as patient outcome and early tumor recurrence. Absence of stromal caveolin-1 expression was detected in 18.7% of cases, while 25.3% of cases revealed tumor epithelial caveolin-1 expression. Combined stromal and tumor caveolin-1 immunopositivity was seen in 24.2% of cases. Absence of stromal cav-1 associated with larger tumor size, higher grade, higher nodal stage, higher number of positive nodes, higher TNM stage, positive HER2 status, higher recurrence rate, and shorter mean progression free survival (PFS). Stromal cav-1 status was a significant predictor of PFS in ER+, PR +, and HER2 + tumors. In tamoxifen-treated patients, absence of stromal Cav-1 was a significant predictor of poor clinical outcome, suggestive of tamoxifen resistance. Conversely, tumor epithelial and combined caveolin-1 expression, didnot associate with patient outcome. In multivariate analysis, only TNM stage independently associated with survival. Loss of stromal caveolin-1 is a novel breast cancer biomarker that can predict early tumor recurrence, short PFS, and tamoxifen- resistance. Thus, its use as a predictive biomarker, especially in lower grade, lower stage, ER+, PR+, HER2+, and tamoxifen treated patients may allow for early interventions with more aggressive therapies. Thus, stromal marker expression and epithelial-stromal cross talk may be critical for tumor progression and metastasis.

Biomechanical Remodeling of the Microenvironment by Stromal Caveolin-1 Favors Tumor Invasion and Metastasis

Mechanotransduction is a key determinant of tissue homeostasis and tumor progression. It is driven by intercellular adhesions, cell contractility, and forces generated within the microenvironment and is dependent on extracellular matrix composition, organization, and compliance. We show that caveolin-1 (Cav1) favors cell elongation in three-dimensional cultures and promotes Rho- and force-dependent contraction, matrix alignment, and microenvironment stiffening through regulation of p190RhoGAP. In turn, microenvironment remodeling by Cav1 fibroblasts forces cell elongation. Cav1-deficient mice have disorganized stromal tissue architecture. Stroma associated with human carcinomas and melanoma metastases is enriched in Cav1-expressing carcinoma-associated fibroblasts (CAFs). Cav1 expression in breast CAFs correlates with low survival, and Cav1 depletion in CAFs decreases CAF contractility. Consistently, fibroblast expression of Cav1, through p190RhoGAP regulation, favors directional migration and invasiveness of carcinoma cells in vitro. In vivo, stromal Cav1 remodels peri- and intratumoral microenvironments to facilitate tumor invasion, correlating with increased metastatic potency. Thus, Cav1 modulates tissue responses through force-dependent architectural regulation of the microenvironment.

Matrix remodeling stimulates stromal autophagy, “fueling” cancer cell mitochondrial metabolism and metastasis

We have previously demonstrated that loss of stromal caveolin-1 (Cav-1) in cancer-associated fibroblasts is a strong and independent predictor of poor clinical outcome in human breast cancer patients. However, the signaling mechanism(s) by which Cav-1 downregulation leads to this tumor-promoting microenvironment are not well understood. To address this issue, we performed an unbiased comparative proteomic analysis of wild-type (WT) and Cav-1-/- null mammary stromal fibroblasts (MSFs). Our results show that plasminogen activator inhibitor type 1 and type 2 (PAI-1 and PAI-2) expression is significantly increased in Cav-1-/- MSFs. To establish a direct cause-effect relationship, we next generated immortalized human fibroblast lines stably overexpressing either PAI-1 or PAI-2. Importantly, PAI-1/2(+) fibroblasts promote the growth of MDA-MB-231 tumors (a human breast cancer cell line) in a murine xenograft model, without any increases in angiogenesis. Similarly, PAI-1/2(+) fibroblasts stimulate experimental metastasis of MDA-MB-231 cells using an in vivo lung colonization assay. Further mechanistic studies revealed that fibroblasts overexpressing PAI-1 or PAI-2 display increased autophagy (“self-eating”) and are sufficient to induce mitochondrial biogenesis/activity in adjacent cancer cells, in co-culture experiments. In xenografts, PAI-1/2(+) fibroblasts significantly reduce the apoptosis of MDA-MB-231 tumor cells. The current study provides further support for the “Autophagic Tumor Stroma Model of Cancer” and identifies a novel “extracellular matrix”-based signaling mechanism, by which a loss of stromal Cav-1 generates a metastatic phenotype. Thus, the secretion and remodeling of extracellular matrix components (such as PAI-1/2) can directly regulate both (1) autophagy in stromal fibroblasts and (2) epithelial tumor cell mitochondrial metabolism.

Molecular profiling of a lethal tumor microenvironment, as defined by stromal caveolin-1 status in breast cancers

Breast cancer progression and metastasis are driven by complex and reciprocal interactions, between epithelial cancer cells and their surrounding stromal microenvironment. We have previously shown that a loss of stromal Cav-1 expression is associated with an increased risk of early tumor recurrence, metastasis and decreased overall survival. To identify and characterize the signaling pathways that are activated in Cav-1 negative tumor stroma, we performed gene expression profiling using laser microdissected breast cancer-associated stroma. Tumor stroma was laser capture microdissected from 4 cases showing high stromal Cav-1 expression and 7 cases with loss of stromal Cav-1. Briefly, we identified 238 gene transcripts that were upregulated and 232 gene transcripts that were downregulated in the stroma of tumors showing a loss of Cav-1 expression (p ≤ 0.01 and fold-change ≥1.5). Gene set enrichment analysis (GSEA) revealed “stemness,” inflammation, DNA damage, aging, oxidative stress, hypoxia, autophagy and mitochondrial dysfunction in the tumor stroma of patients lacking stromal Cav-1. Our findings are consistent with the recently proposed “Reverse Warburg Effect” and the “Autophagic Tumor Stroma Model of Cancer Metabolism.” In these two complementary models, cancer cells induce oxidative stress in adjacent stromal cells, which then forces these stromal fibroblasts to undergo autophagy/mitophagy and aerobic glycolysis. This, in turn, produces recycled nutrients (lactate, ketones and glutamine) to feed anabolic cancer cells, which are undergoing oxidative mitochondrial metabolism. Our results are also consistent with previous biomarker studies showing that the increased expression of known autophagy markers (such as ATG16L and the cathepsins) in the tumor stroma is specifically associated with metastatic tumor progression and/or poor clinical outcome.

The impact of caveolin protein expression in tumor stroma on prognosis of breast cancer

We aimed to investigate the expression of caveolin-1, -2, -3, and platelet-derived growth factor (PDGF) β receptor in breast cancer cells and stroma by immunohistochemistry and to analyze their implications. The expression rates of stromal caveolin-2 and PDGF β receptor increased as the tumor progressed from ductal carcinoma in situ to microinvasive ductal carcinoma, intraductal component of invasive ductal carcinoma (IDC), and IDC (p<0.001). The expression loss of caveolin-1 in tumor stroma of IDC correlated with high tumor stage (p<0.001), high nodal stage (p=0.011), high cancer stage (p=0.005), estrogen receptor negativity (p=0.003), and tumor recurrence (p=0.003). In addition, the expression loss of caveolin-1 in tumor stroma was correlated with a shorter disease-free survival and an overall survival (p<0.001). In conclusion, the loss of stromal caveolin-1 is related to poor prognosis in IDC.

Abstract P3-10-42: Stromal Caveolin-1 Is a Powerful Marker That Further Enhances a Multi-Marker Prognostic Profile

Abstract Background: We previously reported development and validation of a prognostic profile with a continuous risk of recurrence algorithm for patients with operable, hormone receptor-positive breast cancer to help guide the appropriate level of adjuvant treatment. The prognostic profile includes six IHC markers (ER, PR, HER2, EGFR, BCL2, and p53), one FISH marker (MYC/8q24), and three clinicopathologic risk factors (tumor size, tumor grade, and nodal status). p27 is an additional marker that is predictive of chemotherapy benefit. When a single risk threshold is chosen for the prognostic profile to divide patients into high and low risk categories, a hazard ratio of ∼4-10 is consistently achieved with a ∼4-6% 10-year recurrence rate (95% CI, ∼0-9%). Several additional markers, including caveolin-1, MTA1, and TIMP1, were identified in previous pilot studies as candidates to add prognostic power to the profile. This was based on their ability to stratify patients and their implicated roles in invasion and metastasis pathways that are not covered by the current markers. The goal of this study was to validate these additional candidates. Methods: TMAs of patients from Royal Perth Hospital (RPH), on which the current profile had already been run, were stained by IHC for the new markers, caveolin-1, MTA1, and TIMP1. Pathologists recorded intensity (0, 1, 2, or 3) and percentage (0-100%) scores, and the products of the scores were calculated to generate immunoscores. Membranous/cytoplasmic caveolin-1 was scored separately in tumor epithelial and stromal cells. Nuclear MTA1, nuclear TIMP1, and cytoplasmic TIMP1 were scored in tumor cells. Kaplan-Meier survival and Cox proportional hazards analyses were then conducted using various marker thresholds and known patient outcome. Results: In univariate analyses, only stromal caveolin-1 reached statistical significance. In patients treated with hormone therapy with or without chemotherapy (n=173), stromal caveolin-1 (immunoscore threshold of 200) achieved a hazard ratio of 5.6 (95% CI, 2.4-13) for time to recurrence. Similar results were achieved in the hormone therapy-only group (n=131). However, the caveolin-1 low-risk group had a 10-year recurrence rate that exceeded 10%, so its clinical utility is limited as a single marker. When caveolin-1 was assessed within the profile high-risk patients of the hormone therapy with or without chemotherapy group (baseline 10-year recurrence rate of ∼50%), it separated them into sub-groups with -30% and -80% recurrence rates (P&amp;lt;0.0001). And, when it was assessed in the profile low-risk group, it identified two of the three recurrence events (previously false negatives). Conclusion: Stromal caveolin-1 appears to be a powerful independent marker, and it will be incorporated into the current profile for future validation studies. Such stromal markers cannot easily be assessed in transcript-based prognostics due to tissue processing requirements. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P3-10-42.

Caveolin-1 P132L Mutation in Human Cancers: 1 CAVeat to be Voiced

Caveolae are 50–100 nm flask-shaped invaginations of the plasma membrane that function as membrane organizing centers and play important roles in intracellular trafficking of cellular components (eg, lipid and cholesterol) and signal transduction.1 The CAV1 gene maps to 7q31.1 and encodes the ubiquitously expressed caveolin-1, which is an essential component of caveolae. Caveolin-1 is believed to regulate the activity of a plethora of signaling molecules highly represented in caveolae, such as H-ras, c-src, and src-like kinases; endothelial nitric oxide synthase; G-proteins and membrane-associated receptors coupled to G-proteins; or tyrosine kinase receptors including epidermal growth factor receptor.1 In normal breast tissue, caveolin-1 is expressed at high levels in adipocytes, endothelial cells, fibroblasts, and myoepithelial cells of the breast.1 Initial studies have suggested that caveolin-1 would have properties consistent with those of a tumor suppressor protein.2,3,4 A dominant negative mutation of the CAV1 gene was reported to be found in up to 20% of estrogen receptor (ER)-positive breast cancers.2,4 Surprisingly, however, CAV1 knock-out mice failed to develop any tumor.1,3 Furthermore, the presence of CAV1 gene mutations could not be independently validated.5,6,7,8,9 Concurrent with the controversies about the role of CAV1 as a tumor suppressor gene, evidence of caveolin-1 as a potential oncogene has been mounting.7,8,9,10,11,12,13,14,15 In fact, multiple independent groups have reported overexpression of caveolin-1 in aggressive subtypes of cancer,10,11,13,14,15 and even CAV1 gene amplification has been documented,14,16 although this has been shown to be a rare phenomenon. Furthermore, expression of caveolin-1 in stromal cells of breast cancer has also been shown to be associated with the outcome of breast cancer patients.17,18 Despite the interest in the tumor suppressive and oncogenic functions of caveolin-1, numerous issues remain unanswered. For instance, caveolin-1 expression in normal luminal epithelial cells of normal breast lobules and ducts remains a matter of controversy, as does the existence and prevalence of the CAV1 P132L dominant-negative mutation. In this issue of The Journal of Molecular Diagnostics, Koike et al19 have addressed the question of the prevalence of the CAV1 P132L mutation in human cancer. Using three different methods for mutation detection (ie, reverse-transcriptase direct sequencing, DNA-based direct sequencing, and cycleave PCR assay), the authors failed to detect the CAV1 P132L mutation in a large series of 409 human tumors, including 139 breast cancers [114 estrogen receptor (ER)-positive and 25 ER-negative tumors] and 270 cancers from other anatomical sites (ie, gastrointestinal tract, lung, ovary, and pancreas). Taken together, the findings reported by Koike et al19 and those by others5,6,7,8,9 call into question the presence of the CAV1 P132L mutation in breast cancers and other cancer types and heat up the debate about the roles of caveolin-1 in human cancer.

Autophagy in cancer associated fibroblasts promotes tumor cell survival

Recently, using a co-culture system, we demonstrated that MCF7 epithelial cancer cells induce oxidative stress in adjacent cancer-associated fibroblasts, resulting in the autophagic/lysosomal degradation of stromal caveolin-1 (Cav-1). However, the detailed signaling mechanism(s) underlying this process remain largely unknown. Here, we show that hypoxia is sufficient to induce the autophagic degradation of Cav-1 in stromal fibroblasts, which is blocked by the lysosomal inhibitor chloroquine. Concomitant with the hypoxia-induced degradation of Cav-1, we see the upregulation of a number of well-established autophagy/mitophagy markers, namely LC3, ATG16L, BNIP3, BNIP3L, HIF-1α and NFκB. In addition, pharmacological activation of HIF-1α drives Cav-1 degradation, while pharmacological inactivation of HIF-1 prevents the downregulation of Cav-1. Similarly, pharmacological inactivation of NFκB-another inducer of autophagy-prevents Cav-1 degradation. Moreover, treatment with an inhibitor of glutathione synthase, namely BSO, which induces oxidative stress via depletion of the reduced glutathione pool, is sufficient to induce the autophagic degradation of Cav-1. Thus, it appears that oxidative stress mediated induction of HIF1- and NFκB-activation in fibroblasts drives the autophagic degradation of Cav-1. In direct support of this hypothesis, we show that MCF7 cancer cells activate HIF-1α- and NFκB-driven luciferase reporters in adjacent cancer-associated fibroblasts, via a paracrine mechanism. Consistent with these findings, acute knock-down of Cav-1 in stromal fibroblasts, using an siRNA approach, is indeed sufficient to induce autophagy, with the upregulation of both lysosomal and mitophagy markers. How does the loss of stromal Cav-1 and the induction of stromal autophagy affect cancer cell survival? Interestingly, we show that a loss of Cav-1 in stromal fibroblasts protects adjacent cancer cells against apoptotic cell death. Thus, autophagic cancer-associated fibroblasts, in addition to providing recycled nutrients for cancer cell metabolism, also play a protective role in preventing the death of adjacent epithelial cancer cells. We demonstrate that cancer-associated fibroblasts upregulate the expression of TIGAR in adjacent epithelial cancer cells, thereby conferring resistance to apoptosis and autophagy. Finally, the mammary fat pads derived from Cav-1 (-/-) null mice show a hypoxia-like response in vivo, with the upregulation of autophagy markers, such as LC3 and BNIP3L. Taken together, our results provide direct support for the "Autophagic Tumor Stroma Model of Cancer Metabolism," and explain the exceptional prognostic value of a loss of stromal Cav-1 in cancer patients. Thus, a loss of stromal fibroblast Cav-1 is a biomarker for chronic hypoxia, oxidative stress and autophagy in the tumor microenvironment, consistent with its ability to predict early tumor recurrence, lymph node metastasis and tamoxifen-resistance in human breast cancers. Our results imply that cancer patients lacking stromal Cav-1 should benefit from HIF-inhibitors, NFκB-inhibitors, anti-oxidant therapies, as well as autophagy/lysosomal inhibitors. These complementary targeted therapies could be administered either individually or in combination, to prevent the onset of autophagy in the tumor stromal compartment, which results in a "lethal" tumor microenvironment.

Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution

Loss of stromal fibroblast caveolin-1 (Cav-1) is a powerful single independent predictor of poor prognosis in human breast cancer patients, and is associated with early tumor recurrence, lymph node metastasis, and tamoxifen-resistance. We developed a novel co-culture system to understand the mechanism(s) by which a loss of stromal fibroblast Cav-1 induces a "lethal tumor micro-environment". Here, we propose a new paradigm to explain the powerful prognostic value of stromal Cav-1. In this model, cancer cells induce oxidative stress in cancer associated fibroblasts, which then acts as a "metabolic" and "mutagenic" motor to drive tumor-stroma co-evolution, DNA damage, and aneuploidy in cancer cells. More specifically, we show that an acute loss of Cav-1 expression leads to mitochondrial dysfunction, oxidative stress, and aerobic glycolysis in cancer associated fibroblasts. Also, we propose that defective mitochondria are removed from cancer-associated fibroblasts by autophagy/mitophagy that is induced by oxidative stress. As a consequence, cancer associated fibroblasts provide nutrients (such as lactate) to stimulate mitochondrial biogenesis and oxidative metabolism in adjacent cancer cells (the "Reverse Warburg Effect"). We provide evidence that oxidative stress in cancer associated fibroblasts is sufficient to induce genomic instability in adjacent cancer cells, via a bystander effect, potentially increasing their aggressive behavior. Finally, we directly demonstrate that nitric oxide (NO) over-production, secondary to Cav-1 loss, is the root cause for mitochondrial dysfunction in cancer associated fibroblasts. In support of this notion, treatment with anti-oxidants (such as N-acetyl-cysteine, metformin, and quercetin), or NO inhibitors (L-NAME) was sufficient to reverse many of the cancer-associated fibroblast phenotypes that we describe. Thus, cancer cells use "oxidative stress" in adjacent fibroblasts i) as an "engine" to fuel their own survival via the stromal production of nutrients, and ii) to drive their own mutagenic evolution towards a more aggressive phenotype, by promoting genomic instability. We also present evidence that the "field effect" in cancer biology could also be related to the stromal production of ROS and NO species. eNOS-expressing fibroblasts have the ability to down-regulate Cav-1 and induce mitochondrial dysfunction in adjacent fibroblasts that do not express eNOS. As such, the effects of stromal oxidative stress can be laterally propagated, amplified, and are effectively "contagious"-spread from cell-to-cell like a virus-creating an "oncogenic/mutagenic" field promoting widespread DNA damage.

Loss of stromal caveolin-1 expression predicts poor clinical outcome in triple negative and basal-like breast cancers

Here, we investigated the possible predictive value of stromal caveolin-1 (Cav-1) as a candidate biomarker for clinical outcome in triple negative (TN) breast cancer patients. A cohort of 85 TN breast cancer patients was available, with the necessary annotation and nearly 12 years of follow-up data. Our primary outcome of interest in this study was overall survival. Interestingly, TN patients with high-levels of stromal Cav-1, had a good clinical outcome, with >50% of the patients remaining alive during the follow-up period. In contrast, the median survival for TN patients with moderate stromal Cav-1 staining was 33.5 months. Similarly, the median survival for TN patients with absent stromal Cav-1 staining was 25.7 months. A comparison of 5-year survival rates yields a similar pattern. TN patients with high stromal Cav-1 had a good 5-year survival rate, with 75.5% of the patients remaining alive. In contrast, TN patients with moderate or absent stromal Cav-1 levels had progressively worse 5-year survival rates, with 40% and 9.4% of the patients remaining alive. In contrast, in a parallel analysis, the levels of tumor epithelial Cav-1 had no prognostic significance. As such, the prognostic value of Cav-1 immunostaining in TN breast cancer patients is compartment-specific, and selective for an absence of Cav-1 staining in the stromal fibroblast compartment. A recursive-partitioning algorithm was used to assess which factors are most predictive of overall survival in TN breast cancer patients. In this analysis, we included tumor size, histologic grade, whether the patient received surgery, radiotherapy or chemotherapy, CK5/6, EGFR, p53 and Ki67 status, as well as the stromal Cav-1 score. This analysis indicated that stromal Cav-1 expression was the most important prognostic factor for overall survival in TN breast cancer. Virtually identical results were obtained with CK5/6 (+) and/or EGFR (+) TN breast cancer cases, demonstrating that a loss of stromal Cav-1 is also a strong prognostic factor for basal-like breast cancers. Our current findings may have important implications for the close monitoring and treatment stratification of TN and basal-like breast cancer patients.

Loss of stromal caveolin-1 leads to oxidative stress, mimics hypoxia and drives inflammation in the tumor microenvironment, conferring the “reverse Warburg effect”: A transcriptional informatics analysis with validation

Cav-1 (-/-) deficient stromal cells are a new genetic model for myofibroblasts and cancer-associated fibroblasts. Using an unbiased informatics analysis of the transcriptional profile of Cav-1 (-/-) deficient mesenchymal stromal cells, we have now identified many of the major signaling pathways that are activated by a loss of Cav-1, under conditions of metabolic restriction (with low glucose media). Our informatics analysis suggests that a loss of Cav-1 induces oxidative stress, which mimics a constitutive pseudo-hypoxic state, leading to 1) aerobic glycolysis and 2) inflammation in the tumor stromal microenvironment. This occurs via the activation of 2 major transcription factors, namely HIF (aerobic glycolysis) and NF-kB (inflammation) in Cav-1 (-/-) stromal fibroblastic cells. Experimentally, we show that Cav-1 deficient stromal cells may possess defective mitochondria, due to the over-production of nitric oxide (NO), resulting in the tyrosine nitration of the mitochondrial respiratory chain components (such as complex I). Elevated levels of nitro-tyrosine were observed both in Cav-1 (-/-) stromal cells, and via acute knock-down with siRNA targeting Cav-1. Finally, metabolic restriction with mitochondrial (complex I) and glycolysis inhibitors was synthetically lethal with a Cav-1 (-/-) deficiency in mice. As such, Cav-1 deficient mice show a dramatically reduced mitochondrial reserve capacity. Thus, a mitochondrial defect in Cav-1 deficient stromal cells could drive oxidative stress, leading to aerobic glycolysis, and inflammation, in the tumor microenvironment. These stromal alterations may underlie the molecular basis of the “Reverse Warburg Effect”, and could provide the key to targeted anti-cancer therapies using metabolic inhibitors. In direct support of these findings, the transcriptional profile of Cav-1 (-/-) stromal cells overlaps significantly with Alzheimer’s disease, which is characterized by oxidative stress, NO over-production (peroxynitrite formation), inflammation, hypoxia, and mitochondrial dysfunction. We conclude that Cav-1 (-/-) deficient mice are a new whole-body animal model for an activated lethal tumor micro-environment, i.e., “tumor stroma” without the tumor. Since Cav-1 (-/-) mice are also an established animal model for pro-fibrotic disease, our current results may have implications for understanding the pathogenesis of scleroderma (systemic sclerosis) and pulmonary fibrosis, which are also related to abnormal mesenchymal stem cell function.

The reverse Warburg Effect: Glycolysis inhibitors prevent the tumor promoting effects of caveolin-1 deficient cancer associated fibroblasts

We and others have previously identified a loss of stromal caveolin-1 (Cav-1) in cancer-associated fibroblasts (CAFs) as a powerful single independent predictor of breast cancer patient tumor recurrence, metastasis, tamoxifen-resistance, and poor clinical outcome. However, it remains unknown how loss of stromal Cav-1 mediates these effects clinically. To mechanistically address this issue, we have now generated a novel human tumor xenograft model. In this two-component system, nude mice are co-injected with i) human breast cancer cells (MDA-MB-231), and ii) stromal fibroblasts (wild-type (WT) versus Cav-1 (-/-) deficient). This allowed us to directly evaluate the effects of a Cav-1 deficiency solely in the tumor stromal compartment. Here, we show that Cav-1-deficient stromal fibroblasts are sufficient to promote both tumor growth and angiogenesis, and to recruit Cav-1 (+) micro-vascular cells. Proteomic analysis of Cav-1-deficient stromal fibroblasts indicates that these cells upregulate the expression of glycolytic enzymes, a hallmark of aerobic glycolysis (the Warburg effect). Thus, Cav-1-deficient stromal fibroblasts may contribute towards tumor growth and angiogenesis, by providing energy-rich metabolites in a paracrine fashion. We have previously termed this new idea the “Reverse Warburg Effect”. In direct support of this notion, treatment of this xenograft model with glycolysis inhibitors functionally blocks the positive effects of Cav-1-deficient stromal fibroblasts on breast cancer tumor growth. Thus, pharmacologically-induced metabolic restriction (via treatment with glycolysis inhibitors) may be a promising new therapeutic strategy for breast cancer patients that lack stromal Cav-1 expression. We also identify the stromal expression of PKM2 and LDH-B as new candidate biomarkers for the “Reverse Warburg Effect” or “Stromal-Epithelial Metabolic Coupling” in human breast cancers.

Stromal Caveolin-1 Levels Predict Early Ductal Carcinoma In Situ Progression to Invasive Breast Carcinoma.

Abstract With the increase use of screening mammography the incidence of ductal carcinoma in situ (DCIS) increased and DCIS currently accounts for 20-30% of newly diagnosed breast cancers.The current classification for DCIS based on nuclear grade, architectural differentiation and presence of necrosis, does not adequately predict the likelihood of recurrence after breast conservingtherapy. Therefore, there is a critical need to identify novel predictors of DCIS progression and potential targets for therapy. Recently, we showed that an absence of stromal caveolin-1 (Cav-1)expression in invasive breast cancers is a powerful single independent predictor of early disease recurrence and metastasis. The aim of this study was to evaluate the stromal expression of Cav-1 in a cohort of DCIS patients treated with wide-excision and close follow-up, but without any form of chemo- or radiotherapy.Material and MethodsSeventy eighty DCIS cases were included in the study. Cav-1 expression in the tumor stroma was assessed by employing a standard immunoperoxidase method with monoclonal ani-Cav-1 antibody (BD Biosciences, San Jose, CA; 1:50 dilution).The staining was scored semi-quantitatively as negative (0; no staining), weak (1; either diffuse weak staining or strong staining in less than 30% of stromal cells) and strong (2; defined as strong staining of 30% of stromal cells).Statistical analysis was performed using the Fisher exact test or the Kruskal-Wallis test.ResultsThe overall recurrence rate was twenty-eight percent (50% recurred as DCIS, and the other 50% recurred with progression to invasive breast cancer). Nearly seventy percent of DCIS patients with any type of recurrence had reduced levels of stromal Cav-1 (score = 1 or 0). Eighty nine percent of DCIS patients that underwent recurrence with progression to invasive breast cancer had reduced or absent levels of stromal Cav-1. Conversely, ninety-seven percent of DCIS patients with high levels of stromal Cav-1 (score = 2) did not show any invasive recurrence over the duration of follow-up (4-208 months).Our results indicate that an absence of stromal Cav-1 is a novel biomarker that predicts early DCIS progression to invasive breast carcinoma. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2136.

An absence of stromal caveolin-1 is associated with advanced prostate cancer, metastatic disease spread and epithelial Akt activation

Here, we examined the status of stromal Cav-1 expression in patients with benign prostatic hypertrophy (BPH), primary prostate cancers (PCa), and prostate-cancer metastases (Mets). Interestingly, an absence of stromal Cav-1 directly correlated with prostate cancer disease progression. For example, virtually all BPH samples showed abundant stromal Cav-1 immunostaining. In contrast, in a subset of patients with primary prostate cancer, the stromal levels of Cav-1 were significantly decreased, and this correlated with a high Gleason score, indicative of a worse prognosis and poor clinical outcome. Remarkably, all metastatic tumors (either from lymph node or bone) were completely negative for stromal Cav-1 staining. Thus, stromal Cav-1 expression may be considered as a new biomarker of prostate cancer disease progression and metastasis. Mechanistically, stromal Cav-1 levels were inversely correlated with the epithelial expression levels of Cav-1 and epithelial phospho-Akt. Thus, loss of stromal Cav-1 is predictive of elevated levels of epithelial Cav-1 and epithelial Akt-activation. This provides important new clinical evidence for paracrine signaling between prostate cancer epithelial cells and the tumor stromal micro-environment, especially related to disease progression and metastasis.

Stromal caveolin-1 levels predict early DCIS progression to invasive breast cancer

Here, we determined the possible association of stromal caveolin-1 (Cav-1) levels with DCIS recurrence and/or progression to invasive breast cancer. An initial cohort of 78 DCIS patients with follow-up data was examined. As ER-positivity was associated with recurrence, we focused our analysis on this subset of 56 patients. In this group, we observed that DCIS progressed to invasive breast cancer in ~14% of the patient population (8/56), in accordance with an expected progression rate of 12-15%. Nearly ninety percent of DCIS patients (7/8) that underwent recurrence to invasive breast cancer had reduced or absent levels of stromal Cav-1. Remarkably, an absence of stromal Cav-1 (score = 0) was specifically associated with early disease progression to invasive breast cancer, with reduced time to recurrence and higher recurrence rate. All DCIS patients with an absence of stromal Cav-1 underwent some form of recurrence (5/5) and the majority (4/5) underwent progression to invasive breast cancer. This represents an overall cumulative incidence rate of 100% for recurrence and 80% for progression. An absence of stromal Cav-1 in DCIS lesions was also specifically associated with the presence of inflammatory cells. Conversely, ninety-seven percent of ER(+) DCIS patients (35/36) with high levels of stromal Cav-1 (score = 2) did not show any invasive recurrence over the duration of follow-up (4-208 months), and 89% of such patients are estimated to remain free of invasive recurrence, even after 15 years. Thus, determination of stromal Cav-1 levels may be a useful new biomarker for guiding the treatment of ER(+) DCIS patients.

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.

Stromal Cell Expression of Caveolin-1 Predicts Outcome in Breast Cancer

Caveolin-1 has been linked to tumor progression and clinical outcome in breast cancer, but a clear resolution of its role as a prognostic marker is lacking. We assessed caveolin-1 levels in normal breast tissue and two breast cancer cohorts for which outcome data were available. We found that caveolin-1 was not expressed in normal breast luminal epithelium but was present in the epithelial compartment of some tumors. We found no association between caveolin-1 expression in the epithelial compartment and clinical outcome. However, high levels of caveolin-1 in the stromal tissue surrounding the tumor, rather than within tumor cells, associated strongly with reduced metastasis and improved survival (P < 0.0001). The onset of mammary tumors driven by Her2/neu overexpression was accelerated in mice lacking caveolin-1, thereby supporting the observation that the presence of caveolin-1 in the tumor microenvironment modulates tumor development. These studies suggest that stromal caveolin-1 expression may be a potential therapeutic target and a valuable prognostic indicator of breast cancer progression.