Role Of Cav-1 Research Articles

Cav1.3 L-Type Calcium Channel Dysfunction in Human Disease

Voltage-gated calcium-channels are key molecular regulators of calcium-dependent signaling processes in electrically excitable cells. The family of L-type calcium-channels (LTCCs, Cav1.1-Cav1.4 pore-forming α1-subunits) are sensitive to organic calcium-channel blockers (e.g. dihydropyridines). The in vivo pharmacological effects of these widely used antihypertensives are limited to inhibition of Cav1.2 LTCCs in the cardiovascular system, despite the (often overlapping) expression of both Cav1.2 and Cav1.3 in most excitable tissues. Important insight into the physiological relevance of these channels therefore came from mutant mice and human disease pinpointing their distinct functions not only in the heart but also for hearing, brain and endocrine function. Cav1.2 and Cav1.3 α1 share high structural similarity and are frequently expressed in the same cell but exhibit important functional differences. Their activity is fine-tuned by auto-inhibitory domains within their long C-terminal tails but they use different mechanisms to adjust the extent of auto-inhibition. Moreover, Cav1.3 channels activate at more negative potentials allowing them to serve unique roles for sinoatrial node pacemaking and cochlear inner hair cell neurotransmitter release. Accordingly, a loss-of-function mutation in Cav1.3 α1 (CACNA1D) in humans causes sinoatrial node dysfunction and congenital deafness (SANDD). Human mutations also revealed a novel role for Cav1.3 in adrenal aldosterone-producing adenomas (APA). In APA somatic gain-of-function mutations can drive calcium-dependent aldosterone production causing secondary hypertension. SANDD and APA mutations also provided valuable insight into the structure-function relationship of Cav1.3 α1.Both channels have recently also been implicated as risk genes for psychiatric diseases, including autism spectrum disorders. It will therefore be important to understand how subtle changes in the function of these channels is associated with human disease and how calcium-channel blockers can be further optimized to specifically target them to LTCCs outside the cardiovascular system.(Support: Austrian Science Fund, F44020).

Loss of Caveolin-1 Causes Blood–Retinal Barrier Breakdown, Venous Enlargement, and Mural Cell Alteration

Blood-retinal barrier (BRB) breakdown and related vascular changes are implicated in several ocular diseases. The molecules and mechanisms regulating BRB integrity and pathophysiology are not fully elucidated. Caveolin-1 (Cav-1) ablation results in loss of caveolae and microvascular pathologies, but the role of Cav-1 in the retina is largely unknown. We examined BRB integrity and vasculature in Cav-1 knockout mice and found a significant increase in BRB permeability, compared with wild-type controls, with branch veins being frequent sites of breakdown. Vascular hyperpermeability occurred without apparent alteration in junctional proteins. Such hyperpermeability was not rescued by inhibiting eNOS activity. Veins of Cav-1 knockout retinas exhibited additional pathological features, including i) eNOS-independent enlargement, ii) altered expression of mural cell markers (eg, down-regulation of NG2 and up-regulation of αSMA), and iii) dramatic alterations in mural cell phenotype near the optic nerve head. We observed a significant NO-dependent increase in retinal artery diameter in Cav-1 knockout mice, suggesting that Cav-1 plays a role in autoregulation of resistance vessels in the retina. These findings implicate Cav-1 in maintaining BRB integrity in retinal vasculature and suggest a previously undefined role in the retinal venous system and associated mural cells. Our results are relevant to clinically significant retinal disorders with vascular pathologies, including diabetic retinopathy, uveoretinitis, and primary open-angle glaucoma.

CAV1.2 calcium channel is involved in the circadian regulation of sleep

Two L-type Ca 2+ channels (Cav1.2 & Cav1.3) are expressed in the mouse brain with Cav1.2 contributing to a major proportion (85%). The expression of L-type Ca 2+ channels is greatly densed in the suprachiasmatic nuclei, a neuronal area capable of functioning as autonomous clock and as a generator of circadian rhythms. Although one of the most important aspects regarding the circadian rhythm is the sleep–wake cycle, yet the role of Cav1.2 in the sleep–wake cycle is unclear. Therefore, we investigated whether depletion of Cav1.2 in a transgenic mouse line Cav1.2 (+/−) would alter spontaneous sleep–wake activities. Sleep–wake patterns were monitored by means of EEG and EMG recordings in Cav1.2 (+/−) mice and their wild-type littermates under baseline and sleep deprivation conditions (6 h by gentle handling). Under basal condition, Cav1.2 (+/−) mice showed increased sleep onset latency but subsequent hypersomnolence as compared to wild-type. The hypersomnolence observed in these mice was characterized by higher slow wave activity. Moreover, these heterozygous mice also exhibited drastically shorter wake episodes in the dark period, due to an increased number of NREM sleep episodes. Analysis of sleep architecture further revealed that these mice showed frequent transitions from wake to NREM sleep and vice versa. After sleep deprivation, the sleep onset latency decreased drastically and the trend for higher NREM sleep was observed in Cav1.2 (+/−) mice. Our results demonstrate that depletion of Cav1.2 significantly impacts circadian sleep regulation indicated by increased NREM sleep and decreased time spent in wake in the dark period. However, homeostatic regulation of sleep was unaltered. It has been reported that L-type Ca 2+ channels are involved in wake-promoting effects of hypocretin1. Therefore, decreased wakefulness in Cav1.2 (+/−) mice suggests that a depletion of Cav1.2 might attenuate the hypocretin 1 mediated excitation of wake-related neurons. The alpha-1 subunit of L-type Ca 2+ channels (Cav1.2) is encoded by the CACNA1C gene. Genome-wide association studies (GWAS) have suggested that polymorphisms in the CACNA1C gene are associated with sleep disorders, e.g., insomnia and narcolepsy. Increased sleep onset latency and fragmented sleep architecture displayed by Cav1.2 (+/−) mice might be signs of symptomatic sleep disorders but further studies are needed to elucidate this. Nevertheless, several GWAS studies have significantly associated the CACNA1C gene with psychiatric disorders.

IGF-IR internalizes with CAVEOLIN-1 and PTRF/CAVIN in HACAT cells

Background: Insulin-like growth factor-I receptor (IGF-IR) is a tyrosine kinase receptor (RTK) associated with caveolae, invaginations of the plasma membrane that regulate vesicular transport, endocytosis and intracellular signaling. IGF-IR internalization represents a key mechanism of down-modulation of receptors number on plasma membrane. IGF-IR interacts directly with Caveolin-1 (Cav-1), the most relevant protein of caveolae. Recently it has been demonstrated that the Polymerase I and Transcript Release Factor I (PTRF/Cavin) is required for caveolae biogenesis and function. The role of Cav-1 and PTRF/Cavin in IGF-IR internalization is still to be clarified. Methodology/Principal Findings: We have investigated the interaction of IGF-IR with Cav-1 and PTRF/Cavin in the presence of IGF1in human Hacat cells. We show that IGF-IR internalization triggers Cav-1 and PTRF/Cavin translocation from plasma membrane to cytosol and increases IGF-IR interaction with these proteins. In fact, Cav-1 and PTRF/Cavin coimmunoprecipitate with IGF-IR during receptor internalization. We found a different time course of co-immunoprecipitation between IGF-IR and Cav-1 compared to IGF-IR and PTRF/Cavin. Cav-1 and PTRF/Cavin silencing by siRNA differently affect surface IGF-IR levels following IGF1 treatment: Cav-1 and PTRF/Cavin silencing significantly affect IGF-IR rate of internalization, while PTRF/Cavin silencing also decreases IGF-IR plasma membrane recovery. Since Cav-1 phosphorylation could have a role in IGF-IR internalization, the mutant Cav-1Y14F lacking Tyr14 was transfected. Cav-1Y14F transfected cells showed a reduced internalization of IGF-IR compared with cells expressing wild type Cav-1. Receptor internalization was not impaired by Clathrin silencing. These findings support a critical role of caveolae in IGF-IR intracellular traveling. Conclusions/Significance: These data indicate that Caveolae play a role in IGF-IR internalization. Based on these findings, Cav-1 and PTRF/Cavin could represent two relevant and distinct targets to modulate IGF-IR function.

Caveolin-1 Is Required For T Cell-Mediated Acute GvHD

Caveolin-1 (Cav-1) is a key organizer of membrane specializations that coordinate membrane and protein traffic in different cells including T cells. Cav-1 promotes the formation and stability of plasma membrane raft microdomains that serve as platforms for signal transduction and binds a wide array of signal transducers through interactions with its phosphorylated tyrosine 14 or the so-called scaffolding domain. Several of the proteins identified as Cav-1 binding partners have been suggested to play a role in TCR-regulated membrane dynamics and intracellular signaling. Therefore, we aimed to determine the requirement of Cav-1 for T cells during acute graft-versus-host disease (GvHD). We observed upregulation of Cav-1 in the T cells of mice developing acute GvHD which was dependent on both tissue damage and homeostatic signals as investigated in conditioned WT recipients or RAG-/-C gamma chain-/- non-conditioned mice. By using gene targeted mice we found that Cav-1 deficiency of the donor led to improved survival (p=0.02), lower GvHD histopathology scores (p=0.004) and reduced serum levels of IL-6 (p=0.02) while Treg numbers increased when Cav-1 deficient donors were used. Comparative microarray analysis of WT compared to Cav-1 deficient T cells indicated a reduced IL-17A production in Cav-1 T cells exposed to allogeneic dendritic cells. These observations were confirmed on the protein level. In summary we show a role for Cav-1 in T cells during GvHD as an alloantigen driven T cell response. Regarding the mechanism we describe the relevance of Cav-1 for Th-17 lineage commitment of alloreactive T cells during GvHD. Disclosures: No relevant conflicts of interest to declare.

Developmental alterations in the biophysical properties of Cav1.3 Ca2+ channels in mouse inner hair cells

Prior to hearing onset, spontaneous action potentials activate voltage-gated Cav1.3 Ca2+ channels in mouse inner hair cells (IHCs), which triggers exocytosis of glutamate and excitation of afferent neurons. In mature IHCs, Cav1.3 channels open in response to evoked receptor potentials, causing graded changes in exocytosis required for accurate sound transmission. Developmental alterations in Cav1.3 properties may support distinct roles of Cav1.3 in IHCs in immature and mature IHCs, and have been reported in various species. It is not known whether such changes in Cav1.3 properties occur in mouse IHCs, but this knowledge is necessary for understanding the roles of Cav1.3 in developing and mature IHCs. Here, we describe age-dependent differences in the biophysical properties of Cav1.3 channels in mouse IHCs. In mature IHCs, Cav1.3 channels activate more rapidly and exhibit greater Ca2+-dependent inactivation (CDI) than in immature IHCs. Consistent with the properties of Cav1.3 channels in heterologous expression systems, CDI in mature IHCs is not affected by increasing intracellular Ca2+ buffering strength. However, CDI in immature IHCs is significantly reduced by strong intracellular Ca2+ buffering, which both slows the onset of, and accelerates recovery from, inactivation. These results signify a developmental decline in the sensitivity of CDI to global elevations in Ca2+, which restricts negative feedback regulation of Cav1.3 channels to incoming Ca2+ ions in mature IHCs. Together with faster Cav1.3 activation kinetics, increased reliance of Cav1.3 CDI on local Ca2+ may sharpen presynaptic Ca2+ signals and improve temporal aspects of sound coding in mature IHCs.

Caveolin-1 and polymerase I and transcript release factor: new players in insulin-like growth factor-I receptor signaling.

Caveolae are plasma membrane regions enriched in Caveolin proteins which regulate vesicular transport, endocytosis, and cell signaling. IGF-I receptor (IGF-IR) localizes in caveolae and tyrosine phosphorylates Caveolin-1 (Cav-1), the most represented caveolar protein. Cav-1 participates to IGF-IR internalization and signaling directly interacting with IGF-IR and its substrates. Recently, polymerase I and transcript release factor (PTRF) or Cavin-1, has been identified in the caveolar backbone. PTRF does not play a Cav-1 ancillary role and emerging data support a direct role of PTRF in IGF-IR signaling. PTRF and Cav-1 can bind IGF-IR and regulate IGF-IR internalization and plasma membrane replacement, mechanisms frequently deregulated in cancer cells. Although the exact roles of Cav-1 and IGF-IR in human cancer continue to be a matter of some debate, there is a strong evidence for an association between Cav-1 and IGF-IR in cancer development. With the discovery of IGF-IR interaction with PTRF in caveolae, new insight emerged to understand the growing functions of these domains in IGF-I action.

In MMTV-Her-2/neu transgenic mammary tumors the absence of caveolin-1−/− alters PTEN and NHERF1 but not β-catenin expression

In a recent study, we have shown that in mammary tumors from mice lacking the Cav-1 gene, there are alterations in specific heat shock proteins as well as in tumor development. With this in mind, we have now investigated other proteins in the same mammary mouse tumor model (Her-2/neu expressing mammary tumors from Cav-1 wild type and Cav-1 null mice), to further comprehend the complex tumor-stroma mechanisms involved in regulating stress responses during tumor development. In this tumor model the cancer cells always lacked of Cav-1, so the KO influenced the Cav-1 in the stroma. By immunohistochemistry, we have found a striking co-expression of β-catenin and Her-2/neu in the tumor cells. The absence of Cav-1 in the tumor stroma had no effect on expression or localization of β-catenin and Her-2/neu. Both proteins appeared co-localized at the cell surface during tumor development and progression. Since Her-2/neu activation induces MTA1, we next evaluated MTA1 in the mouse tumors. Although this protein was found in numerous nuclei, the absence of Cav-1 did not alter its expression level. In contrast, significantly more PTEN protein was noted in the tumors lacking Cav-1 in the stroma, with the protein localized mainly in the nuclei. P-Akt levels were relatively low in tumors from both Cav-1 WT and Cav-1 KO mice. There was also an increase in nuclear NHERF1 expression levels in the tumors arising from Cav-1 KO mice. The data obtained in the MMTV-neu model are consistent with a role for Cav-1 in adjacent breast cancer stromal cells in modulating the expression and localization of important proteins implicated in tumor cell behavior.

Cav1.1 controls frequency-dependent events regulating adult skeletal muscle plasticity

An important pending question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype as slow or fast twitch muscle fibers. We have previously shown that voltage-gated L-type calcium channel (Cav1.1) acts as a voltage sensor for activation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P₃]-dependent Ca(2+) signals that regulates gene expression. ATP released by muscle cells after electrical stimulation through pannexin-1 channels plays a key role in this process. We show now that stimulation frequency determines both ATP release and Ins(1,4,5)P₃ production in adult skeletal muscle and that Cav1.1 and pannexin-1 colocalize in the transverse tubules. Both ATP release and increased Ins(1,4,5)P₃ was seen in flexor digitorum brevis fibers stimulated with 270 pulses at 20 Hz, but not at 90 Hz. 20 Hz stimulation induced transcriptional changes related to fast-to-slow muscle fiber phenotype transition that required ATP release. Addition of 30 µM ATP to fibers induced the same transcriptional changes observed after 20 Hz stimulation. Myotubes lacking the Cav1.1-α1 subunit released almost no ATP after electrical stimulation, showing that Cav1.1 has a central role in this process. In adult muscle fibers, ATP release and the transcriptional changes produced by 20 Hz stimulation were blocked by both the Cav1.1 antagonist nifedipine (25 µM) and by the Cav1.1 agonist (-)S-BayK 8644 (10 µM). We propose a new role for Cav1.1, independent of its calcium channel activity, in the activation of signaling pathways allowing muscle fibers to decipher the frequency of electrical stimulation and to activate specific transcriptional programs that define their phenotype.

Abstract 531: Role of Insulin Resistance in the Altered Vascular Relaxation Mechanisms and Hypertension in Caveolin-1 Deficient Mice

Insulin resistance (IR) and endothelial dysfunction are often associated with hypertension; however, the specific causality and genetic underpinnings of these associations are unclear. Caveolin-1 (cav-1) is a transmembrane anchoring protein and potential modulator of vascular function and insulin sensitivity of the vascular and metabolic pathways. Our recent findings point to a role for cav-1 in IR, endothelial dysfunction and hypertension. In humans, cav-1 gene variants are associated with IR in hypertensive individuals. In mice, cav-1 deficiency leads to changes in endothelial function, IR and blood pressure (BP). To test whether the vascular changes associated with cav-1 deficiency are related to or are independent of IR, we assessed BP, blood glucose and vascular function in WT and cav-1 KO mice treated with placebo or metformin 400 mg/Kg/day for 21 days. BP was greater in cav-1 KO vs WT (140.1±2.1 vs 116.5±1.3 mmHg), and metformin treatment did not change BP in cav-1 KO (140.7±1.4) or WT (112.5±1.6 mmHg). Fasting blood glucose was greater in cav-1 KO vs WT (112±3.9 vs. 83.1±4.4 mg/dl), supporting a link between cav-1 deficiency and IR. Metformin did not change blood glucose levels in cav-1 KO (110.4±4.1) or WT (86.7±8.2 mg/dl). Acetylcholine (ACh)-induced maximal aortic relaxation was greater in cav-1 KO vs WT (85.3±5.7 vs. 17.5±4.4%), and was abolished by endothelium removal or the NOS inhibitor L-NAME, supporting changes in endothelial NO production/signaling. Metformin reduced ACh relaxation in cav-1 KO to 60.7±6.6%, but had no effect in WT. The exogenous NO donor sodium nitroprusside (SNP) produced similar maximal relaxation in cav-1 KO and WT (98.4±1.6 and 98.2±1.1%), but was more potent in cav-1 KO vs WT (pEC50, 8.6 vs 8.1). Metformin reduced SNP relaxation in cav-1 KO (84.9±6.4%) but not in WT, and reversed the enhanced sensitivity to SNP in cav-1 KO mice. The metformin-induced decrease in vascular relaxation and sensitivity to NO in cav-1 KO mice suggests a role for vascular IR in the changes in endothelial function. The lack of effect of metformin on fasting blood glucose in cav-1 KO mice suggests recalcitrant IR of the metabolic pathways. Together, these data support partial independence of vascular function from systemic IR in cav-1 deficiency states.

Lymph node‐induced immune tolerance in chronic lymphocytic leukaemia: a role for caveolin‐1

Emerging evidence indicates that the tumour microenvironment (TME) regulates the behaviour of chronic lymphocytic leukaemia (CLL). However, the precise mechanism and molecules involved in this process remain unknown. Gene expression profiles of CLL cells from lymph node (LN), bone marrow (BM) and peripheral blood (PB) indicate overexpression of a tolerogenic signature in CLL cells in lymph nodes (LN-CLL). Based on their role in B cell biology, the progression of CLL, or immune regulation, a few genes of this 83-gene signature were selected for further analyses. We observed a significant correlation between the clinical outcomes and the expression of CAV1 (P = 0·041), FGFR1 isoform 8 (P = 0·032), PTPN6 (P = 0·031) and ZWINT (P < 0·001). CAV1, a molecule involved in the regulation of tumour progression in other cancers, was seven-fold higher in LN-CLL cells compared to BM- and PB-CLL cells. Knockdown of CAV1 expression in CLL cells resulted in significantly decreased migration (P = 0·016) and proliferation (P = 0·04). When CAV1 was knocked down in B and T cell lines, we observed an inability to form immune synapses. Furthermore, CAV1 knockdown in CLL cells impaired their ability to form immune synapses with autologous T lymphocytes and allogeneic, healthy T cells. Subsequent analyses of microarray data showed differential expression of cytoskeletal genes, specifically those involved in actin polymerization. Therefore, we report a novel role for CAV1 in tumour-induced immunosuppression during the progression of CLL.

Genetic Ablation of Cav1 Differentially Affects Melanoma Tumor Growth and Metastasis in Mice: Role of Cav1 in Shh Heterotypic Signaling and Transendothelial Migration

Both cell-autonomous and non-cell-autonomous factors contribute to tumor growth and metastasis of melanoma. The function of caveolin-1 (Cav1), a multifunctional scaffold protein known to modulate several biologic processes in both normal tissue and cancer, has been recently investigated in melanoma cancer cells, but its role in the melanoma microenvironment remains largely unexplored. Here, we show that orthotopic implantation of B16F10 melanoma cells in the skin of Cav1KO mice increases tumor growth, and co-injection of Cav1-deficient dermal fibroblasts with melanoma cells is sufficient to recapitulate the tumor phenotype observed in Cav1KO mice. Using indirect coculture experiments with fibroblasts and melanoma cells combined with cytokine analysis, we found that Cav1-deficient fibroblasts promoted the growth of melanoma cells via enhanced paracrine cytokine signaling. Specifically, Cav1-deficient fibroblasts displayed increased ShhN expression, which heterotypically enhanced the Shh signaling pathway in melanoma cells. In contrast to primary tumor growth, the ability of B16F10 melanoma cells to form lung metastases was significantly reduced in Cav1KO mice. This phenotype was associated mechanistically with the inability of melanoma cells to adhere to and to transmigrate through a monolayer of endothelial cells lacking Cav1. Together, our findings show that Cav1 may regulate different mechanisms during primary melanoma tumor growth and metastatic dissemination.

Abstract 5013: CpG island shore methylation regulates caveolin-1 expression in breast cancer

Abstract Caveolin-1 (Cav1), a multifunctional membrane protein, regulates caveolae-dependent lipid trafficking, vesicular transport and signal transduction. Cav1 expression is decreased in breast carcinomas compared to normal tissue, and loss of Cav1 expression promotes mammary tumorigenesis. However, recent reports indicate that Cav1 is overexpressed in a subset of basal-like breast carcinomas and consider it as a basal marker. To investigate the mechanism that results in differential Cav1 expression in breast cancer, we performed genome-wide profiling of DNA methylation using affinity purification and subsequent next-generation sequencing of eluted DNA (MethylCap-seq) on 30 breast cancer cell lines. In breast cancer cell lines displaying low Cav1 expression (“Cav1-low;” n=15, mostly luminal subtype), Cav1 promoter CpG island (CGI) hypermethylation was observed in only four cell lines. In the remaining Cav1-low lines, dense methylation at “CGI shores” (methylation at regions outside of CGI) was seen, while Cav1 promoter CGI methylation was strikingly low or absent. In “Cav1-high” cell lines (n=15 of basal-like subtype), hypomethylation of both promoter CGI and CGI shores was observed. By integrating the methylome data with Cav1 mRNA expression profiles, we found a significant negative correlation between Cav1 CGI shore methylation and Cav1 gene expression. A similar DNA methylation pattern was observed in a panel of antiestrogen-sensitive and -resistant cell lines previously generated by our lab, with hypermethylation at CGI shores for a Cav1-low, tamoxifen-resistant line, and CGI shore hypomethylation in Cav1-high, fulvestrant-resistant lines (basal-like). Treatment of Cav1-low cells with the DNA methyltransferase inhibitor 5-aza-deoxycytidine resulted in demethylation of CGI shores and Cav1 re-expression, further confirming the regulation of Cav1 expression by CGI shore methylation. ChIP-seq results of Cav1-low cells indicated that the Cav1 CGI hypomethylation was due to H3K4me2 occupancy. SiRNA-mediated Cav1 knockdown resulted in a significant inhibition of cell growth and invasion, indicating an important role of Cav1 in supporting breast cancer cell progression. Furthermore, by using an on-line survival analysis tool (Kaplan Meier Plotter), we determined a significant negative correlation between Cav1 levels and overall survival for patients with ERα-negative breast tumors. The results of this study demonstrate that DNA methylation at Cav1 CGI shores is associated with tumor progression, displaying hypomethylation in normal breast epithelial cells, hypermethylation in luminal breast cancer cells, and hypomethylation in basal-like breast cancer cells. In addition, Cav1 CGI shore methylation may represent a novel prognostic factor for ERα-negative, fulvestrant-resistant breast cancer. 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 5013. doi:1538-7445.AM2012-5013

Caveolin-1 regulates Mcl-1 stability and anoikis in lung carcinoma cells

Both caveolin-1 (Cav-1) and Mcl-1 have been implicated in the regulation of cancer cell anoikis, but their relationship and underlying mechanisms of regulation are not known. The present study demonstrated for the first time that Cav-1 regulates Mcl-1 through protein-protein interaction and inhibits its downregulation during cell anoikis in human lung cancer cells. Immunoprecipitation and immunocytochemistry studies showed that Cav-1 interacted with Mcl-1 and prevented it from degradation via the ubiquitin-proteasome pathway. Mcl-1 and Mcl-1-Cav-1 complex were highly elevated in Cav-1-overexpressing cells but were greatly reduced in Cav-1 knockdown cells. Consistent with this finding, we found that Mcl-1 ubiquitination was significantly attenuated by Cav-1 overexpression but increased by Cav-1 knockdown. Together, our results indicate a novel role of Cav-1 in anoikis regulation through Mcl-1 interaction and stabilization, which provides a new insight to the pathogenesis of metastatic lung cancer and its potential treatment.

Abstract C22: Next-generation sequencing reveals CpG island shore methylation regulates caveolin-1 expression in breast cancer

Abstract Caveolin-1 (Cav1), a multifunctional membrane protein associated with cell surface caveolae, regulates caveolae-dependent lipid trafficking, vesicular transport and signal transduction. While reported to function as a tumor suppressor at early stages of cancer progression, a growing body of evidence indicates that Cav1 also performs a tumor-promoting role in various cancer types. Cav1 expression level is decreased in breast carcinomas compared to normal tissue, and loss of Cav1 expression promotes mammary tumorigenesis and is associated with the development of tamoxifen resistance. However, gene expression profiling of breast cancer cell lines revealed an inverse relationship between expression of Cav1 and estrogen receptor-alpha (ERα), and Cav1 overexpression was observed in a subset of basal-like breast carcinomas. To investigate the mechanism regulating Cav1 expression, we performed genome-wide profiling of DNA methylation using affinity purification and subsequent next-generation sequencing of eluted DNA (MethylCap-seq) on 30 breast cancer cell lines. In breast cancer cell lines displaying low Cav1 expression (“Cav1-low;” n=15 of primarily luminal subtype), Cav1 promoter CpG island (CGI) hypermethylation was observed in only four cell lines. In the remaining Cav1-low lines, dense methylation at “CGI shores” (methylation at regions outside of CGI) was seen, and Cav1 promoter CGI methylation was strikingly low or absent. In “Cav1-high” cell lines (n=15 of basal-like subtype), hypomethylation of both promoter CGI and CGI shores was observed. By integrating the methylome data with Cav1 mRNA expression profiles, we found a significant negative correlation between Cav1 CGI shore methylation and Cav1 gene expression. Treatment of Cav1-low cells with the DNA methyltransferase inhibitor 5-aza-deoxycytidine resulted in demethylation of CGI shores and Cav1 re-expression, further confirming the regulation of Cav1 expression by CGI shore methylation. MethylCap-seq results were validated by pyrosequencing, and we observed a similar phenomenon in a panel of antiestrogen-sensitive and -resistant cell lines previously generated by our lab, with hypermethylation at CGI shores for a Cav1-low, tamoxifen-resistant line, and CGI shore hypomethylation in Cav1-high, fulvestrant-resistant lines (basal-like). SiRNA-mediated Cav1 knockdown resulted in a significant cell growth inhibition, indicating an important role of Cav1 in supporting breast cancer cell proliferation. Furthermore, by using an on-line survival analysis tool (Kaplan Meier Plotter), we determined a significant positive correlation between Cav1 levels and poor overall survival for patients with ERα-negative breast tumors. The results of this study demonstrate that DNA methylation at Cav1 CGI shores is associated with tumor progression, displaying hypomethylation in normal breast epithelial cells, hypermethylation in luminal breast cancer cells, and hypomethylation in basal-like breast cancer cells. In addition, Cav1 CGI shore methylation may represent a novel prognostic factor for ERα-negative, fulvestrant-resistant breast cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C22.

The Ras Inhibitors Caveolin-1 and Docking Protein 1 Activate Peroxisome Proliferator-Activated Receptor γ through Spatial Relocalization at Helix 7 of Its Ligand-Binding Domain

Peroxisome proliferator-activated receptor γ (PPARγ) is a transcription factor that promotes differentiation and cell survival in the stomach. PPARγ upregulates and interacts with caveolin-1 (Cav1), a scaffold protein of Ras/mitogen-activated protein kinases (MAPKs). The cytoplasmic-to-nuclear localization of PPARγ is altered in gastric cancer (GC) patients, suggesting a so-far-unknown role for Cav1 in spatial regulation of PPARγ signaling. We show here that loss of Cav1 accelerated proliferation of normal stomach and GC cells in vitro and in vivo. Downregulation of Cav1 increased Ras/MAPK-dependent phosphorylation of serine 84 in PPARγ and enhanced nuclear translocation and ligand-independent transcription of PPARγ target genes. In contrast, Cav1 overexpression sequestered PPARγ in the cytosol through interaction of the Cav1 scaffolding domain (CSD) with a conserved hydrophobic motif in helix 7 of PPARγ's ligand-binding domain. Cav1 cooperated with the endogenous Ras/MAPK inhibitor docking protein 1 (Dok1) to promote the ligand-dependent transcriptional activity of PPARγ and to inhibit cell proliferation. Ligand-activated PPARγ also reduced tumor growth and upregulated the Ras/MAPK inhibitors Cav1 and Dok1 in a murine model of GC. These results suggest a novel mechanism of PPARγ regulation by which Ras/MAPK inhibitors act as scaffold proteins that sequester and sensitize PPARγ to ligands, limiting proliferation of gastric epithelial cells.

Caveolin-1 sensitizes cisplatin-induced lung cancer cell apoptosis via superoxide anion-dependent mechanism

Caveolin-1 (Cav-1) expression frequently found in lung cancer was linked with disease prognosis and progression. This study reveals for the first time that Cav-1 sensitizes cisplatin-induced lung carcinoma cell death by the mechanism involving oxidative stress modulation. We established stable Cav-1 overexpressed (H460/Cav-1) cells and investigated their cisplatin susceptibility in comparison with control-transfected cells and found that Cav-1 expression significantly enhanced cisplatin-mediated cell death. Results indicated that the different response to cisplatin between these cells was resulted from different level of superoxide anion induced by cisplatin. Inhibitory study revealed that superoxide anion inhibitor MnTBAP could inhibit cisplatin-mediated toxicity only in H460/Cav-1 cells while had no effect on H460 cells. Further, superoxide anion detected by DHE probe indicated that H460/Cav-1 cells generated significantly higher superoxide anion level in response to cisplatin than that of control cells. The role of Cav-1 in regulating cisplatin sensitivity was confirmed in shRNA-mediated Cav-1 down-regulated (H460/shCav-1) cells and the cells exhibited decreased cisplatin susceptibility and superoxide generation. In summary, these findings reveal novel aspects regarding role of Cav-1 in modulating oxidative stress induced by cisplatin, possibly providing new insights for cancer biology and cisplatin-based chemotherapy.

Prognostic significance of tumor/stromal caveolin‐1 expression in breast cancer patients

Caveolin-1 (Cav-1) has been extensively characterized in cancer biological research. However, the role of Cav-1 in the interaction between tumor and stromal cells remains unclear. In the present study, we examined Cav-1 expression in tumor cells and stromal cells in breast cancer tissue by immunohistochemical analysis and evaluated its prognostic value in a training cohort. Immunohistochemical analysis of Cav-1 expression was scored as (++), (+) or (-) according to the proportion of positively stained tumor cells (T) and stromal cells (S). Correlation analysis between tumor/stromal Cav-1 expression and clinicopathological parameters revealed that only T(++) Cav-1 status was positively associated with tumor size and histological nodal status (P = 0.019 and 0.021, respectively). Univariate analysis revealed that combined T(++)/S(-) status was significantly correlated with unfavorable prognostic outcomes (P < 0.001). Multivariate analysis demonstrated that this combined status is an independent prognostic factor for primary breast cancer (P = 0.002). Clinical outcomes in different subgroups of breast cancer patients were also strictly dependent on this combined status (P < 0.05). The prognostic value of T(++)/S(-) Cav-1 status was also validated in the testing cohort. Collectively, our data indicate that high Cav-1 expression in tumor cells and lack of this expression in stromal cells could help identify a particular subgroup of breast cancer patients with potentially poor survival. Further studies are required to understand the regulatory mechanism of Cav-1 in the tumor microenvironment.

Are Cav1.3 pacemaker channels in chromaffin cells? Possible bias from resting cell conditions and DHP blockers usage

Mouse and rat chromaffin cells (MCCs, RCCs) fire spontaneously at rest and their activity is mainly supported by the two L-type Ca2+ channels expressed in these cells (Cav1.2 and Cav1.3). Using Cav1.3-/- KO MCCs we have shown that Cav1.3 possess all the prerequisites for carrying subthreshold currents that sustain low frequency cell firing near resting (0.5 to 2 Hz at -50 mV)1: low-threshold and steep voltage dependence of activation, slow and incomplete inactivation during pulses of several hundreds of milliseconds. Cav1.2 contributes also to pacemaking MCCs and possibly even Na+ channels may participate in the firing of a small percentage of cells. We now show that at potentials near resting (–50 mV), Cav1.3 carries equal amounts of Ca2+ current to Cav1.2 but activates at 9 mV more negative potentials. MCCs express only TTX-sensitive Nav1 channels that activate at 24 mV more positive potentials than Cav1.3 and are fully inactivating. Their blockade prevents the firing only in a small percentage of cells (13%). This suggests that the order of importance with regard to pacemaking MCCs is: Cav1.3, Cav1.2 and Nav1. The above conclusions, however, rely on the proper use of DHPs, whose blocking potency is strongly holding potential dependent. We also show that small increases of KCl concentration steadily depolarize the MCCs causing abnormally increased firing frequencies, lowered and broadened AP waveforms and an increased facility of switching “non-firing” into “firing” cells that may lead to erroneous conclusions about the role of Cav1.3 and Cav1.2 as pacemaker channels in MCCs.2

Abstract 2049: Role of hypoxia-induced upregulation of caveolin-1 in hepatocellular carcinoma cell migration and invasion

Abstract Intratumoral hypoxia occurs when cells in the inner core of solid tumor mass become deprived of oxygen. It correlates with an increased risk to develop metastasis. However, the molecular basis about the adaptation and survival of tumor cells in hypoxic environment remains obscure. Our recent data revealed that Cav1, a component of focal adhesion, is dramatically increased in metastatic hepatocellular carcinoma (HCC) cell lines and tissues, suggesting a role of Cav1 in HCC metastasis. In this study, we further investigated the potential role of Cav1 in HCC metastasis under hypoxia. Our data revealed that Cav1 mRNA and protein expressions were enhanced in various HCC cells lines when placed in hypoxic chamber or treated with hypoxic mimetic drug. Such upregulation of Cav1 was abolished with the addition of HIF1α inhibitor or depletion of HIF1α by siRNA. This suggests that hypoxia-induced Cav1 expression is HIF1α-dependent. Our findings also showed that HCC cells possessed enhanced motility under hypoxia. However, knockdown of Cav1 in HCC cells attenuated the capacity of cells to migrate and invade. Collectively, our study suggests that the increased motility of HCC cells conferred by the hypoxia-induced Cav1 expression is an adaptive mechanism of cells to survive under hypoxic stress. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2049. doi:10.1158/1538-7445.AM2011-2049