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Michael Lisanti

    Michael Lisanti

    • I began my education at New York University, graduating Magna Cum Laude in Chemistry. I obtained my MD-PhD degrees at... moreedit
    MitoTracker Deep Red (MTDR) is a relatively non-toxic, carbocyanine-based, far-red, fluorescent probe that is routinely used to chemically mark and visualize mitochondria in living cells. Previously, we used MTDR at low nano-molar... more
    MitoTracker Deep Red (MTDR) is a relatively non-toxic, carbocyanine-based, far-red, fluorescent probe that is routinely used to chemically mark and visualize mitochondria in living cells. Previously, we used MTDR at low nano-molar concentrations to stain and metabolically fractionate breast cancer cells into Mito-high and Mito-low cell sub-populations, by flow-cytometry. Functionally, the Mito-high cell population was specifically enriched in cancer stem cell (CSC) activity, i) showing increased levels of ESA cell surface expression and ALDH activity, ii) elevated 3D anchorage-independent growth, iii) larger overall cell size (>12-μm) and iv) Paclitaxel-resistance. The Mito-high cell population also showed enhanced tumor-initiating activity, in an in vivo preclinical animal model. Here, we explored the hypothesis that higher nano-molar concentrations of MTDR could also be used to therapeutically target and eradicate CSCs. For this purpose, we employed an ER(+) cell line (MCF7) an...
    Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells... more
    Fluid shear stress activates a member of the mitogen-activated protein (MAP) kinase family, extracellular signal-regulated kinase (ERK), by mechanisms dependent on cholesterol in the plasma membrane in bovine aortic endothelial cells (BAEC). Caveolae are microdomains of the plasma membrane that are enriched with cholesterol, caveolin, and signaling molecules. We hypothesized that caveolin-1 regulates shear activation of ERK. Because caveolin-1 is not exposed to the outside, cells were minimally permeabilized by Triton X-100 (0.01%) to deliver a neutralizing, polyclonal caveolin-1 antibody (pCav-1) inside the cells. pCav-1 then bound to caveolin-1 and inhibited shear activation of ERK but not c-Jun NH2-terminal kinase. Epitope mapping studies showed that pCav-1 binds to caveolin-1 at two regions (residues 1–21 and 61–101). When the recombinant proteins containing the epitopes fused to glutathione- S-transferase (GST-Cav1–21 or GST-Cav61–101) were preincubated with pCav-1, only GST-Ca...
    Macrophages are a major cellular constituent of the tumour stroma and contribute to breast cancer prognosis. The precise role and treatment strategies to target macrophages remain elusive. As macrophage infiltration is associated with... more
    Macrophages are a major cellular constituent of the tumour stroma and contribute to breast cancer prognosis. The precise role and treatment strategies to target macrophages remain elusive. As macrophage infiltration is associated with poor prognosis and high grade tumours we used the THP-1 cell line to model monocyte-macrophage differentiation in co-culture with four breast cancer cell lines (MCF7, T47D, MDA-MB-231, MDA-MB-468) to model in vivo cellular interactions. Polarisation into M1 and M2 subtypes was confirmed by specific cell marker expression of ROS and HLA-DR, respectively. Co-culture with all types of macrophage increased migration of ER-positive breast cancer cell lines, while M2-macrophages increased mammosphere formation, compared to M1-macrophages, in all breast cancer cells lines. Treatment of cells with Zoledronate in co-culture reduced the "pro-tumourigenic" effects (increased mammospheres/migration) exerted by macrophages. Direct treatment of breast canc...
    Tumor-initiating cells (TICs), a.k.a. cancer stem cells (CSCs), are difficult to eradicate with conventional approaches to cancer treatment, such as chemo-therapy and radiation. As a consequence, the survival of residual CSCs is thought... more
    Tumor-initiating cells (TICs), a.k.a. cancer stem cells (CSCs), are difficult to eradicate with conventional approaches to cancer treatment, such as chemo-therapy and radiation. As a consequence, the survival of residual CSCs is thought to drive the onset of tumor recurrence, distant metastasis, and drug-resistance, which is a significant clinical problem for the effective treatment of cancer. Thus, novel approaches to cancer therapy are needed urgently, to address this clinical need. Towards this end, here we have investigated the therapeutic potential of graphene oxide to target cancer stem cells. Graphene and its derivatives are well-known, relatively inert and potentially non-toxic nano-materials that form stable dispersions in a variety of solvents. Here, we show that graphene oxide (of both big and small flake sizes) can be used to selectively inhibit the proliferative expansion of cancer stem cells, across multiple tumor types. For this purpose, we employed the tumor-sphere a...
    Chagas disease, caused by the parasite Trypanosoma cruzi, is an important cause of cardiac disease in endemic areas of Latin America. It is now being diagnosed in nonendemic areas because of immigration. Typical cardiac manifestations of... more
    Chagas disease, caused by the parasite Trypanosoma cruzi, is an important cause of cardiac disease in endemic areas of Latin America. It is now being diagnosed in nonendemic areas because of immigration. Typical cardiac manifestations of Chagas disease include dilated cardiomyopathy, congestive heart failure, arrhythmias, cardioembolism, and stroke. Clinical and laboratory-based research to define the pathology resulting from T. cruzi infection has shed light on many of the cellular and molecular mechanisms leading to these manifestations. Antiparasitic treatment may not be appropriate for patients with advanced cardiac disease. Clinical management of Chagas heart disease is similar to that used for cardiomyopathies caused by other processes. Cardiac transplantation has been successfully performed in a small number of patients with Chagas heart disease.
    Caveolin, a 21- to 24-kDa integral membrane protein, is a principal component of caveolae membranes. Caveolin interacts directly with heterotrimeric guanine nucleotide binding proteins (G proteins) and can functionally regulate their... more
    Caveolin, a 21- to 24-kDa integral membrane protein, is a principal component of caveolae membranes. Caveolin interacts directly with heterotrimeric guanine nucleotide binding proteins (G proteins) and can functionally regulate their activity. Here, an approximately 20-kDa caveolin-related protein, caveolin-2, was identified through microsequencing of adipocyte-derived caveolin-enriched membranes; caveolin was retermed caveolin-1. Caveolins 1 and 2 are similar in most respects. mRNAs for both caveolin-1 and caveolin-2 are most abundantly expressed in white adipose tissue and are induced during adipocyte differentiation. Caveolin-2 colocalizes with caveolin-1, indicating that caveolin-2 also localizes to caveolae. However, caveolin-1 and caveolin-2 differ in their functional interactions with heterotrimeric G proteins, possibly explaining why caveolin-1 and -2 are coexpressed within a single cell.
    The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Here, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1 , in the mouse demonstrates a... more
    The serine threonine kinase Akt1 has been implicated in the control of cellular metabolism, survival and growth. Here, disruption of the ubiquitously expressed member of the Akt family of genes, Akt1 , in the mouse demonstrates a requirement for Akt1 in ErbB2-induced mammary tumorigenesis. Akt1 deficiency delayed tumor growth and reduced lung metastases, correlating with a reduction in phosphorylation of the Akt1 target, tuberous sclerosis 2 (TSC2) at Ser-939. Akt1 -deficient mammary epithelial tumor cells (MEC) were reduced in size and proliferative capacity, with reduced cyclin D1 and p27 KIP1 abundance. Akt1 deficiency abrogated the oncogene-induced changes in polarization of MEC in three-dimensional culture and reverted oncogene-induced relocalization of the phosphorylated ezrin–radixin–moesin proteins. Akt1 increased MEC migration across an endothelial cell barrier, enhancing the persistence of migratory directionality. An unbiased proteomic analysis demonstrated Akt1 mediated ...
    Caveolae and their principal component caveolin have been implicated in playing a major role in G protein-mediated transmembrane signaling. We examined whether caveolin interacts with adenylyl cyclase, an effector of G protein signaling,... more
    Caveolae and their principal component caveolin have been implicated in playing a major role in G protein-mediated transmembrane signaling. We examined whether caveolin interacts with adenylyl cyclase, an effector of G protein signaling, using a 20-mer peptide derived from the N-terminus scaffolding domain of caveolin-1. When tissue adenylyl cyclases were examined, cardiac adenylyl cyclase was inhibited more potently than other tissue adenylyl cyclases. The caveolin-1 peptide inhibited type V, as well as type III adenylyl cyclase, overexpressed in insect cells, whereas the same peptide had no effect on type II. The caveolin-3 scaffolding domain peptide similarly inhibited type V adenylyl cyclase. In contrast, peptides derived from the caveolin-2 scaffolding domain and a caveolin-1 nonscaffolding domain had no effect. Kinetic studies showed that the caveolin-1 peptide decreased the maximal rate (Vmax) value of type V without changing the Michaelis constant (Km) value for the substrat...
    The c-jun gene regulates cellular proliferation and apoptosis via direct regulation of cellular gene expression. Alternative splicing of pre-mRNA increases the diversity of protein functions, and alternate splicing events occur in tumors.... more
    The c-jun gene regulates cellular proliferation and apoptosis via direct regulation of cellular gene expression. Alternative splicing of pre-mRNA increases the diversity of protein functions, and alternate splicing events occur in tumors. Here, by targeting the excision of the endogenous c-jun gene within the mouse mammary epithelium, we have identified its selective role as an inhibitor of RNA splicing. Microarray-based assessment of gene expression, on laser capture microdissected c-jun−/− mammary epithelium, showed that endogenous c-jun regulates the expression of approximately 50 genes governing RNA splicing. In addition, genome-wide splicing arrays showed that endogenous c-jun regulated the alternate exon of approximately 147 genes, and 18% of these were either alternatively spliced in human tumors or involved in apoptosis. Endogenous c-jun also was shown to reduce splicing activity, which required the c-jun dimerization domain. Together, our findings suggest that c-jun directl...
    Expression of the cyclin-dependent kinase (Cdk) inhibitor (p27Kip1) is frequently reduced in human tumors, often correlating with poor prognosis. p27Kip1 functions as a haploinsufficient tumor suppressor; however, the mechanism by which... more
    Expression of the cyclin-dependent kinase (Cdk) inhibitor (p27Kip1) is frequently reduced in human tumors, often correlating with poor prognosis. p27Kip1 functions as a haploinsufficient tumor suppressor; however, the mechanism by which one allele of p27Kip1 regulates oncogenic signaling in vivo is not well understood. We therefore investigated the mechanisms by which p27Kip1 inhibits mammary tumor onset. Using the common background strain of FVB, p27Kip1 heterozygosity (p27+/−) accelerated ErbB2-induced mammary tumorigenesis. We conducted microarray analyses of mammary tumors developing in mice with genetic haploinsufficiency for p27Kip1 expressing a mammary-targeted ErbB2 oncogene. Global gene expression profiling and Western blot analysis of ErbB2/p27+/− tumors showed that the loss of p27Kip1 induced genes promoting lymphangiogenesis, cellular proliferation, and collaborative oncogenic signaling (Wnt/β-catenin/Tcf, Cdc25a, Smad7, and Skp2). Skp2 expression was induced by ErbB2 an...
    The cyclin D1 gene is amplified and overexpressed in human breast cancer, functioning as a collaborative oncogene. As the regulatory subunit of a holoenzyme phosphorylating Rb, cyclin D1 promotes cell cycle progression and a noncatalytic... more
    The cyclin D1 gene is amplified and overexpressed in human breast cancer, functioning as a collaborative oncogene. As the regulatory subunit of a holoenzyme phosphorylating Rb, cyclin D1 promotes cell cycle progression and a noncatalytic function has been described to sequester the cyclin-dependent kinase inhibitor protein p27. Cyclin D1 overexpression correlates with tumor metastasis and cyclin D1–deficient fibroblasts are defective in migration. The genetic mechanism by which cyclin D1 promotes migration and movement is poorly understood. Herein, cyclin D1 promoted cellular migration and cytokinesis of mammary epithelial cells. Cyclin D1 enhanced cellular migratory velocity. The induction of migration by cyclin D1 was abolished by mutation of K112 or deletion of NH2-terminal residues 46 to 90. These mutations of cyclin D1 abrogated physical interaction with p27KIP1. Cyclin D1−/− cells were p27KIP1 deficient and the defect in migration was rescued by p27KIP1 reintroduction. Convers...
    Caveolae are 50- to 100-nm cell surface plasma membrane invaginations present in terminally differentiated cells. They are characterized by the presence of caveolin-1, sphingolipids, and cholesterol. Caveolin-1 is thought to play an... more
    Caveolae are 50- to 100-nm cell surface plasma membrane invaginations present in terminally differentiated cells. They are characterized by the presence of caveolin-1, sphingolipids, and cholesterol. Caveolin-1 is thought to play an important role in the regulation of cellular cholesterol homeostasis, a process that needs to be properly controlled to limit and prevent cholesterol accumulation and eventually atherosclerosis. We have recently generated caveolin-1-deficient [Cav-1(−/−)] mice in which caveolae organelles are completely eliminated from all cell types, except cardiac and skeletal muscle. In the present study, we examined the metabolism of cholesterol in wild-type (WT) and Cav-1(−/−) mouse embryonic fibroblasts (MEFs) and mouse peritoneal macrophages (MPMs). We observed that Cav-1(−/−) MEFs are enriched in esterified cholesterol but depleted of free cholesterol compared with their wild-type counterparts. Similarly, Cav-1(−/−) MPMs also contained less free cholesterol and w...
    The potential role of caveolin-1 in apoptosis remains controversial. Here, we investigate whether caveolin-1 expression is proapoptotic or antiapoptotic using a well-defined antisense approach. We show that NIH/3T3 cells harboring... more
    The potential role of caveolin-1 in apoptosis remains controversial. Here, we investigate whether caveolin-1 expression is proapoptotic or antiapoptotic using a well-defined antisense approach. We show that NIH/3T3 cells harboring antisense caveolin-1 are resistant to staurosporine-induced apoptosis, as assessed using cell morphology, DNA content, caspase 3 activation, and focal adhesion kinase cleavage. Importantly, sensitivity to apoptosis is recovered when caveolin-1 levels are restored. Conversely, recombinant stable expression of caveolin-1 in T24 bladder carcinoma cells sensitizes these cells to caspase 3 activation. Consistent with the observations using NIH/3T3 cells, downregulation of caveolin-1 in T24 cells substantially diminishes caspase 3-like activity. Loss of sensitivity to apoptotic stimulation is recovered by inhibition of the phosphatidylinositol 3-kinase pathway using LY-294002, suggesting a possible mechanism for the sensitizing effect of caveolin-1. Thus our res...
    Cancer cells grow in highly complex stromal microenvironments, which through metabolic remodelling, catabolism, autophagy and inflammation nurture them and are able to facilitate metastasis and resistance to therapy. However, these... more
    Cancer cells grow in highly complex stromal microenvironments, which through metabolic remodelling, catabolism, autophagy and inflammation nurture them and are able to facilitate metastasis and resistance to therapy. However, these changes in the metabolic profile of stromal cancer-associated fibroblasts and their impact on cancer initiation, progression and metastasis are not well-known. This is the first study to provide a comprehensive proteomic portrait of the azathioprine and taxol-induced catabolic state on human stromal fibroblasts, which comprises changes in the expression of metabolic enzymes, myofibroblastic differentiation markers, antioxidants, proteins involved in autophagy, senescence, vesicle trafficking and protein degradation, and inducers of inflammation. Interestingly, many of these features are major contributors to the aging process. A catabolic stroma signature, generated with proteins found differentially up-regulated in taxol-treated fibroblasts, strikingly c...
    DNA-PK is an enzyme that is required for proper DNA-repair and is thought to confer radio-resistance in cancer cells. As a consequence, it is a high-profile validated target for new pharmaceutical development. However, no FDA-approved... more
    DNA-PK is an enzyme that is required for proper DNA-repair and is thought to confer radio-resistance in cancer cells. As a consequence, it is a high-profile validated target for new pharmaceutical development. However, no FDA-approved DNA-PK inhibitors have emerged, despite many years of drug discovery and lead optimization. This is largely because existing DNA-PK inhibitors suffer from poor pharmacokinetics. They are not well absorbed and/or are unstable, with a short plasma half-life. Here, we identified the first FDA-approved DNA-PK inhibitor by "chemical proteomics". In an effort to understand how doxycycline targets cancer stem-like cells (CSCs), we serendipitously discovered that doxycycline reduces DNA-PK protein expression by nearly 15-fold (> 90%). In accordance with these observations, we show that doxycycline functionally radio-sensitizes breast CSCs, by up to 4.5-fold. Moreover, we demonstrate that DNA-PK is highly over-expressed in both MCF7- and T47D-deriv...
    Caveolae are specialized membrane microdomains present within the plasma membrane of the vast majority of cell types. They have a unique composition in that they are highly enriched in cholesterol, sphingolipids, and their coat proteins... more
    Caveolae are specialized membrane microdomains present within the plasma membrane of the vast majority of cell types. They have a unique composition in that they are highly enriched in cholesterol, sphingolipids, and their coat proteins the caveolins (-1, -2, and -3). In recent years it has been recognized that caveolae act as signaling platforms, serving as a concentrating point for numerous signaling molecules, as well as regulating flux through many distinct signaling cascades. Although caveolae are found in a variety of cell types, they are most abundant in adipose tissue. This fact has led to the intense study of the function of these organelles in adipocytes. It has now become apparent that effective insulin signaling in the adipocyte may be strictly dependent on localization of at least two insulin-responsive elements to caveolae (insulin receptor and GLUT4), as well as on a direct functional interaction between caveolin-1 and the insulin receptor. We present a critical discu...
    Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a “transformation... more
    Caveolae are vesicular invaginations of the plasma membrane. Caveolin-1 is the principal structural component of caveolae in vivo. Several lines of evidence are consistent with the idea that caveolin-1 functions as a “transformation suppressor” protein. In fact, caveolin-1 mRNA and protein expression are lost or reduced during cell transformation by activated oncogenes. Interestingly, the human caveolin-1 gene is localized to a suspected tumor suppressor locus (7q31.1). We have previously demonstrated that overexpression of caveolin-1 arrests mouse embryonic fibroblasts in the G0/G1 phase of the cell cycle through activation of a p53/p21-dependent pathway, indicating a role of caveolin-1 in mediating growth arrest. However, it remains unknown whether overexpression of caveolin-1 promotes cellular senescence in vivo. Here, we demonstrate that mouse embryonic fibroblasts transgenically overexpressing caveolin-1 show: 1) a reduced proliferative lifespan; 2) senescence-like cell morphol...
    Reactive oxygen species (ROS) can induce premature cellular senescence, which is believed to contribute to aging and age-related diseases. The nuclear erythroid 2 p45–related factor-2 (Nrf2) is a transcription factor that mediates... more
    Reactive oxygen species (ROS) can induce premature cellular senescence, which is believed to contribute to aging and age-related diseases. The nuclear erythroid 2 p45–related factor-2 (Nrf2) is a transcription factor that mediates cytoprotective responses against stress. We demonstrate that caveolin-1 is a direct binding partner of Nrf2, as shown by the binding of the scaffolding domain of caveolin-1 (amino acids 82–101) to the caveolin-binding domain of Nrf2 (amino acids 281–289). Biochemical studies show that Nrf2 is concentrated into caveolar membranes in human and mouse fibroblasts, where it colocalizes with caveolin-1, under resting conditions. After oxidative stress, caveolin-1 limits the movement of Nrf2 from caveolar membranes to the nucleus. In contrast, Nrf2 is constitutively localized to the nucleus before and after oxidative stress in caveolin-1–null mouse embryonic fibroblasts (MEFs), which do not express caveolin-1. Functional studies demonstrate that caveolin-1 acts a...
    It is generally well accepted that caveolin-3 expression is muscle specific, whereas caveolin-1 and -2 are coexpressed in a variety of cell types, including adipocytes, endothelial cells, epithelial cells, and fibroblasts. Caveolin-1 and... more
    It is generally well accepted that caveolin-3 expression is muscle specific, whereas caveolin-1 and -2 are coexpressed in a variety of cell types, including adipocytes, endothelial cells, epithelial cells, and fibroblasts. Caveolin-1 and -2 are known to form functional hetero-oligomeric complexes in cells where they are coexpressed, whereas caveolin-3 forms homo-oligomeric high molecular mass complexes. Although caveolin-2 might be expected to interact in a similar manner with caveolin-3, most studies indicate that this is not the case. However, this view has recently been challenged as it has been demonstrated that caveolin-2 and -3 are coexpressed in primary cultures of cardiac myocytes, where these two proteins can be coimmunoprecipitated. Thus it remains controversial whether caveolin-2 interacts with caveolin-3. Here, we directly address the issue of caveolin isoform protein-protein interactions by means of three distinct molecular genetic approaches. First, using caveolin-1-de...

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