Overexpression Of Caveolin-1 Research Articles

Neuron-targeted caveolin-1 improves neuromuscular function and extends survival in SOD1G93A mice.

Interventions that preserve motor neurons or restore functional motor neuroplasticity may extend longevity in amyotrophic lateral sclerosis (ALS). Delivery of neurotrophins may potentially revive degenerating motor neurons, yet this approach is dependent on the proper subcellular localization of neurotrophin receptor (NTR) to plasmalemmal signaling microdomains, termed membrane/lipid rafts (MLRs). We previously showed that overexpression of synapsin-driven caveolin-1 (Cav-1) (SynCav1) increases MLR localization of NTR [e.g., receptor tyrosine kinase B (TrkB)], promotes hippocampal synaptic and neuroplasticity, and significantly improves learning and memory in aged mice. The present study crossed a SynCav1 transgene-positive (SynCav1+) mouse with the mutant human superoxide dismutase glycine to alanine point mutation at amino acid 93 (hSOD1G93A) mouse model of ALS. When compared with hSOD1G93A, hSOD1G93A/SynCav1+ mice exhibited greater body weight and longer survival as well as better motor function. Microscopic analyses of hSOD1G93A/SynCav1+ spinal cords revealed preserved spinal cord α-motor neurons and preserved mitochondrial morphology. Moreover, hSOD1G93A/SynCav1+ spinal cords contained more MLRs (cholera toxin subunit B positive) and MLR-associated TrkB and Cav-1 protein expression. These findings demonstrate that SynCav1 delays disease progression in a mouse model of ALS, potentially by preserving or restoring NTR expression and localization to MLRs.-Sawada, A., Wang, S., Jian, M., Leem, J., Wackerbarth, J., Egawa, J., Schilling, J. M., Platoshyn, O., Zemljic-Harpf, A., Roth, D. M., Patel, H. H., Patel, P. M., Marsala, M., Head, B. P. Neuron-targeted caveolin-1 improves neuromuscular function and extends survival in SOD1G93A mice.

Cardiac-specific overexpression of caveolin-3 preserves t-tubular ICa during heart failure in mice.

New Findings What is the central question of this study? What is the cellular basis of the protection conferred on the heart by overexpression of caveolin‐3 (Cav‐3 OE) against many of the features of heart failure normally observed in vivo? What is the main finding and its importance? Cav‐3 overexpression has little effect in normal ventricular myocytes but reduces cellular hypertrophy and preserves t‐tubular I Ca, but not local t‐tubular Ca2+ release, in heart failure induced by pressure overload in mice. Thus Cav‐3 overexpression provides specific but limited protection following induction of heart failure, although other factors disrupt Ca2+ release. Caveolin‐3 (Cav‐3) is an 18 kDa protein that has been implicated in t‐tubule formation and function in cardiac ventricular myocytes. During cardiac hypertrophy and failure, Cav‐3 expression decreases, t‐tubule structure is disrupted and excitation–contraction coupling (ECC) is impaired. Previous work has suggested that Cav‐3 overexpression (OE) is cardio‐protective, but the effect of Cav‐3 OE on these cellular changes is unknown. We therefore investigated whether Cav‐3 OE in mice is protective against the cellular effects of pressure overload induced by 8 weeks’ transverse aortic constriction (TAC). Cav‐3 OE mice developed cardiac dilatation, decreased stroke volume and ejection fraction, and hypertrophy and pulmonary congestion in response to TAC. These changes were accompanied by cellular hypertrophy, a decrease in t‐tubule regularity and density, and impaired local Ca2+ release at the t‐tubules. However, the extent of cardiac and cellular hypertrophy was reduced in Cav‐3 OE compared to WT mice, and t‐tubular Ca2+ current (I Ca) density was maintained. These data suggest that Cav‐3 OE helps prevent hypertrophy and loss of t‐tubular I Ca following TAC, but that other factors disrupt local Ca2+ release.

Clinical implications of hypoxia-inducible factor-1α and caveolin-1 overexpression in isocitrate dehydrogenase-wild type glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common type of primary brain tumour in adults, and presents a very low survival rate. Isocitrate dehydrogenase (IDH)1/2 mutations have been found in ~12% of glioblastomas and are associated with long-term GBM survival. However, the risk factors that influence the prognosis of IDH-wild type GBM remain unclear. Hypoxia-inducible factor (HIF)-1α, an important oxygen-regulated transcription factor, has been demonstrated to serve a crucial role in tumour development and to be associated with a poor prognosis. In addition, caveolin-1 (CAV1) is a plasma membrane organizing protein, the expression of which can also be regulated by a hypoxic microenvironment. The present study therefore aimed to examine the expression levels of HIF-1α and CAV1, and their association with GBM prognosis. Reverse transcription-quantitative polymerase chain reaction and western blotting were performed to analyse the expression levels of HIF-1α and CAV1 in paired GBM tumour and adjacent non-tumour tissues. Immunohistochemistry was used to analyse the expression of the two proteins in paraffin-embedded tissues obtained from 42 patients with IDH-wild type GBM. Statistical analyses were performed to examine the correlation between HIF-1α and CAV1 expression and patient prognosis. The results revealed hat the expression levels of HIF-1α and CAV1 were upregulated in IDH-wild type GBM tissues compared to their paired non-tumour tissues (P<0.001). The expression of CAV1 was significantly correlated with high HIF-1α expression (P<0.01). In addition, overexpression of HIF-1α and CAV1 was markedly associated with a poor prognosis (P<0.001). In conclusion, HIF-1α and CAV1 may represent potential biomarkers for IDH-wild type GBM prognosis and potential targets for the development of therapies extending GBM survival.

Role of caveolin-1 in epidermal stem cells during burn wound healing in rats

Local transplantation of stem cells has therapeutic effects on skin damage but cannot provide satisfactory wound healing. Studies on the mechanisms underlying the therapeutic effects of stem cells on skin wound healing will be needed. Hence, in the present study, we explored the role of Caveolin-1 in epidermal stem cells (EpiSCs) in the modulation of wound healing. We first isolated EpiSCs from mouse skin tissues and established stable EpiSCs with overexpression of Caveolin-1 using a lentiviral construct. We then evaluated the epidermal growth factor (EGF)-induced cell proliferation ability using cell counting Kit-8 (CCK-8) assay and assessed EpiSC pluripotency by examining Nanog mRNA levels in EpiSCs. Furthermore, we treated mice with skin burn injury using EpiSCs with overexpression of Caveolin-1. Histological examinations were conducted to evaluate re-epithelialization, wound scores, cell proliferation and capillary density in wounds. We found that overexpression of Caveolin-1 in EpiSCs promoted EGF-induced cell proliferation ability and increased wound closure in a mouse model of skin burn injury. Histological evaluation demonstrated that overexpression of Caveolin-1 in EpiSCs promoted re-epithelialization in wounds, enhanced cellularity, and increased vasculature, as well as increased wound scores. Taken together, our results suggested that Caveolin-1 expression in the EpiSCs play a critical role in the regulation of EpiSC proliferation ability and alteration of EpiSC proliferation ability may be an effective approach in promoting EpiSC-based therapy in skin wound healing.

Intermittent hypoxia regulates vasoactive molecules and alters insulin-signaling in vascular endothelial cells

Vascular dysfunction and insulin resistance (IR) are associated with obstructive sleep apnea (OSA), which is characterized by frequent episodes of nocturnal intermittent hypoxia (IH). While it is recognized that the balance between vasoconstrictive (endothelin-1) and vasodilatory molecules (nitric oxide, NO) determine vascular profile, molecular mechanisms contributing to vascular dysfunction and IR in OSA are not completely understood. Caveolin-1 is a membrane protein which regulates endothelial nitric oxide synthase (eNOS) activity which is responsible for NO generation and cellular insulin-signaling. Hence, we examined the effects of IH on caveolin-1, eNOS, and endothelin-1 in human coronary artery endothelial cells in the context of IR. Chronic 3-day IH exposure up-regulated caveolin-1 and endothelin-1 expression while reducing NO. Also, IH altered insulin-mediated activation of AKT but not ERK resulting in increased endothelin-1 transcription. Similarly, caveolin-1 overexpression attenuated basal and insulin-stimulated NO synthesis along with impaired insulin-dependent activation of AKT and eNOS, with no effect on insulin-stimulated ERK1/2 phosphorylation and endothelin-1 transcription. Our data suggest that IH contributes to a vasoconstrictive profile and to pathway-selective vascular IR, whereby insulin potentiates ET-1 expression. Moreover, IH may partly mediate its effects on NO and insulin-signaling via upregulating caveolin-1 expression.

Astragaloside IV enhances taxol chemosensitivity of breast cancer via caveolin-1-targeting oxidant damage.

Accumulating evidence suggests that caveolin-1 (CAV-1) is a stress-related oncotarget and closely correlated to chemoresistance. Targeting CAV-1 might be a promising strategy to improve chemosensitivity for breast cancer treatment. Astragaloside IV (AS-IV), a bioactive compound purified from Astragalus membranaceus, has been shown to exhibit multiple bioactivities, including anticancer. However, the involved molecular targets are still ambiguous. In this study, we investigated the critical role of CAV-1 in mediating the chemosensitizing effects of AS-IV to Taxol on breast cancer. We found that AS-IV could enhance the chemosensitivity of Taxol with minimal direct cytotoxicity on breast cancer cell lines MCF-7 and MDA-MB-231, as well as the nontumor mammary epithelial cell line MCF-10A. AS-IV was further demonstrated to aggravate Taxol-induced apoptosis and G2/M checkpoint arrest. The phosphorylation of mitogen-activated protein kinase (MAPK) signaling extracellular signal-regulated kinase (ERK) and c-Jun N-terminal Kinase (JNK), except p38, was also abrogated by a synergistic interaction between AS-IV and Taxol. Moreover, AS-IV inhibited CAV-1 expression in a dose-dependent manner and reversed CAV-1 upregulation induced by Taxol administration. Mechanism study further demonstrated that AS-IV treatment triggered the eNOS/NO/ONOO- pathway via inhibiting CAV-1, which led to intense oxidant damage. CAV-1 overexpression abolished the chemosensitizing effects of AS-IV to Taxol by inhibiting oxidative stress. In vivo experiments further validated that AS-IV increased Taxol chemosensitivity on breast cancer via inhibiting CAV-1 expression, followed by activation of the eNOS/NO/ONOO- pathway. Taken together, our findings not only suggested the potential of AS-IV as a promising candidate to enhance chemosensitivity, but also highlighted the significance of CAV-1 as the target to reverse cancer drug resistance.

Caveolin enhances hepatocellular carcinoma cell metabolism, migration, and invasion in vitro via a hexokinase 2-dependent mechanism.

The development and progression of hepatocellular carcinoma (HCC) have been associated with abnormal cellular metabolism. Gene Expression Profiling Interactive Analysis RNA sequencing data revealed caveolin-1 (CAV-1) and hexokinase 2 (HK2) messenger RNA (mRNA) were significantly upregulated in human HCC compared with normal tissues, and high HK2 expression was associated with significantly poorer overall survival in HCC ( p < 0.05). CAV-1 and HK2 mRNA and protein expression were upregulated and positively correlated in 42 fresh human HCC tissues compared with tumor-adjacent normal tissues. Overexpression of CAV-1 or HK2 in SMMC-7721 and HepG2 HCC cells enhanced glucose and lactate metabolism and increased cell migration and invasion in transwell assays; knocking down CAV-1 or HK2 had the opposite effects. Overexpression of CAV-1 increased HK2 expression; overexpression of HK2 did not affect CAV-1 expression. Knocking down HK2 partially reversed the ability of CAV-1 to promote cellular metabolism, invasion, and migration in HCC, indicating CAV-1 enhances glycolysis, invasion, and metastasis in HCC cells via HK2-dependent mechanism. Further studies of the function and relationship between CAV-1 or HK2 expression are warranted to explore the potential of these proteins as metabolic targets for the treatment of HCC.

MicroRNA-124 inhibits cell proliferation, invasion and migration by targeting CAV1 in bladder cancer.

MicroRNAs (miRs) may have promotive or suppressive roles in various human cancers types, but the molecular mechanisms underlying the role of miR-124 in bladder cancer (BC) progression have remained largely elusive. In the present study, it was observed that miR-124 was significantly downregulated in BC tissues compared with that in adjacent non-neoplastic tissues. Furthermore, its expression was also reduced in several human BC cell lines (T24, HT-1376 and 5637) compared with that in the normal bladder epithelial SV-HUC-1 cell line. A low expression of miR-124 in BC patients was significantly associated with advanced malignancy and a poor prognosis. Caveolin 1 (CAV1) was identified as a novel target gene of miR-124, and the expression of CAV1 was negatively regulated by miR-124 in T24 cells. Furthermore, CAV1 was identified to be significantly upregulated in BC tissues and cell lines, and a negative correlation was observed between the expression of miR-124 and CAV1 in BC tissues. Furthermore, restoration of miR-124 expression significantly inhibited the proliferation, migration and invasion of T24 cells, and these effects were impaired following overexpression of CAV1. Taken together, the present results demonstrate that miR-124 has a suppressive role in the proliferation, migration and invasion of BC cells by targeting CAV1, which suggests that miR-124 is a potential therapeutic candidate for BC.

Cell growth potential drives ferroptosis susceptibility in rhabdomyosarcoma and myoblast cell lines.

Ferroptosis is a programmed form of iron-dependent cell death caused by lipid hydroperoxide accumulation, which can be prevented by glutathione peroxidase 4 (GPx4) activity. Here we investigated the effects of ferroptosis inducers called erastin and RSL3, which act by glutathione depletion and GPx4 inactivation, respectively, on muscle-derived cell lines of embryonal and alveolar rhabdomyosarcoma (RMS), and mouse normal skeletal C2C12 myoblasts. Myogenic lines were exposed to stepwise increasing concentrations of ferroptosis inducers either alone or in combination with iron supplementation, iron chelating agents (bathophenanthrolinedisulfonic acid, BPS), antioxidant molecules (glutathione, N-acetylcysteine), lipid peroxidation inhibitors (ferrostatin-1), and chemotherapeutic agents (doxorubicin and actinomycin D). Drug susceptibility was quantified by measuring cell viability, proliferation and differentiation via neutral red assay, crystal violet assay and Giemsa staining, respectively. The detection of lipid hydroperoxide and protein levels was performed by immunofluorescence and Western blot analysis, respectively. Erastin and RSL3 increased lipid hydroperoxide levels preferentially in the embryonal U57810 and myoblast C2C12 lines, leading to ferroptosis that was accentuated by iron supplementation or prevented by co-treatment with BPS, glutathione, N-acetylcysteine and ferrostatin-1. The inhibition of extracellular regulated kinases (ERK) pathway prevented ferroptosis in U57810 and C2C12 cells, whereas its increased activation in the embryonal RD cells mediated by caveolin-1 (Cav-1) overexpression led to augmented ferroptosis susceptibility. Finally, we observed the combination of erastin or RSL3 with chemotherapeutic doxorubicin and actinomycin D agents to be effective in increasing cell death in all RMS lines. Erastin and RSL3 triggerferroptosis in highly proliferatingmyogenic lines through a ERK pathway-dependent fashion.

Abstract 4352: High caveolin-1 expression in African American women with early-stage triple-negative breast cancer

Abstract In the U.S., the incidence rates of breast cancer (BC) among Caucasian (CA) women are lower than those of African American (AA); however, AA women have a significantly higher mortality rate. It is imperative that we continue to investigate the underlying molecular biology that may contribute to the cause of health disparities between AA and CA with TNBC. In this study, gene expression profiling, using the Almac BC DSA Research Tool, was performed on archived FFPE samples, obtained from CA and AA women diagnosed with early-stage (Node 0) TNBC. Unsupervised hierarchical clustering revealed a pattern of differential gene expression in the AA cohort compared to CA. Using a subtyping Tool for TNBC, we found a distinct distribution pattern of TNBC molecular subtypes in the AA cohort, which was very different than the CA cohort: basal-like (14%), immunomodulatory (43%) and mesenchymal (43%). Gene expression analyses, comparing the AA and CA cohort (fold change &amp;gt; 2.0, p-value &amp;lt;.05), resulted in 190 differentially expressed genes (DEG). Pathway enrichment analysis conducted in MetaCore GeneGo revealed that the DEGs were over-represented in cytoskeletal remodeling, cell adhesion, tight junctions, and immune response in the AA TNBC cohort. Furthermore, several genes in the Wnt/β-catenin pathways were overexpressed in the top 10 enrichment pathways. We validated our results using RT-qPCR and identified Caveolin-1 (CAV1) as being significantly expressed in the AA-TNBC cohort (p-value 1.22 x 10-05). An independent cohort of FFPE samples, from AA and CA women with early stage TNBC, was used to create a tissue microarray (TMA). Immunohistochemistry results showed no difference in subcelluar localization of Cav1 between the AA and CA cohorts; however, the AA cohort had significantly higher levels of Cav1 staining (p-value 0.04). Additionally, using RT-qPCR, we demonstrated that CAV1 mRNA was significantly higher in the AA TNBC cohort (p-value 0.48). Furthermore, endogenous Cav1 was shown to be highly expressed in a cell line panel of TNBC, in particular, those of the mesenchymal and basal-like molecular subtypes. Finally, using siRNA, we demonstrated that CAV1 silencing resulted in a significant decrease in cell proliferation for each of the TNBC cell lines while it showed no effect on the luminal ER+ cell lines. Our combined study results suggest that CAV1 overexpression may be a biologic contributor to the observed health disparity between AA women and CA diagnosed with early-stage TNBC. Citation Format: Lisa L. Baumbach-Reardon, Julie Getz, Mary E. Ahearn, John Carpten, Mark Pegram. High caveolin-1 expression in African American women with early-stage triple-negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4352.

Abstract 4042: Metformin-induced caveolin-1 expression promotes T-DM1 drug efficacy in breast cancer cells

Abstract Trastuzumab emtansine (T-DM1, Kadcyla) is an antibody-drug conjugate (ADC) that was recently approved by the Federal Drug Administration (FDA) of the USA for the treatment of HER-2-positive metastatic breast cancer. The drug sensitivity of ADCs depends in the main on the internalization efficiency of the drug. Endocytic protein caveolin-1 was shown to promote T-DM1 internalization and enhance drug sensitivity. Whether caveolin-1 can be overexpressed to improve T-DM1 efficacy is interesting and has the potential for clinical application. In this study, the first-line diabetes drug metformin was investigated in terms of induction of caveolin-1 expression for increased efficacy of subsequent T-DM1 application. HER-2-positive BT-474 cells were pretreated with metformin, followed by combined therapy with metformin and T-DM1. The T-DM1 internalization and drug efficacy were determined, and the protein expressions for signal transduction were also monitored by Western blot to ascertain the molecular mechanism. Caveolin-1 shRNA was applied to suppress endogenous cellular caveolin-1 expression, and the ability of metformin to promote T-DM1 efficacy was investigated. The results showed that in BT-474 cells pretreated with metformin, cellular caveolin-1 overexpression was induced, which then promoted drug efficacy by enhancing T-DM1 internalization. As cellular caveolin-1 was suppressed by shRNA, the effect of metformin-enhanced T-DM1 cytotoxicity was decreased. This study demonstrated that metformin can be applied prior to T-DM1 treatment to improve the clinical efficacy of T-DM1 by enhancing caveolin-1-mediated endocytosis. Citation Format: Wan-Chen Wei, Yuan-Chiang Chung, Ling-Yi Kao, Hsiang-Ling Chiu, Yi-Che Wu, Wei-Ting Chao. Metformin-induced caveolin-1 expression promotes T-DM1 drug efficacy in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4042.

Caveolin‐1 Phosphorylation is Essential for iPSC‐derived Human Neuronal Axonal Growth during Early Stage of Differentiation

Proper axonal growth and guidance is essential for functional neuronal connections. Formation of aberrant neuronal connections due to genetic or epigenetic influences can contribute to neurological and mental disorders. Identification and targeting of the molecular events that promote normal neuronal growth and differentiation may promote structural and functional neuroplasticity and improve behavior in neurodegenerative diseases such as Alzheimer's disease (AD), Down syndrome, schizophrenia, and depression. Using human neurons derived from induced pluripotent stem cells (iPSCs), we show that neuron‐specific targeted (synapsin promoted, Syn) caveolin‐1 (Cav‐1) overexpression (SynCav1) significantly augmented axonal growth (SMI 31), findings akin to effects from SynCav1 murine neurons in vitro and in vivo. In contrast, neurons infected with a Cav‐1 phosphorylation mutant (SynCav1(Y14A)) exhibited stunted axonal growth. In addition, SynCav1(Y14A) significantly impaired Rac1/Cdc42CA‐mediated axonal growth. These results suggest that 1) P‐Cav‐1 is necessary for proper axonal growth during its early differentiation stage, 2) SynCav1‐mediated axonal growth is dependent upon Cav‐1 phosphorylation, 3) Rac1/Cdc42CA‐mediated neuronal growth may in part be dependent upon P‐Cav‐1. In conclusion, phosphorylation of Cav‐1 is crucial for axonal growth during early stage of differentiation in human neurons.Support or Funding InformationNS073653 (B. P. Head); Veteran Affairs Merit Award from the Department of Veterans Affairs BX003671This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Tubeimoside V sensitizes human triple negative breast cancer MDA-MB-231 cells to anoikis via regulating caveolin-1-related signaling pathways

Metastatic triple-negative breast cancer (TNBC) has poor outcome with conventional chemotherapy regimens due to its aggressive behavior. The acquisition of anoikis resistance, a programmed cell death process triggered by substratum detachment, is an important mechanism in TNBC metastasis. Therefore, agents that can restore the sensitivity of cancer cells to anoikis may be helpful for the treatment of metastatic TNBC. In this study, we investigated the inhibitory effect of Tubeimosides V (TBMS-V), a cyclic bisdesmoside isolated from the ethanol extracts of tubers of B. paniculatum., on anoikis resistance and the involvement of caveolin-1(CAV-1)-related signaling pathways in such process in MDA-MB-231 cells. The results showed that the treatment of TBMS-V could sensitize cancer cells to anoikis, which was associated with suppression of anchorage-independent culture-induced CAV-1 overexpression, EGFR activation as well as ITGB1-FAK activation. The data from this study might contribute to providing a potential therapeutic target for metastatic TNBC and suggest the possibility of TBMS-V and its derivatives for metastatic TNBC therapy.

Protein arginine methyltransferase 5 mediates enolase-1 cell surface trafficking in human lung adenocarcinoma cells

ObjectivesEnolase-1-dependent cell surface proteolysis plays an important role in cell invasion. Although enolase-1 (Eno-1), a glycolytic enzyme, has been found on the surface of various cells, the mechanism responsible for its exteriorization remains elusive. Here, we investigated the involvement of post-translational modifications (PTMs) of Eno-1 in its lipopolysaccharide (LPS)-triggered trafficking to the cell surface. ResultsWe found that stimulation of human lung adenocarcinoma cells with LPS triggered the monomethylation of arginine 50 (R50me) within Eno-1. The Eno-1R50me was confirmed by its interaction with the tudor domain (TD) from TD-containing 3 (TDRD3) protein recognizing methylarginines. Substitution of R50 with lysine (R50K) reduced Eno-1 association with epithelial caveolar domains, thereby diminishing its exteriorization. Similar effects were observed when pharmacological inhibitors of arginine methyltransferases were applied. Protein arginine methyltransferase 5 (PRMT5) was identified to be responsible for Eno-1 methylation. Overexpression of PRMT5 and caveolin-1 enhanced levels of membrane-bound extracellular Eno-1 and, conversely, pharmacological inhibition of PRMT5 attenuated Eno-1 cell-surface localization. Importantly, Eno-1R50me was essential for cancer cell motility since the replacement of Eno-1 R50 by lysine or the suppression of PRMT 5 activity diminished Eno-1-triggered cell invasion. ConclusionsLPS-triggered Eno-1R50me enhances Eno-1 cell surface levels and thus potentiates the invasive properties of cancer cells. Strategies to target Eno-1R50me may offer novel therapeutic approaches to attenuate tumor metastasis in cancer patients.

Metformin-induced caveolin-1 expression promotes T-DM1 drug efficacy in breast cancer cells

Trastuzumab emtansine (T-DM1) is an antibody drug conjugate (ADC) that was recently approved for the treatment of HER-2-positive metastatic breast cancer. The drug sensitivity of ADCs depends mainly on the internalization efficiency of the drug. Caveolin-1 was shown to promote T-DM1 internalization and enhance drug sensitivity. Whether caveolin-1 can be overexpressed to improve T-DM1 efficacy is interesting and has the potential for clinical application. In this study, diabetes drug metformin was investigated in terms of induction of caveolin-1 expression for increased efficacy of subsequent T-DM1 application. BT-474 cells were pretreated with metformin, followed by combined therapy with metformin and T-DM1. The T-DM1 internalization and drug efficacy were determined, and the protein expressions for signal transduction were also monitored. Caveolin-1 shRNA was applied to suppress endogenous caveolin-1 expression, and the ability of metformin to promote T-DM1 efficacy was investigated. Result showed that in BT-474 cells pretreated with metformin, cellular caveolin-1 overexpression was induced, which then promoted drug efficacy by enhancing T-DM1 internalization. As cellular caveolin-1 was suppressed by shRNA, the effect of metformin-enhanced T-DM1 cytotoxicity was decreased. This study demonstrated that metformin can be applied prior to T-DM1 treatment to improve the clinical efficacy of T-DM1 by enhancing caveolin-1-mediated endocytosis.

Caveolin-1 promotes the tumor suppressor properties of oncogene-induced cellular senescence

Oncogene-induced senescence (OIS) is considered a powerful tumor suppressor mechanism. Caveolin-1 acts as a scaffolding protein to functionally regulate signaling molecules. We demonstrate that a lack of caveolin-1 expression inhibits oncogenic K-Ras (K-RasG12V)-induced premature senescence in mouse embryonic fibroblasts and normal human bronchial epithelial cells. Oncogenic K-Ras induces senescence by limiting the detoxification function of MTH1. We found that K-RasG12V promotes the interaction of caveolin-1 with MTH1, which results in inhibition of MTH1 activity. Lung cancer cells expressing oncogenic K-Ras have bypassed the senescence barrier. Interestingly, overexpression of caveolin-1 restores cellular senescence in both A549 and H460 lung cancer cells and inhibits their transformed phenotype. In support of these findings, our in vivo data demonstrate that overexpression of oncogenic K-Ras (K-RasG12D) induces cellular senescence in the lung of wildtype but not caveolin-1-null mice. A lack of K-RasG12D-induced premature senescence in caveolin-1-null mice results in the formation of more abundant lung tumors. Consistent with these data, caveolin-1-null mice overexpressing K-RasG12D display accelerated mortality. Finally, our animal data were supported by human sample analysis in which we show that caveolin-1 expression is dramatically down-regulated in lung adenocarcinomas from lung cancer patients, both at the mRNA and protein levels, and that low caveolin-1 expression is associated with poor survival. Together, our data suggest that lung cancer cells escape oncogene-induced premature senescence through down-regulation of caveolin-1 expression to progress from premalignant lesions to cancer.

Bone marrow adipocytes resist lipolysis and remodeling in response to β-adrenergic stimulation.

Bone marrow adipose tissue (BMAT) is preserved or increased in states of caloric restriction. Similarly, we found that BMAT in the tail vertebrae, but not the red marrow in the tibia, resists loss of neutral lipid with acute, 48-hour fasting in rats. The mechanisms underlying this phenomenon and its seemingly distinct regulation from peripheral white adipose tissue (WAT) remain unknown. To test the role of β-adrenergic stimulation, a major regulator of adipose tissue lipolysis, we examined the responses of BMAT to β-adrenergic agonists. Relative to inguinal WAT, BMAT had reduced phosphorylation of hormone sensitive lipase (HSL) after treatment with pan-β-adrenergic agonist isoproterenol. Phosphorylation of HSL in response to β3-adrenergic agonist CL316,243 was decreased by an additional ~90% (distal tibia BMAT) or could not be detected (tail vertebrae). Ex vivo, adrenergic stimulation of lipolysis in purified BMAT adipocytes was also substantially less than iWAT adipocytes and had site-specific properties. Specifically, regulated bone marrow adipocytes (rBMAs) from proximal tibia and femur underwent lipolysis in response to both CL316,243 and forskolin, while constitutive BMAs from the tail responded only to forskolin. This occurred independently of changes in gene expression of β-adrenergic receptors, which were similar between adipocytes from iWAT and BMAT, and could not be explained by defective coupling of β-adrenergic receptors to lipolytic machinery through caveolin 1. Specifically, we found that whereas caveolin 1 was necessary to mediate maximal stimulation of lipolysis in iWAT, overexpression of caveolin 1 was insufficient to rescue impaired BMAT signaling. Lastly, we tested the ability of BMAT to respond to 72-hour treatment with CL316,243 in vivo. This was sufficient to cause beiging of iWAT adipocytes and a decrease in iWAT adipocyte cell size. By contrast, adipocyte size in the tail BMAT and distal tibia remained unchanged. However, within the distal femur, we identified a subpopulation of BMAT adipocytes that underwent lipid droplet remodeling. This response was more pronounced in females than in males and resembled lipolysis-induced lipid partitioning rather than traditional beiging. In summary, BMAT has the capacity to respond to β-adrenergic stimuli, however, its responses are muted and BMAT generally resists lipid hydrolysis and remodeling relative to iWAT. This resistance is more pronounced in distal regions of the skeleton where the BMAT adipocytes are larger with little intervening hematopoiesis, suggesting that there may be a role for both cell-autonomous and microenvironmental determinants. Resistance to β-adrenergic stimuli further separates BMAT from known regulators of energy partitioning and contributes to our understanding of why BMAT is preserved in states of fasting and caloric restriction.

The Effects of Aging on the Regulation of T-Tubular ICa by Caveolin in Mouse Ventricular Myocytes.

Aging is associated with diminished cardiac function in males. Cardiac excitation-contraction coupling in ventricular myocytes involves Ca influx via the Ca current (ICa) and Ca release from the sarcoplasmic reticulum, which occur predominantly at t-tubules. Caveolin-3 regulates t-tubular ICa, partly through protein kinase A (PKA), and both ICa and caveolin-3 decrease with age. We therefore investigated ICa and t-tubule structure and function in cardiomyocytes from male wild-type (WT) and caveolin-3-overexpressing (Cav-3OE) mice at 3 and 24 months of age. In WT cardiomyocytes, t-tubular ICa-density was reduced by ~50% with age while surface ICa density was unchanged. Although regulation by PKA was unaffected by age, inhibition of caveolin-3-binding reduced t-tubular ICa at 3 months, but not at 24 months. While Cav-3OE increased cardiac caveolin-3 protein expression ~2.5-fold at both ages, the age-dependent reduction in caveolin-3 (WT ~35%) was preserved in transgenic mice. Overexpression of caveolin-3 reduced t-tubular ICa density at 3 months but prevented further ICa loss with age. Measurement of Ca release at the t-tubules revealed that the triggering of local Ca release by t-tubular ICa was unaffected by age. In conclusion, the data suggest that the reduction in ICa density with age is associated with the loss of a caveolin-3-dependent mechanism that augments t-tubular ICa density.

Abstract 24070: Cardiac-Specific Overexpression Of Caveolin-3 Expedites Cardiac Relaxation After Adrenergic Stimulation

Introduction: Caveolae are membrane localized signaling platforms that compartmentalize signal transduction proteins such as GPCRs. Previous studies from our laboratory have demonstrated that overexpression of caveolin-3 in cardiac myocytes (Cav3 OE) protects against pressure-overload induced heart failure. Additionally, Cav3 OE mice present increased heart rate variability with lower nocturnal heart rates, but the mechanisms behind these phenotypes remain unclear. Hypothesis: Since Cav3 OE mice are protected from stressful cardiac stimuli, we tested the rationale that Cav3 OE hearts may show altered parasympathetic control of cardiac responses to adrenergic stimulation. Methods: Cav3 OE mice and transgene negative littermate controls (Ctrl) (12-16-week-old, n=10-11 each) were anesthetized with isoflurane and cardiac contractility was assessed by echocardiography at baseline. Isoprenaline (Isuprel USP [Iso], 300 nM i.p.) was administered ~10 min after baseline recordings and cardiac function recorded at 2 min, 5 min, and 10-15 min after Iso. The same animal cohort received a single atropine injection (2 mg/kg i.p) 20 min before isoflurane anesthesia and Iso challenge echocardiography was performed as described above. Results: At baseline, no differences in cardiac contractility (% ejection fraction, %EF±SD) were detected between the two groups ~68±8 %EF. After Iso injection cardiac contractility was increased to ~91±3 %EF in both genotypes at 2 min; however, in Cav3 OE mice cardiac contractility recovered to 83±3 %EF by 5 min post Iso challenge whereas Ctrl animals maintained increased contractility of 92±3 %EF (p &lt; 0.001). Importantly, Cav3 OE mice pre-treated with atropine no longer showed increased recovery at 5 min or 10-15 post Iso (p = 0.007). A three-way ANOVA of the time-course after Iso injection found a significant effect of atropine on the responses of Cav3 OE versus Ctrl hearts to Iso (p = 0.007). Conclusion: We show for the first time that Cav3 OE mice show a faster recovery from hypercontractility after isoproterenol stimulation. Since atropine abrogated this recovery, these data suggest that Cav3 OE mice exhibit increased parasympathetic tone that may be responsible for improved stress adaptation and heart rate variability.

The level of caveolin-1 expression determines response to TGF-\u03b2 as a tumour suppressor in hepatocellular carcinoma cells

Hepatocellular carcinoma (HCC) is a heterogeneous tumour associated with poor prognostic outcome. Caveolin-1 (CAV1), a membrane protein involved in the formation of caveolae, is frequently overexpressed in HCC. Transforming growth factor-beta (TGF-β) is a pleiotropic cytokine having a dual role in hepatocarcinogenesis: inducer of apoptosis at early phases, but pro-tumourigenic once cells acquire mechanisms to overcome its suppressor effects. Apoptosis induced by TGF-β is mediated by upregulation of the NADPH oxidase NOX4, but counteracted by transactivation of the epidermal growth factor receptor (EGFR) pathway. Previous data suggested that CAV1 is required for the anti-apoptotic signals triggered by TGF-β in hepatocytes. Whether this mechanism is relevant in hepatocarcinogenesis has not been explored yet. Here we analysed the TGF-β response in HCC cell lines that express different levels of CAV1. Accordingly, stable CAV1 knockdown or overexpressing cell lines were generated. We demonstrate that CAV1 is protecting HCC cells from TGF-β-induced apoptosis, which attenuates its suppressive effect on clonogenic growth and increases its effects on cell migration. Downregulation of CAV1 in HLE cells promotes TGF-β-mediated induction of the pro-apoptotic BMF, which correlates with upregulation of NOX4, whereas CAV1 overexpression in Huh7 cells shows the opposite effect. CAV1 silenced HLE cells show attenuation in TGF-β-induced EGFR transactivation and activation of the PI3K/AKT pathway. On the contrary, Huh7 cells, which do not respond to TGF-β activating the EGFR pathway, acquire the capacity to do so when CAV1 is overexpressed. Analyses in samples from HCC patients revealed that tumour tissues presented higher expression levels of CAV1 compared with surrounding non-tumoural areas. Furthermore, a significant positive correlation among the expression of CAV1 and TGFB1 was observed. We conclude that CAV1 has an essential role in switching the response to TGF-β from cytostatic to tumourigenic, which could have clinical meaning in patient stratification.