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Caveolin-1 and Accelerated Host Aging in the Breast Tumor Microenvironment

Chemoprevention with Rapamycin, an mTOR Inhibitor and Anti-Aging Drug
  • Isabelle Mercier
    Correspondence
    Address reprint requests to Isabelle Mercier, Ph.D., or Michael P. Lisanti, M.D., Ph.D., Department of Stem Cell Biology and Regenerative Medicine, Thomas Jefferson University, 233 S 10th St, Bluemle Bldg, Room 933, Philadelphia, PA 19107
    Affiliations
    Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania

    The Jefferson Stem Cell Biology and Regenerative Medicine Center, Thomas Jefferson University, Philadelphia, Pennsylvania
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  • Jeanette Camacho
    Affiliations
    Department of Pathology, Cooper University Hospital, Camden, New Jersey
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  • Kanani Titchen
    Affiliations
    Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
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  • Donna M. Gonzales
    Affiliations
    Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
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  • Kevin Quann
    Affiliations
    Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
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  • Kelly G. Bryant
    Affiliations
    Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
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  • Alexander Molchansky
    Affiliations
    Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
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  • Janet N. Milliman
    Affiliations
    Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
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  • Diana Whitaker-Menezes
    Affiliations
    Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania

    Department of Pathology, Cooper University Hospital, Camden, New Jersey
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  • Federica Sotgia
    Affiliations
    Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania

    The Jefferson Stem Cell Biology and Regenerative Medicine Center, Thomas Jefferson University, Philadelphia, Pennsylvania

    Breakthrough Breast Cancer Research Unit, Manchester Breast Center, Paterson Institute for Cancer Research, and the School of Cancer, Enabling Sciences and Technology, Manchester Academic Health Science Center, University of Manchester, Manchester, United Kingdom
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  • Jean-François Jasmin
    Affiliations
    Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania

    The Jefferson Stem Cell Biology and Regenerative Medicine Center, Thomas Jefferson University, Philadelphia, Pennsylvania
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  • Roland Schwarting
    Affiliations
    Department of Pathology, Cooper University Hospital, Camden, New Jersey
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  • Richard G. Pestell
    Affiliations
    Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania

    The Jefferson Stem Cell Biology and Regenerative Medicine Center, Thomas Jefferson University, Philadelphia, Pennsylvania

    Department of Cancer Biology and Medical Oncology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
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  • Mikhail V. Blagosklonny
    Affiliations
    Roswell Park Cancer Institute, Buffalo, New York
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  • Michael P. Lisanti
    Affiliations
    Department of Stem Cell Biology and Regenerative Medicine, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania

    The Jefferson Stem Cell Biology and Regenerative Medicine Center, Thomas Jefferson University, Philadelphia, Pennsylvania

    Breakthrough Breast Cancer Research Unit, Manchester Breast Center, Paterson Institute for Cancer Research, and the School of Cancer, Enabling Sciences and Technology, Manchester Academic Health Science Center, University of Manchester, Manchester, United Kingdom

    Department of Cancer Biology and Medical Oncology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, Pennsylvania
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Open AccessPublished:June 15, 2012DOI:https://doi.org/10.1016/j.ajpath.2012.03.017
      Increasing chronological age is the most significant risk factor for human cancer development. To examine the effects of host aging on mammary tumor growth, we used caveolin (Cav)-1 knockout mice as a bona fide model of accelerated host aging. Mammary tumor cells were orthotopically implanted into these distinct microenvironments (Cav-1+/+ versus Cav-1−/− age-matched young female mice). Mammary tumors grown in a Cav-1–deficient tumor microenvironment have an increased stromal content, with vimentin-positive myofibroblasts (a marker associated with oxidative stress) that are also positive for S6-kinase activation (a marker associated with aging). Mammary tumors grown in a Cav-1–deficient tumor microenvironment were more than fivefold larger than tumors grown in a wild-type microenvironment. Thus, a Cav-1–deficient tumor microenvironment provides a fertile soil for breast cancer tumor growth. Interestingly, the mammary tumor-promoting effects of a Cav-1–deficient microenvironment were estrogen and progesterone independent. In this context, chemoprevention was achieved by using the mammalian target of rapamycin (mTOR) inhibitor and anti-aging drug, rapamycin. Systemic rapamycin treatment of mammary tumors grown in a Cav-1–deficient microenvironment significantly inhibited their tumor growth, decreased their stromal content, and reduced the levels of both vimentin and phospho-S6 in Cav-1–deficient cancer-associated fibroblasts. Since stromal loss of Cav-1 is a marker of a lethal tumor microenvironment in breast tumors, these high-risk patients might benefit from treatment with mTOR inhibitors, such as rapamycin or other rapamycin-related compounds (rapalogues).
      Caveolin (Cav)-1 knockout (KO) mice represent an established animal model of accelerated aging.
      • Park D.S.
      • Cohen A.W.
      • Frank P.G.
      • Razani B.
      • Lee H.
      • Williams T.M.
      • Chandra M.
      • Shirani J.
      • De Souza A.P.
      • Tang B.
      • Jelicks L.A.
      • Factor S.M.
      • Weiss L.M.
      • Tanowitz H.B.
      • Lisanti M.P.
      Caveolin-1 null (−/−) mice show dramatic reductions in life span.
      • Head B.P.
      • Peart J.N.
      • Panneerselvam M.
      • Yokoyama T.
      • Pearn M.L.
      • Niesman I.R.
      • Bonds J.A.
      • Schilling J.M.
      • Miyanohara A.
      • Headrick J.
      • Ali S.S.
      • Roth D.M.
      • Patel P.M.
      • Patel H.H.
      Loss of caveolin-1 accelerates neurodegeneration and aging.
      Cav-1 KO mice have a significantly reduced life span,
      • Park D.S.
      • Cohen A.W.
      • Frank P.G.
      • Razani B.
      • Lee H.
      • Williams T.M.
      • Chandra M.
      • Shirani J.
      • De Souza A.P.
      • Tang B.
      • Jelicks L.A.
      • Factor S.M.
      • Weiss L.M.
      • Tanowitz H.B.
      • Lisanti M.P.
      Caveolin-1 null (−/−) mice show dramatic reductions in life span.
      and exhibit many signs of premature aging, such as increased neurodegeneration, astrogliosis, reduced synapses, and increased β-amyloid production.
      • Head B.P.
      • Peart J.N.
      • Panneerselvam M.
      • Yokoyama T.
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      • Ali S.S.
      • Roth D.M.
      • Patel P.M.
      • Patel H.H.
      Loss of caveolin-1 accelerates neurodegeneration and aging.
      Cav-1 KO mice also exhibit other age-related pathological conditions, such as benign prostatic hypertrophy,
      • Woodman S.E.
      • Cheung M.W.
      • Tarr M.
      • North A.C.
      • Schubert W.
      • Lagaud G.
      • Marks C.B.
      • Russell R.G.
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      • Factor S.M.
      • Christ G.J.
      • Lisanti M.P.
      Urogenital alterations in aged male caveolin-1 knockout mice.
      glucose intolerance, insulin resistance, and other key features of metabolic syndrome, but remain lean and are resistant to diet-induced obesity.
      • Razani B.
      • Combs T.P.
      • Wang X.B.
      • Frank P.G.
      • Park D.S.
      • Russell R.G.
      • Li M.
      • Tang B.
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      • Scherer P.E.
      • Lisanti M.P.
      Caveolin-1-deficient mice are lean, resistant to diet-induced obesity, and show hypertriglyceridemia with adipocyte abnormalities.
      • Cohen A.W.
      • Razani B.
      • Schubert W.
      • Williams T.M.
      • Wang X.B.
      • Iyengar P.
      • Brasaemle D.L.
      • Scherer P.E.
      • Lisanti M.P.
      Role of caveolin-1 in the modulation of lipolysis and lipid droplet formation.
      • Cohen A.W.
      • Razani B.
      • Wang X.B.
      • Combs T.P.
      • Williams T.M.
      • Scherer P.E.
      • Lisanti M.P.
      Caveolin-1-deficient mice show insulin resistance and defective insulin receptor protein expression in adipose tissue.
      • Cohen A.W.
      • Schubert W.
      • Brasaemle D.L.
      • Scherer P.E.
      • Lisanti M.P.
      Caveolin-1 expression is essential for proper nonshivering thermogenesis in brown adipose tissue.
      These phenotypic changes in Cav-1 KO mice have been mechanistically attributed to systemic metabolic defects.
      • Pavlides S.
      • Tsirigos A.
      • Migneco G.
      • Whitaker-Menezes D.
      • Chiavarina B.
      • Flomenberg N.
      • Frank P.G.
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      • Wang C.
      • Pestell R.G.
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      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      The autophagic tumor stroma model of cancer: role of oxidative stress and ketone production in fueling tumor cell metabolism.
      For example, Cav-1 KO mice show evidence of increased oxidative stress and mitochondrial dysfunction.
      • Pavlides S.
      • Tsirigos A.
      • Migneco G.
      • Whitaker-Menezes D.
      • Chiavarina B.
      • Flomenberg N.
      • Frank P.G.
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      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      The autophagic tumor stroma model of cancer: role of oxidative stress and ketone production in fueling tumor cell metabolism.
      • Bosch M.
      • Mari M.
      • Herms A.
      • Fernandez A.
      • Fajardo A.
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      • Pol A.
      Caveolin-1 deficiency causes cholesterol-dependent mitochondrial dysfunction and apoptotic susceptibility.
      In fact, knockdown of Cav-1 in fibroblasts, using a small-interfering RNA approach, is sufficient to induce reactive oxygen species production and DNA damage and to drastically reduce mitochondrial membrane potential.
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      • Mari M.
      • Herms A.
      • Fernandez A.
      • Fajardo A.
      • Kassan A.
      • Giralt A.
      • Colell A.
      • Balgoma D.
      • Barbero E.
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      • Matias N.
      • Tebar F.
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      • Camps M.
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      • Gross S.P.
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      • Perez-Navarro E.
      • Fernandez-Checa J.C.
      • Pol A.
      Caveolin-1 deficiency causes cholesterol-dependent mitochondrial dysfunction and apoptotic susceptibility.
      • Martinez-Outschoorn U.E.
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      • Rivadeneira D.B.
      • Chiavarina B.
      • Pavlides S.
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      • Whitaker-Menezes D.
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      • Sotgia F.
      • Lisanti M.P.
      Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution: a new paradigm for understanding tumor metabolism, the field effect and genomic instability in cancer cells.
      • Martinez-Outschoorn U.E.
      • Trimmer C.
      • Lin Z.
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      • Zhou J.
      • Wang C.
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      • Flomenberg N.
      • Howell A.
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      • Caro J.
      • Lisanti M.P.
      • Sotgia F.
      Autophagy in cancer associated fibroblasts promotes tumor cell survival: role of hypoxia: HIF1 induction and NFkappaB activation in the tumor stromal microenvironment.
      Thus, we and others have concluded that Cav-1 KO mice are a new model for mitochondrial oxidative stress and accelerated host aging.
      • Park D.S.
      • Cohen A.W.
      • Frank P.G.
      • Razani B.
      • Lee H.
      • Williams T.M.
      • Chandra M.
      • Shirani J.
      • De Souza A.P.
      • Tang B.
      • Jelicks L.A.
      • Factor S.M.
      • Weiss L.M.
      • Tanowitz H.B.
      • Lisanti M.P.
      Caveolin-1 null (−/−) mice show dramatic reductions in life span.
      • Head B.P.
      • Peart J.N.
      • Panneerselvam M.
      • Yokoyama T.
      • Pearn M.L.
      • Niesman I.R.
      • Bonds J.A.
      • Schilling J.M.
      • Miyanohara A.
      • Headrick J.
      • Ali S.S.
      • Roth D.M.
      • Patel P.M.
      • Patel H.H.
      Loss of caveolin-1 accelerates neurodegeneration and aging.
      • Pavlides S.
      • Tsirigos A.
      • Migneco G.
      • Whitaker-Menezes D.
      • Chiavarina B.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      The autophagic tumor stroma model of cancer: role of oxidative stress and ketone production in fueling tumor cell metabolism.
      • Bosch M.
      • Mari M.
      • Herms A.
      • Fernandez A.
      • Fajardo A.
      • Kassan A.
      • Giralt A.
      • Colell A.
      • Balgoma D.
      • Barbero E.
      • Gonzalez-Moreno E.
      • Matias N.
      • Tebar F.
      • Balsinde J.
      • Camps M.
      • Enrich C.
      • Gross S.P.
      • Garcia-Ruiz C.
      • Perez-Navarro E.
      • Fernandez-Checa J.C.
      • Pol A.
      Caveolin-1 deficiency causes cholesterol-dependent mitochondrial dysfunction and apoptotic susceptibility.
      • Trimmer C.
      • Sotgia F.
      • Whitaker-Menezes D.
      • Balliet R.
      • Eaton G.
      • Martinez-Outschoorn U.E.
      • Pavlides S.
      • Howell A.
      • Iozzo R.V.
      • Pestell R.G.
      • Scherer P.E.
      • Capozza F.
      • Lisanti M.P.
      Caveolin-1 and mitochondrial SOD2 (MnSOD) function as tumor suppressors in the stromal microenvironment: a new genetically tractable model for human cancer associated fibroblasts.
      Because Cav-1 is a critical regulator of nitric oxide production (via its interactions with nitric oxide synthase) and cholesterol transport, increased nitric oxide production and/or abnormal cholesterol transport have been implicated in generating mitochondrial oxidative stress in Cav-1–deficient fibroblasts.
      • Bosch M.
      • Mari M.
      • Herms A.
      • Fernandez A.
      • Fajardo A.
      • Kassan A.
      • Giralt A.
      • Colell A.
      • Balgoma D.
      • Barbero E.
      • Gonzalez-Moreno E.
      • Matias N.
      • Tebar F.
      • Balsinde J.
      • Camps M.
      • Enrich C.
      • Gross S.P.
      • Garcia-Ruiz C.
      • Perez-Navarro E.
      • Fernandez-Checa J.C.
      • Pol A.
      Caveolin-1 deficiency causes cholesterol-dependent mitochondrial dysfunction and apoptotic susceptibility.
      • Martinez-Outschoorn U.E.
      • Balliet R.M.
      • Rivadeneira D.B.
      • Chiavarina B.
      • Pavlides S.
      • Wang C.
      • Whitaker-Menezes D.
      • Daumer K.M.
      • Lin Z.
      • Witkiewicz A.K.
      • Flomenberg N.
      • Howell A.
      • Pestell R.G.
      • Knudsen E.S.
      • Sotgia F.
      • Lisanti M.P.
      Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution: a new paradigm for understanding tumor metabolism, the field effect and genomic instability in cancer cells.
      • Martinez-Outschoorn U.E.
      • Trimmer C.
      • Lin Z.
      • Whitaker-Menezes D.
      • Chiavarina B.
      • Zhou J.
      • Wang C.
      • Pavlides S.
      • Martinez-Cantarin M.P.
      • Capozza F.
      • Witkiewicz A.K.
      • Flomenberg N.
      • Howell A.
      • Pestell R.G.
      • Caro J.
      • Lisanti M.P.
      • Sotgia F.
      Autophagy in cancer associated fibroblasts promotes tumor cell survival: role of hypoxia: HIF1 induction and NFkappaB activation in the tumor stromal microenvironment.
      • Trimmer C.
      • Sotgia F.
      • Whitaker-Menezes D.
      • Balliet R.
      • Eaton G.
      • Martinez-Outschoorn U.E.
      • Pavlides S.
      • Howell A.
      • Iozzo R.V.
      • Pestell R.G.
      • Scherer P.E.
      • Capozza F.
      • Lisanti M.P.
      Caveolin-1 and mitochondrial SOD2 (MnSOD) function as tumor suppressors in the stromal microenvironment: a new genetically tractable model for human cancer associated fibroblasts.
      • Pavlides S.
      • Tsirigos A.
      • Vera I.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Fortina P.
      • Addya S.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Sotgia F.
      • Lisanti M.P.
      Loss of stromal caveolin-1 leads to oxidative stress, mimics hypoxia and drives inflammation in the tumor microenvironment, conferring the “reverse Warburg effect”: a transcriptional informatics analysis with validation.
      Recently, it has been proposed that oxidative stress in the tumor microenvironment may lead to accelerated host aging, with accompanying DNA damage, inflammation, and a shift toward aerobic glycolysis (due to the autophagic destruction of mitochondria).
      • Lisanti M.P.
      • Martinez-Outschoorn U.E.
      • Pavlides S.
      • Whitaker-Menezes D.
      • Pestell R.G.
      • Howell A.
      • Sotgia F.
      Accelerated aging in the tumor microenvironment: connecting aging, inflammation and cancer metabolism with personalized medicine.
      • Lisanti M.P.
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      • Pestell R.G.
      • Howell A.
      • Sotgia F.
      Hydrogen peroxide fuels aging, inflammation, cancer metabolism and metastasis: the seed and soil also needs “fertilizer.”.
      As a consequence, oxidative stress and autophagy in the tumor microenvironment produce high-energy nutrients (eg, L-lactate and ketones) that can fuel tumor growth via oxidative mitochondrial metabolism in cancer cells.
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      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      The autophagic tumor stroma model of cancer: role of oxidative stress and ketone production in fueling tumor cell metabolism.
      • Pavlides S.
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      The reverse Warburg effect: aerobic glycolysis in cancer associated fibroblasts and the tumor stroma.
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      Understanding the “lethal” drivers of tumor-stroma co-evolution: emerging role(s) for hypoxia, oxidative stress and autophagy/mitophagy in the tumor micro-environment.
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      Understanding the Warburg effect and the prognostic value of stromal caveolin-1 as a marker of a lethal tumor microenvironment.
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      Herein, we have used Cav-1 KO mice as a new breast cancer stromal model to assess the potential effects of oxidative stress and accelerated host aging on mammary tumor growth in vivo. Tumors grown in Cav-1–deficient mammary fat pads were significantly larger (more than fivefold) and showed increased stromal content, as predicted.
      Oxidative stress is sufficient to drive myofibroblast differentiation
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      In accordance with these findings, we show that Cav-1–deficient cancer-associated fibroblasts (CAFs) have increased levels of vimentin (a myofibroblast marker) and phospho-S6 (a marker of increased aging). In fact, deletion of S6-kinase is sufficient to dramatically increase life span in mice, indicating that S6-kinase activation is also a functional marker of accelerated aging.
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      • Li P.W.
      • Thomas E.L.
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      With TOR, less is more: a key role for the conserved nutrient-sensing TOR pathway in aging.
      In this context, rapamycin has been proposed to function as an anti-aging drug by experimentally extending life span in both normal mice and tumor-bearing mice.
      • Blagosklonny M.V.
      Rapamycin and quasi-programmed aging: four years later.
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      A longer and healthier life with TOR down-regulation: genetics and drugs.
      Thus, we next examined whether systemically shutting down mTOR/S6-kinase signaling was sufficient to prevent the formation of mammary tumors in a Cav-1–deficient microenvironment.
      Remarkably, rapamycin treatment significantly inhibited the growth of mammary tumors in a Cav-1–deficient tumor microenvironment. Rapamycin effectively reduced the stromal content of these tumors; the levels of vimentin and phospho-S6 were also significantly decreased in Cav-1–deficient CAFs. Thus, we should consider using rapamycin (or its close relatives, the rapalogues) for the chemoprevention of recurrence in breast cancer patients who show an absence of stromal Cav-1. In direct support of this notion of chemoprevention, rapamycin and its analogues have significantly reduced the risk of developing multiple malignancies in transplant patients.
      • Kauffman H.M.
      • Cherikh W.S.
      • Cheng Y.
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      Maintenance immunosuppression with target-of-rapamycin inhibitors is associated with a reduced incidence of de novo malignancies.
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      Prevention of cancer by inhibiting aging.
      Importantly, a loss of stromal Cav-1 is a powerful predictive biomarker that is associated with early tumor recurrence, lymph node metastasis, and tamoxifen resistance, driving poor clinical outcome in breast cancer patients.
      • Witkiewicz A.K.
      • Dasgupta A.
      • Sotgia F.
      • Mercier I.
      • Pestell R.G.
      • Sabel M.
      • Kleer C.G.
      • Brody J.R.
      • Lisanti M.P.
      An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor clinical outcome in human breast cancers.
      • Sloan E.K.
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      • Natoli A.
      • Restall C.
      • Henderson M.A.
      • Fanelli M.A.
      • Cuello-Carrion F.D.
      • Gago F.E.
      • Anderson R.L.
      Stromal cell expression of caveolin-1 predicts outcome in breast cancer.
      • Ghajar C.M.
      • Meier R.
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      Quis custodiet ipsos custodies: who watches the watchmen?.
      • Qian N.
      • Ueno T.
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      • Mikami Y.
      • Wakasa T.
      • Shintaku M.
      • Tsuyuki S.
      • Inamoto T.
      • Toi M.
      Prognostic significance of tumor/stromal caveolin-1 expression in breast cancer patients.
      • Koo J.S.
      • Park S.
      • Kim S.I.
      • Lee S.
      • Park B.W.
      The impact of caveolin protein expression in tumor stroma on prognosis of breast cancer.
      Similarly, a loss of stromal Cav-1 in patients with ductal carcinoma in situ is predictive of recurrence and progression to invasive breast cancer, up to 20 years in advance.
      • Witkiewicz A.K.
      • Dasgupta A.
      • Nguyen K.H.
      • Liu C.
      • Kovatich A.J.
      • Schwartz G.F.
      • Pestell R.G.
      • Sotgia F.
      • Rui H.
      • Lisanti M.P.
      Stromal caveolin-1 levels predict early DCIS progression to invasive breast cancer.
      Similar results were also obtained with triple-negative breast cancer patients.
      • Witkiewicz A.K.
      • Dasgupta A.
      • Sammons S.
      • Er O.
      • Potoczek M.B.
      • Guiles F.
      • Sotgia F.
      • Brody J.R.
      • Mitchell E.P.
      • Lisanti M.P.
      Loss of stromal caveolin-1 expression predicts poor clinical outcome in triple negative and basal-like breast cancers.
      In TN patients, a loss of stromal Cav-1 was associated with a 5-year survival rate of <10%. In the same patient cohort, TN patients with high stromal Cav-1 had a survival rate of >75% at up to 12 years after diagnosis.
      • Witkiewicz A.K.
      • Dasgupta A.
      • Sammons S.
      • Er O.
      • Potoczek M.B.
      • Guiles F.
      • Sotgia F.
      • Brody J.R.
      • Mitchell E.P.
      • Lisanti M.P.
      Loss of stromal caveolin-1 expression predicts poor clinical outcome in triple negative and basal-like breast cancers.
      Finally, in prostate cancer patients, a loss of stromal Cav-1 is associated with advanced prostate cancer and metastatic disease, as well as a high Gleason score, which is the current gold standard for predicting prostate cancer prognosis.
      • Di Vizio D.
      • Morello M.
      • Sotgia F.
      • Pestell R.G.
      • Freeman M.R.
      • Lisanti M.P.
      An absence of stromal caveolin-1 is associated with advanced prostate cancer, metastatic disease and epithelial Akt activation.
      As such, Cav-1–deficient mice are a valid model for a lethal tumor microenvironment.
      • Pavlides S.
      • Tsirigos A.
      • Migneco G.
      • Whitaker-Menezes D.
      • Chiavarina B.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      The autophagic tumor stroma model of cancer: role of oxidative stress and ketone production in fueling tumor cell metabolism.
      Consistent with these assertions, a loss of stromal Cav-1 is a surrogate functional marker for aging, oxidative stress, DNA damage, hypoxia, autophagy, and inflammation in the tumor microenvironment.
      • Martinez-Outschoorn U.E.
      • Balliet R.M.
      • Rivadeneira D.B.
      • Chiavarina B.
      • Pavlides S.
      • Wang C.
      • Whitaker-Menezes D.
      • Daumer K.M.
      • Lin Z.
      • Witkiewicz A.K.
      • Flomenberg N.
      • Howell A.
      • Pestell R.G.
      • Knudsen E.S.
      • Sotgia F.
      • Lisanti M.P.
      Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution: a new paradigm for understanding tumor metabolism, the field effect and genomic instability in cancer cells.
      • Martinez-Outschoorn U.E.
      • Trimmer C.
      • Lin Z.
      • Whitaker-Menezes D.
      • Chiavarina B.
      • Zhou J.
      • Wang C.
      • Pavlides S.
      • Martinez-Cantarin M.P.
      • Capozza F.
      • Witkiewicz A.K.
      • Flomenberg N.
      • Howell A.
      • Pestell R.G.
      • Caro J.
      • Lisanti M.P.
      • Sotgia F.
      Autophagy in cancer associated fibroblasts promotes tumor cell survival: role of hypoxia: HIF1 induction and NFkappaB activation in the tumor stromal microenvironment.
      • Pavlides S.
      • Tsirigos A.
      • Vera I.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Fortina P.
      • Addya S.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Sotgia F.
      • Lisanti M.P.
      Loss of stromal caveolin-1 leads to oxidative stress, mimics hypoxia and drives inflammation in the tumor microenvironment, conferring the “reverse Warburg effect”: a transcriptional informatics analysis with validation.
      • Whitaker-Menezes D.
      • Martinez-Outschoorn U.E.
      • Lin Z.
      • Ertel A.
      • Flomenberg N.
      • Witkiewicz A.K.
      • Birbe R.C.
      • Howell A.
      • Pavlides S.
      • Gandara R.
      • Pestell R.G.
      • Sotgia F.
      • Philp N.J.
      • Lisanti M.P.
      Evidence for a stromal-epithelial “lactate shuttle” in human tumors: mCT4 is a marker of oxidative stress in cancer-associated fibroblasts.
      • Martinez-Outschoorn U.E.
      • Pavlides S.
      • Whitaker-Menezes D.
      • Daumer K.M.
      • Milliman J.N.
      • Chiavarina B.
      • Migneco G.
      • Witkiewicz A.K.
      • Martinez-Cantarin M.P.
      • Flomenberg N.
      • Howell A.
      • Pestell R.G.
      • Lisanti M.P.
      • Sotgia F.
      Tumor cells induce the cancer associated fibroblast phenotype via caveolin-1 degradation: implications for breast cancer and DCIS therapy with autophagy inhibitors.
      • Chiavarina B.
      • Whitaker-Menezes D.
      • Migneco G.
      • Martinez-Outschoorn U.E.
      • Pavlides S.
      • Howell A.
      • Tanowitz H.B.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Grieshaber P.
      • Caro J.
      • Sotgia F.
      • Lisanti M.P.
      HIF1-alpha functions as a tumor promoter in cancer associated fibroblasts, and as a tumor suppressor in breast cancer cells: autophagy drives compartment-specific oncogenesis.
      • Martinez-Outschoorn U.E.
      • Whitaker-Menezes D.
      • Lin Z.
      • Flomenberg N.
      • Howell A.
      • Pestell R.G.
      • Lisanti M.P.
      • Sotgia F.
      Cytokine production and inflammation drive autophagy in the tumor microenvironment: role of stromal caveolin-1 as a key regulator.
      • Witkiewicz A.K.
      • Kline J.
      • Queenan M.
      • Brody J.R.
      • Tsirigos A.
      • Bilal E.
      • Pavlides S.
      • Ertel A.
      • Sotgia F.
      • Lisanti M.P.
      Molecular profiling of a lethal tumor microenvironment, as defined by stromal caveolin-1 status in breast cancers.
      In fact, genome-wide transcriptional profiling of laser-captured tumor stroma isolated from Cav-1–negative breast cancer patients showed the presence of multiple gene signatures associated with aging, DNA damage, inflammation, and even Alzheimer's disease brain.
      • Witkiewicz A.K.
      • Kline J.
      • Queenan M.
      • Brody J.R.
      • Tsirigos A.
      • Bilal E.
      • Pavlides S.
      • Ertel A.
      • Sotgia F.
      • Lisanti M.P.
      Molecular profiling of a lethal tumor microenvironment, as defined by stromal caveolin-1 status in breast cancers.
      Virtually identical results were also obtained via the transcriptional profiling of bone marrow–derived stromal cells generated from young Cav-1 KO mice, further validating a strict association with accelerated aging.
      • Pavlides S.
      • Tsirigos A.
      • Migneco G.
      • Whitaker-Menezes D.
      • Chiavarina B.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      The autophagic tumor stroma model of cancer: role of oxidative stress and ketone production in fueling tumor cell metabolism.
      • Pavlides S.
      • Tsirigos A.
      • Vera I.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Fortina P.
      • Addya S.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Sotgia F.
      • Lisanti M.P.
      Loss of stromal caveolin-1 leads to oxidative stress, mimics hypoxia and drives inflammation in the tumor microenvironment, conferring the “reverse Warburg effect”: a transcriptional informatics analysis with validation.
      • Pavlides S.
      • Whitaker-Menezes D.
      • Castello-Cros R.
      • Flomenberg N.
      • Witkiewicz A.K.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Fortina P.
      • Addya S.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Sotgia F.
      • Lisanti M.P.
      The reverse Warburg effect: aerobic glycolysis in cancer associated fibroblasts and the tumor stroma.
      • Pavlides S.
      • Tsirigos A.
      • Vera I.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      Transcriptional evidence for the “Reverse Warburg Effect” in human breast cancer tumor stroma and metastasis: similarities with oxidative stress, inflammation, Alzheimer's disease, and “Neuron-Glia Metabolic Coupling.”.
      Thus, our current findings have important translational implications, specifically for the diagnosis and the therapeutic stratification of breast cancer patients (ie, personalized cancer medicine and/or theragnostics).

      Materials and Methods

      Animals

      This study was conducted according to the guidelines of the NIH and the Thomas Jefferson University Institute for Animal Studies. The approval was granted by the Institutional Animal Care and Use Committee at Thomas Jefferson University. Cav-1 KO mice were generated, as previously described.
      • Razani B.
      • Engelman J.A.
      • Wang X.B.
      • Schubert W.
      • Zhang X.L.
      • Marks C.B.
      • Macaluso F.
      • Russell R.G.
      • Li M.
      • Pestell R.G.
      • Di Vizio D.
      • Hou Jr, H.
      • Kneitz B.
      • Lagaud G.
      • Christ G.J.
      • Edelmann W.
      • Lisanti M.P.
      Caveolin-1 null mice are viable but show evidence of hyperproliferative and vascular abnormalities.
      All mice used in this study were in the FVB/N genetic background.

      Materials

      Mammary tumor (Met-1) cells were the generous gift of Dr. Robert D. Cardiff (University of California–Davis). Met-1 cells are a well-characterized TN mammary tumor cell line (Robert D. Cardiff, personal communication).
      • Namba R.
      • Young L.J.
      • Abbey C.K.
      • Kim L.
      • Damonte P.
      • Borowsky A.D.
      • Qi J.
      • Tepper C.G.
      • MacLeod C.L.
      • Cardiff R.D.
      • Gregg J.P.
      Rapamycin inhibits growth of premalignant and malignant mammary lesions in a mouse model of ductal carcinoma in situ.
      Rabbit polyclonal antibodies against vimentin (R28) and phospho-S6 ribosomal protein (Ser235/236; 91B2) were obtained from Cell Signaling Technology (Danvers, MA). A mouse monoclonal antibody against minichromosal maintenance 7 (141.2) and a rabbit polyclonal antibody against Cav-1 (N-20) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). A mouse monoclonal antibody to glyceradehyde-3-phosphate dehydrogenase was obtained from Fitzgerald Industries International (Acton, MA) and was used as loading control. A rabbit polyclonal antibody against CD31 (28364) was obtained from Abcam (Cambridge, MA). A mouse monoclonal antibody against nucleophosmin/B23 (Fc-61991) was obtained from Invitrogen (Carlsbad, CA). The immunohistochemistry (IHC) visualization kit LSAB2 was obtained from Dako (Carpentaria, CA). Nuclear counterstains, such as Hoechst 33342 and hematoxylin, were obtained from Invitrogen and Sigma-Aldrich (St Louis, MO). Horseradish peroxidase–conjugated secondary antibodies [anti-mouse (1:20,000 dilution) (Pierce Chemical, Rockford, IL) or anti-rabbit (1:20,000 dilution) (BD Biosciences, San Jose, CA)] were used to visualize bound primary antibodies with the supersignal chemiluminescence substrate (Pierce Chemical). Fluorescein isothiocyanate– and tetrarhodamine isothiocyanate–conjugated secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA). Placebo and rapamycin pellets (2.5 mg, 60 days, slow release) were obtained from Innovative Research of America (Sarasota, FL).

      Orthotopic Injection of Met-1 Cells

      Met-1 cells are a tumorigenic cell line established from a mammary adenocarcinoma derived from a female MMTV-PyMT mouse, and are, thus, syngeneic to the FVB/N strain. Met-1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mmol/L glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin. For the injections, 6- to 8-week-old female FVB/N wild-type (WT; Cav-1+/+) and Cav-1 KO (Cav-1−/−) mice (n = 11 to 13 per group) were anesthetized with xylazine:ketamine (5 mg/kg:50 mg/kg), and a ventral incision was performed to expose the right fourth (inguinal) mammary gland. Met-1 cells (0.5 million in 50 μL of complete growth media) were then injected into the right mammary fat pad (inguinal 4) using a 26-gauge needle. The incision was subsequently closed with a 5-0 silk suture. Tumor growth was monitored during a 5-week period, and tumor size was determined by weight. To calculate tumor incidence, mice were divided into groups with tumors that weighed <0.35 g (small), 0.35 to 0.8 g (medium), and >0.8 g (large).

      Rapamycin Pellet Implantation

      Implantation of slow-release pellets was performed under anesthesia after the mammary fat pad injections. The site of incision was shaved and scrubbed. The skin was lifted on the lateral side of the neck of the animal, and an incision equal in diameter to that of the pellet was performed. Then, a horizontal pocket of approximately 2 cm beyond the incision site was generated to introduce the pellet with forceps. The mice were randomly assigned to groups receiving two placebo pellets or two rapamycin pellets (2.5 mg, 60 days, slow release).

      Bilateral Ovariectomy Procedure

      Mice underwent ovariectomy, as previously described.
      • Mercier I.
      • Casimiro M.C.
      • Zhou J.
      • Wang C.
      • Plymire C.
      • Bryant K.G.
      • Daumer K.M.
      • Sotgia F.
      • Bonuccelli G.
      • Witkiewicz A.K.
      • Lin J.
      • Tran T.H.
      • Milliman J.
      • Frank P.G.
      • Jasmin J.F.
      • Rui H.
      • Pestell R.G.
      • Lisanti M.P.
      Genetic ablation of caveolin-1 drives estrogen-hypersensitivity and the development of DCIS-like mammary lesions.
      Briefly, 3- to 4-week-old female FVB/N WT and Cav-1 KO mice were anesthetized using xylazine:ketamine (5 mg/kg:50 mg/kg). A single dorsal incision, followed by ligation of the ovarian arteries and veins with a 4-0 silk suture, was performed, followed by the excision of both ovaries. The incision site was subsequently closed with a 5-0 silk suture.

      Preparation and Analysis of Tissues

      After the mice were sacrificed, inguinal mammary gland 4 was excised and fixed in formalin for 24 hours, paraffin embedded, and cut into sections (5 μm thick) for histological analyses and IHC staining. For blood vessel quantitation, five fields of CD31 staining per tumor were taken with a ×40 objective and the CD31-positive vessels were counted and averaged. The tumors from four to six mice were immunostained with CD31 antibody per group.

      Western Blot Analysis

      Western blot analysis was performed as previously described.
      • Williams T.M.
      • Hassan G.S.
      • Li J.
      • Cohen A.W.
      • Medina F.
      • Frank P.G.
      • Pestell R.G.
      • Di Vizio D.
      • Loda M.
      • Lisanti M.P.
      Caveolin-1 promotes tumor progression in an autochthonous mouse model of prostate cancer: genetic ablation of Cav-1 delays advanced prostate tumor development in tramp mice.
      Briefly, mice were euthanized and the mammary fat pad was dissected, weighed, and snap frozen in liquid nitrogen. Samples were homogenized in radioimmunoprecipitation (RIPA) assay [50 mmol/L Tris (pH 7.5), 150 mmol/L NaCl, 1% Nonidet P-40, 0.5% deoxycholate, and 0.1% SDS]
      • Schlegel A.
      • Arvan P.
      • Lisanti M.P.
      Caveolin-1 binding to endoplasmic reticulum membranes and entry into the regulated secretory pathway are regulated by serine phosphorylation: protein sorting at the level of the endoplasmic reticulum.
      lysis buffer with complete miniprotease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN) and phosphatase inhibitor cocktails from Sigma-Aldrich. After homogenization, samples were sonicated and centrifuged at 12,000 × g for 10 minutes. Samples were separated by SDS-PAGE (12% acrylamide) and transferred to a nitrocellulose membrane for probing. Subsequent wash buffers consisted of 10 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, and 0.05% Tween 20. Membranes were blocked in 10 mmol/L Tris (pH 8.0), 150 mmol/L NaCl, and 0.05% Tween 20 supplemented with 5% bovine serum albumin (Sigma-Aldrich) or 5% nonfat dry milk (Carnation, Solon, OH) for 1 hour at room temperature. The membranes were subsequently incubated with a given primary antibody for 3 hours at room temperature. Primary antibodies were used at a range of 1 to 2.5 μg/mL dilution. Horseradish peroxidase–conjugated secondary antibodies were used to visualize bound primary antibodies with the supersignal chemiluminescence substrate (Pierce Chemical).

      IHC Analysis of Tissues

      IHC was performed as previously described.
      • Mercier I.
      • Casimiro M.C.
      • Zhou J.
      • Wang C.
      • Plymire C.
      • Bryant K.G.
      • Daumer K.M.
      • Sotgia F.
      • Bonuccelli G.
      • Witkiewicz A.K.
      • Lin J.
      • Tran T.H.
      • Milliman J.
      • Frank P.G.
      • Jasmin J.F.
      • Rui H.
      • Pestell R.G.
      • Lisanti M.P.
      Genetic ablation of caveolin-1 drives estrogen-hypersensitivity and the development of DCIS-like mammary lesions.
      Briefly, paraffin-embedded sections (5 µm thick) of fat pads (containing tumor cells) were dehydrated in xylene for 10 minutes and rehydrated in a series of graded ethanols and distilled water for 5 minutes. The slides were then incubated in a citric acid–based antigen unmasking solution with an acidic pH (Vector Labs, Burlingame, CA) using an electric pressure cooker on high pressure for 5 to 10 minutes. The slides (six to eight per group) were incubated in 3% hydrogen peroxide (Thermo Fisher Scientific, Hampton, NH) for 30 minutes at room temperature and blocked with 10% goat normal serum (Jackson ImmunoResearch Laboratories) for 1 hour at room temperature and incubated with a given primary antibody overnight at 4°C. The following day, slides were washed with PBS once and incubated with a biotinylated mouse or rabbit secondary antibody included in the IHC visualization kit (LSAB2). The remainder of the protocol was performed according to the manufacturer's instructions. The slides were counterstained using Mayer's hematoxylin. The slides were dehydrated with graded alcohols and left in xylene for 15 minutes before mounting with Permount (Thermo Fisher Scientific). The images were acquired at ×40 and ×60 magnification.

      Immunofluorescence

      For immunofluorescence, the same protocol as IHC was used, with the exception of the peroxidase blocking step, and the secondary antibodies used were fluorescein isothiocyanate and tetrarhodamine isothiocyanate conjugated (1:300). The slides were counterstained with Hoechst 33342 (1:1000) and mounted with Prolong Gold antifade solution (Invitrogen). The tumors were imaged with a confocal microscope (Zeiss LSM 510; Carl Zeiss, Thornwood, NY). Images were acquired with ×63 and ×100 objectives.

      Statistical Analysis

      All of the statistical analysis was performed using a one-way analysis of variance, followed by a Tukey-Kramer multiple comparison test, unless otherwise stated. P < 0.05 was considered significant.

      Mining of Transcriptional Profiling Data

      The transcriptional profiles of laser-captured tumor stroma,
      • Witkiewicz A.K.
      • Kline J.
      • Queenan M.
      • Brody J.R.
      • Tsirigos A.
      • Bilal E.
      • Pavlides S.
      • Ertel A.
      • Sotgia F.
      • Lisanti M.P.
      Molecular profiling of a lethal tumor microenvironment, as defined by stromal caveolin-1 status in breast cancers.
      • Finak G.
      • Bertos N.
      • Pepin F.
      • Sadekova S.
      • Souleimanova M.
      • Zhao H.
      • Chen H.
      • Omeroglu G.
      • Meterissian S.
      • Omeroglu A.
      • Hallett M.
      • Park M.
      Stromal gene expression predicts clinical outcome in breast cancer.
      isolated from human breast cancer patients, were re-examined for evidence of elevated mTOR/S6-kinase signaling, essentially as we previously described for other signaling pathways related to glycolysis, lysosomal degradation, and autophagy.
      • Pavlides S.
      • Tsirigos A.
      • Migneco G.
      • Whitaker-Menezes D.
      • Chiavarina B.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      The autophagic tumor stroma model of cancer: role of oxidative stress and ketone production in fueling tumor cell metabolism.
      • Pavlides S.
      • Tsirigos A.
      • Vera I.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      Transcriptional evidence for the “Reverse Warburg Effect” in human breast cancer tumor stroma and metastasis: similarities with oxidative stress, inflammation, Alzheimer's disease, and “Neuron-Glia Metabolic Coupling.”.
      More specifically, the data presented in Table 1 were from published DNA microarray data from Pavlides et al
      • Pavlides S.
      • Tsirigos A.
      • Vera I.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      Transcriptional evidence for the “Reverse Warburg Effect” in human breast cancer tumor stroma and metastasis: similarities with oxidative stress, inflammation, Alzheimer's disease, and “Neuron-Glia Metabolic Coupling.”.
      using fresh-frozen invasive breast cancer samples with their matching benign control subjected to laser microdissection to specifically isolate the stromal compartment. The data presented in Table 2 were obtained from published DNA microarray data from Witkiewicz et al
      • Witkiewicz A.K.
      • Kline J.
      • Queenan M.
      • Brody J.R.
      • Tsirigos A.
      • Bilal E.
      • Pavlides S.
      • Ertel A.
      • Sotgia F.
      • Lisanti M.P.
      Molecular profiling of a lethal tumor microenvironment, as defined by stromal caveolin-1 status in breast cancers.
      using fresh-frozen breast cancer samples subjected to laser capture microdissection to specifically isolate the stromal RNA from Cav-1–positive (n = 4) and Cav-1–negative (n = 7) breast cancer stromal compartments.
      Table 1Transcriptional Overexpression of S6-Kinase and Ribosomal Proteins in Tumor Stroma Isolated from Breast Cancer Patients
      SymbolGene descriptionTumor stromaRecurrence stromaMetastasis stroma
      Ribosomal protein S6 kinase
      Rps6kl1Ribosomal protein S6 kinase-like 12.59 × 10−169.39 × 10−4
      Rps6kb1Ribosomal protein S6 kinase, polypeptide 16.12 × 10−139.76 × 10−4
      Rps6ka2Ribosomal protein S6 kinase, polypeptide 28.69 × 10−115.82 × 10−3
      Rps6kb2Ribosomal protein S6 kinase, polypeptide 21.10 × 10−101.37 × 10−3
      Rps6ka1Ribosomal protein S6 kinase, polypeptide 11.43 × 10−103.12 × 10−4
      Rps6ka6Ribosomal protein S6 kinase, polypeptide 62.60 × 10−94.52 × 10−2
      Rps6ka5Ribosomal protein S6 kinase, polypeptide 59.60 × 10−4
      Rps6ka4Ribosomal protein S6 kinase, polypeptide 41.55 × 10−2
      Rps6ka3Ribosomal protein S6 kinase, polypeptide 33.01 × 10−2
      Ribosomal proteins
      Rpl3lRibosomal protein L3-like1.37 × 10−19
      Rps9Ribosomal protein S95.64 × 10−152.27 × 10−3
      Rpl22Ribosomal protein L224.39 × 10−12
      Rpl23Ribosomal protein L235.40 × 10−6
      Rpl30Ribosomal protein L307.77 × 10−4
      Rps27lRibosomal protein S27-like3.89 × 10−2
      Rpl21Ribosomal protein L213.16 × 10−4
      Rps7Ribosomal protein S72.86 × 10−3
      Rsl1d1Ribosomal L1 domain-containing 11.74 × 10−2
      Rps14Ribosomal protein S142.45 × 10−2
      Rpl7l1Ribosomal protein L7-like 13.85 × 10−2
      Rps24Ribosomal protein S244.72 × 10−2
      Mitochondrial ribosomal proteins
      Mrp63Mitochondrial ribosomal protein 631.27 × 10−20
      Mrps10Mitochondrial ribosomal protein S109.11 × 10−20
      Mrpl20Mitochondrial ribosomal protein L201.25 × 10−17
      Mrpl43Mitochondrial ribosomal protein L431.71 × 10−171.16 × 10−2
      Mrps18bMitochondrial ribosomal protein S18B4.86 × 10−171.83 × 10−3
      Mrps6Mitochondrial ribosomal protein S69.10 × 10−14
      Mrps12Mitochondrial ribosomal protein S122.31 × 10−112.55 × 10−2
      Mrpl38Mitochondrial ribosomal protein L382.36 × 10−94.20 × 10−2
      Mrpl2Mitochondrial ribosomal protein L22.13 × 10−71.98 × 10−2
      Mrps25Mitochondrial ribosomal protein S258.25 × 10−62.75 × 10−2
      Mrps24Mitochondrial ribosomal protein S243.06 × 10−5
      Mrps34Mitochondrial ribosomal protein S343.72 × 10−23.85 × 10−2
      Mrpl9Mitochondrial ribosomal protein L91.62 × 10−42.03 × 10−3
      Mrpl55Mitochondrial ribosomal protein L551.61 × 10−31.43 × 10−2
      Mrpl49Mitochondrial ribosomal protein L493.97 × 10−2
      Mrpl52Mitochondrial ribosomal protein L524.08 × 10−2
      Mrpl3Mitochondrial ribosomal protein L32.43 × 10−2
      Mrpl33Mitochondrial ribosomal protein L332.50 × 10−2
      Mrpl4Mitochondrial ribosomal protein L42.85 × 10−2
      Mrps17Mitochondrial ribosomal protein S173.87 × 10−2
      Mrpl12Mitochondrial ribosomal protein L123.95 × 10−2
      Mrps5Mitochondrial ribosomal protein S54.14 × 10−2
      Upstream activators of S6 kinase
      Pik3cdPI3K catalytic δ polypeptide3.43 × 10−266.57 × 10−3
      Pik3cgPI3K catalytic γ polypeptide5.82 × 10−224.10 × 10−2
      Pik3ap1PI3K adaptor protein 17.87 × 10−158.30 × 10−3
      Pdpk1PI3–dependent protein kinase-18.76 × 10−148.65 × 10−4
      Pik3c3PI3K, class 33.74 × 10−13
      Pik3c2gPI3K, C2 domain, γ1.31 × 10−5
      Igf1Insulin-like growth factor 19.71 × 10−18
      RragcRas-related GTP binding C2.61 × 10−18
      RragaRas-related GTP binding A2.92 × 10−17
      Akt3V-akt thymoma viral oncogene homolog 32.18 × 10−13
      RhebRAS-homolog enriched in brain2.76 × 10−12
      Other mTOR downstream effectors/regulators
      Prkaa2Kinase, AMP-activated α2 catalytic9.03 × 10−251.50 × 10−4
      Prkag3Kinase, AMP-activated γ3 noncatalytic3.55 × 10−12
      Prkab2Kinase, AMP-activated β2 noncatalytic1.99 × 10−8
      Prkaa1Kinase, AMP-activated α1 catalytic4.67 × 10−82.50 × 10−2
      Prkag2Kinase, AMP-activated γ2 noncatalytic5.02 × 10−81.10 × 10−22.19 × 10−2
      Cdc42Cell division cycle 42 homolog2.46 × 10−221.36 × 10−3
      Eif4ebp2Eif4e-binding protein 23.59 × 10−17
      Eif4ebp1Eif4e-binding protein 11.41 × 10−5
      Eif4eEukaryotic translation initiation factor 4E1.16 × 10−102.10 × 10−2
      VegfbVascular endothelial growth factor B5.13 × 10−12
      Ddit4lDNA damage–inducible transcript 4-like4.76 × 10−11
      Ulk2Unc-51 like kinase 2 (Caenorhabditis elegans)6.16 × 10−11
      Ppp2r2bProtein phosphatase2 regulatory subunit B1.24 × 10−8
      Ppp2r1aProtein phosphatase2 regulatory subunit A4.06 × 10−3
      Ppp2r2cProtein phosphatase2 regulatory subunit B2.76 × 10−3
      Ppp2r2aProtein phosphatase2 regulatory subunit B3.94 × 10−2
      Hif1aHypoxia-inducible factor 1, α subunit2.10 × 10−6
      P values for the overexpressed stromal gene transcripts are as shown. Data were extracted from Supplemental Tables S1–S3 found in Pavlides et al.
      • Pavlides S.
      • Tsirigos A.
      • Vera I.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      Transcriptional evidence for the “Reverse Warburg Effect” in human breast cancer tumor stroma and metastasis: similarities with oxidative stress, inflammation, Alzheimer's disease, and “Neuron-Glia Metabolic Coupling.”.
      See the Materials and Methods for more details. Transcripts highlighted in bold are associated with recurrence and/or metastasis.
      PI3K, phosphatidylinositol 3-kinase.
      Table 2Transcriptional Overexpression of S6-Kinase and Ribosomal Proteins in Cav-1–Deficient Tumor Stroma Isolated from Breast Cancer Patients
      SymbolGene descriptionP valueFold change (Cav-1/Cav-1+)
      Ribosomal protein S6 kinase
      Rps6ka3Ribosomal protein S6 kinase, polypeptide 30.011.90
      Rps6ka3Ribosomal protein S6 kinase, polypeptide 30.041.89
      Ribosomal proteins
      Rps16Ribosomal protein S160.023.86
      Rps16Ribosomal protein S160.053.19
      Rps7Ribosomal protein S70.062.62
      Rps27Ribosomal protein S270.012.57
      Rps15ARibosomal protein S15a0.022.40
      Rps9Ribosomal protein S90.11.92
      LOC100129381Predicted: similar to ribosomal protein S70.041.69
      Rps14Ribosomal protein S140.041.66
      Rpl7l1Ribosomal protein L7-like 10.081.35
      Mitochondrial ribosomal proteins
      LactbMitochondrial 39S ribosomal protein L56; β-lactamase0.0024.62
      LactbMitochondrial 39S ribosomal protein L56; β-lactamase0.041.80
      Mrpl9Mitochondrial ribosomal protein L90.0032.11
      Mrpl9Mitochondrial ribosomal protein L90.0051.87
      Mrpl49Mitochondrial ribosomal protein L490.041.84
      Mrpl2Mitochondrial ribosomal protein L20.061.70
      Mrps7Mitochondrial ribosomal protein S70.051.48
      Upstream activators of S6 kinase
      Akt3V-AKT thymoma viral oncogene homolog 30.032.62
      Pdpk1PI3–dependent protein kinase-10.0072.15
      MtorMammalian target of rapamycin (serine/threonine kinase)0.062.02
      Pik3cgPI3K catalytic γ polypeptide0.11.93
      Pik3ap1PI3K adaptor protein 10.0451.79
      Pik3c2aPI3K class 2 α polypeptide0.051.50
      Rhebl1Ras homolog enriched in brain like 10.11.50
      Other mTOR downstream effectors/regulators
      Ppp2r5cProtein phosphatase2, regulatory subunit B′, γ0.023.00
      Prkag2Kinase AMP-activated γ 2 noncatalytic0.022.23
      Cdc42Cell division cycle 42 (GTP-binding protein 25 kd)0.031.81
      Hif1aHypoxia-inducible factor 1 α subunit0.11.39
      Eif4ebp2Eukaryotic translation initiation factor 4E binding0.11.33
      P values and fold changes in stromal gene transcript expression are shown. Data were extracted from Supplemental Table S2 found in Witkiewicz et al.
      • Witkiewicz A.K.
      • Kline J.
      • Queenan M.
      • Brody J.R.
      • Tsirigos A.
      • Bilal E.
      • Pavlides S.
      • Ertel A.
      • Sotgia F.
      • Lisanti M.P.
      Molecular profiling of a lethal tumor microenvironment, as defined by stromal caveolin-1 status in breast cancers.
      See the Materials and Methods for more details. Transcripts highlighted in bold overlap with the genes listed in Table 1. In some cases, more than one entry is shown for a gene transcript; these represent the results obtained with more than one probe set for a given gene transcript. The specific probe sets used are available in Supplemental Table S2 in Witkiewicz et al.
      • Witkiewicz A.K.
      • Kline J.
      • Queenan M.
      • Brody J.R.
      • Tsirigos A.
      • Bilal E.
      • Pavlides S.
      • Ertel A.
      • Sotgia F.
      • Lisanti M.P.
      Molecular profiling of a lethal tumor microenvironment, as defined by stromal caveolin-1 status in breast cancers.
      n = 4 patients with high stromal Cav-1, and n = 7 patients with absent stromal Cav-1. As a consequence, gene transcripts with a P ≤ 0.1 were included, because of the small sample size.
      PI3K, phosphatidylinositol 3-kinase.

      Results

      Cav-1–Negative Mammary Stroma Accelerates Met-1 Tumor Growth in Vivo

      Recent reports have shown an association between reduced stromal Cav-1 and unfavorable outcome in breast cancer patients.
      • Witkiewicz A.K.
      • Dasgupta A.
      • Sammons S.
      • Er O.
      • Potoczek M.B.
      • Guiles F.
      • Sotgia F.
      • Brody J.R.
      • Mitchell E.P.
      • Lisanti M.P.
      Loss of stromal caveolin-1 expression predicts poor clinical outcome in triple negative and basal-like breast cancers.
      To understand the mechanisms underlying this phenomenon, we injected Met-1 into WT and Cav-1 KO mouse mammary fat pads. As shown in Figure 1A, the tumors grown in Cav-1 KO mammary fat pads were significantly larger and more vascularized than those grown in their WT counterparts. Quantitatively, as shown in Figure 1B, Met-1 tumors grown in the fat pads of Cav-1 KO mice were approximately fivefold larger than those grown in WT fat pads (n = 11 and n = 13, respectively; P < 0.01). When tumor size was further examined, approximately 90% of the Cav-1 KO mice developed medium-to-large tumors compared with only approximately 40% in the WT group (Figure 1C).
      Figure thumbnail gr1
      Figure 1Cav-1–negative mammary stroma increases mammary tumor growth in vivo. A: Representative images of mammary glands after orthotopic injections of Met-1 cells show accelerated tumor growth in Cav-1 KO mice. Images were acquired using an Olympus DP71 camera (Center Valley, PA) with DP manager software (MacKinney Systems, Springfield, MO) version 3.1.1.208, using a ×1.5 objective. B: Quantitative analysis of tumor weight shows an approximately fivefold increase. *P < 0.001 (n = 11 to 13 per group). Mice were injected in the right mammary fat pad, resulting in only one tumor per mouse. C: Approximately 90% of the Cav-1 KO mice developed medium-large tumors compared with only 40% in the WT group.

      Mammary Tumors Grown in Cav-1 KO Mice Display Increased Angiogenesis and More Stromal Content

      For a tumor to grow beyond approximately 2 mm in diameter, the generation of new blood vessels is necessary, a process known as angiogenesis.
      • Olewniczak S.
      • Chosia M.
      • Kwas A.
      • Kram A.
      • Domagala W.
      Angiogenesis and some prognostic parameters of invasive ductal breast carcinoma in women.
      As such, we postulated that increased angiogenesis could contribute to the accelerated tumor growth observed in Cav-1 KO fat pads. In fact, tumors grown in Cav-1 KO mammary fat pads are more vascularized, as reflected by an increased abundance of CD31-positive vessels (Figure 2).
      Figure thumbnail gr2
      Figure 2Cav-1–negative mammary stroma increases tumor vascularization. Representative images of CD31 staining of tumors resulting from Met-1 orthotopic injections show increased vessel formation in tumors grown in Cav-1 KO mammary fat pads. Staining shows results from IHC of CD31 (brown) with nuclear counterstain (blue) and confocal microscopy of CD31 (red) immunostaining and Hoechst (blue). Original magnification, ×40 (CD31) and ×63 (CD31/Hoechst).
      In addition to being larger and more vascularized, Cav-1 KO tumors were also significantly more stromalized (a high stroma/epithelia ratio). This is depicted in Figure 3 by increased Masson's trichrome staining (blue). An increase in collagen secretion is suggestive of the presence of fibroblasts (Figure 3).
      • Sugimoto H.
      • Mundel T.M.
      • Kieran M.W.
      • Kalluri R.
      Identification of fibroblast heterogeneity in the tumor microenvironment.
      In fact, increased vimentin staining, a marker of myofibroblasts, was also observed in tumors grown in Cav-1 KO fat pads (Figure 3).
      Figure thumbnail gr3
      Figure 3Increased amounts of stroma in tumors grown in Cav-1 KO fat pads. A complex collagen network was detected in H&E-stained tumors by an intense pink staining. Masson's trichrome stain also reveals the presence of collagen, as depicted by a blue stain. To detect the presence of fibroblasts embedded within the collagen network, tumors were immunostained with a vimentin antibody. Cav-1 KO tumors have more vimentin-positive cells. Arrows, stromal cells. Original magnification, ×60.

      Tumor Stroma of Cav-1 KO Mice Is Hyperproliferative

      The increased collagen deposition and vimentin-positive staining in Cav-1 KO fat pads suggested the presence of stromal proliferative fibroblasts. As predicted, dual labeling with a proliferation marker (MCM7) and vimentin antibody revealed that Cav-1 KO stromal cells were more proliferative (Figure 4).
      Figure thumbnail gr4
      Figure 4Increased vimentin and stromal MCM7 in tumors grown in Cav-1 KO fat pads. Confocal microscopy demonstrating dual labeling of vimentin and MCM7 is shown. These results demonstrate that tumors grown in a Cav-1 KO fat pad have increased levels of proliferating fibroblasts, as shown by increased expression of nuclear MCM7 staining in vimentin-positive cells. Representative fields were taken using a ×63 oil objective. Hoechst 33342 was used as a nuclear counterstain (blue), along with vimentin (green) and MCM7 (red). Arrows, stromal cells.

      Cav-1 KO CAFs Show Hypertrophied Nucleoli and Elevated Levels of Nucleophosmin/B23

      Cav-1–negative CAFs found in mammary tumors were more proliferative; thus, we predicted that they would have an increased need for protein synthesis. Our IHC results revealed that CAFs in Cav-1–negative tumors showed enhanced B23/nucleophosmin expression, a nucleolar protein involved in ribosomal biosynthesis
      • Lindstrom M.S.
      • Zhang Y.
      Ribosomal protein S9 is a novel B23/NPM-binding protein required for normal cell proliferation.
      • Korgaonkar C.
      • Hagen J.
      • Tompkins V.
      • Frazier A.A.
      • Allamargot C.
      • Quelle F.W.
      • Quelle D.E.
      Nucleophosmin (B23) targets ARF to nucleoli and inhibits its function.
      • Itahana K.
      • Bhat K.P.
      • Jin A.
      • Itahana Y.
      • Hawke D.
      • Kobayashi R.
      • Zhang Y.
      Tumor suppressor ARF degrades B23, a nucleolar protein involved in ribosome biogenesis and cell proliferation.
      (Figure 5, A and B).
      Figure thumbnail gr5
      Figure 5Increased levels and colocalization of nucleophosmin/B23 and phospho-S6 in tumors grown in Cav-1 KO mice. A: Cav-1 KO mice have increased levels of stromal nucleophosmin/B23, as depicted by brown staining. B: The stromal cells in Cav-1 KO tumors that are positive for nucleophosmin/B23 staining (red) are also positive for pS6 staining (green). A merged image is shown (right panel). All images were acquired using a ×100 oil objective.

      Cav-1 KO Tumor Stroma Displays an Activated mTOR Pathway

      To date, few studies have examined the contribution of stromal protein synthesis machinery in tumor growth and progression. In fact, Cav-1 KO mammary tumors showed increased phosphorylation of the ribosomal protein S6 at serine 235/236 (pS6), a downstream effector of mTOR activity.
      • Hay N.
      • Sonenberg N.
      Upstream and downstream of mTOR.
      As depicted in Figure 6, we observed an inverse relationship between Cav-1 and pS6 expression in stromal fibroblasts. Fibroblasts found both inside and outside the Cav-1 KO tumors had more pS6 compared with those in WT tumors (Figure 6, A and B). In contrast, epithelial pS6 remained unaffected by the presence of Cav-1 (Figure 6).
      Figure thumbnail gr6
      Figure 6Tumors grown in Cav-1 KO mice have increased expression of stromal phospho-S6. Tumors grown in Cav-1 KO mice display increased pS6 staining (brown) in the fibroblasts embedded within the tumors (A) and in the fibroblasts surrounding the tumors (B). In contrast, WT fibroblasts express significantly less pS6 staining. Original magnification, ×40.

      Rapamycin, an mTOR Inhibitor, Prevents the Growth of Cav-1 KO Mammary Tumors

      Hyperactivation of the protein synthesis machinery and increases in mTOR pathway signaling in Cav-1 KO CAFs might contribute to the accelerated growth of mammary tumors. To test this hypothesis, mice injected with Met-1 cells were treated with rapamycin (RAPA), a specific mTOR inhibitor. Remarkably, daily rapamycin treatment (2.78 μg/kg per day) for 5 weeks prevented mammary tumor growth in Cav-1 KO mice, as reflected by tumor weight (Figure 7A; n = 11 and n = 12, respectively; P < 0.001). To ensure the dose of rapamycin used was sufficient to inhibit mTOR activity, we assessed the levels of pS6, its downstream effector. As depicted in Figure 7B, although the levels of pS6 were significantly elevated in tumors grown in Cav-1 KO mice, rapamycin treatment prevented mTOR activity. This observation was also independently validated by IHC (Figure 8). Rapamycin treatment prevented tumor growth in Cav-1 KO mice and also decreased collagen deposition, vimentin staining, and angiogenesis (Figure 9, A–C).
      Figure thumbnail gr7
      Figure 7Tumors grown in Cav-1 KO fat pads are rapamycin sensitive. A: Tumor growth in Cav-1 KO mice was reduced by as much as 4.7-fold after rapamycin treatment when compared with placebo-treated mice (P < 0.001). For rapamycin treatment, 11 WT and 12 Cav-1 KO mice received rapamycin treatment, whereas 13 WT and 11 Cav-1 KO mice received placebo only. B: Western blot analysis showing the hyperactivation of pS6 and its complete inhibition by rapamycin treatment. Total S6, GAPDH, and Cav-1 are shown as control (CTL) of equal loading and to demonstrate the lack of Cav-1 in the tumor stroma of Cav-1 KO mice. *P < 0.001 vs WT-placebo; P < 0.001 vs WT-rapamycin; P < 0.001 vs Cav-1 KO-placebo.
      Figure thumbnail gr8
      Figure 8Status of stromal phospho-S6 immunostaining in mammary tumors after rapamycin treatment in WT and Cav-1 KO mice. After a 5-week treatment with rapamycin, the tumor size reverts to that of WT tumors. Mammary tumor sections were immunostained with a phospho-specific antibody directed against phospho-S6. Tumors grown in Cav-1 KO mice display increased pS6 staining (brown) in the tumor-associated fibroblasts (arrows); this staining was ablated by rapamycin treatment (Cav-1 KO + RAP) and appears as tumors grown in a WT microenvironment.
      Figure thumbnail gr9
      Figure 9Morphological characteristics of mammary tumors after rapamycin (RAPA) treatment in WT and Cav-1 KO mice. After a 5-week treatment with rapamycin, the tumor size reverts to that of WT tumors. Rapamycin significantly decreases the levels of angiogenesis (A), collagen deposition (B), and the total number of fibroblasts in the tumor stroma (C), as depicted by CD31 (A), trichrome (B), and vimentin (C) immunostains, respectively. Original magnification, ×60 objective.

      CD31-Positive Vessels Are Decreased in the Tumors of Cav-1 KO Mice Treated with Rapamycin

      As depicted in Figure 9, the tumors grown in Cav-1 KO mice had increased CD31 staining, suggesting an increase in angiogenesis. To quantitatively assess the levels of angiogenesis, the number of blood vessels was counted by assessing CD31-positive vessels in each tumor before and after rapamycin treatment. Although tumors grown in Cav-1 KO fat pads had >30 blood vessels per field, the number of vessels decreased almost twofold after rapamycin treatment, reaching the levels observed in the tumors grown in WT mice (Figure 10).
      Figure thumbnail gr10
      Figure 10Blood vessel quantitation in the tumors grown in WT and Cav-1 KO mice before and after rapamycin (RAPA) treatment, showing the number of blood vessels per field. A total of five fields were photographed at ×40 magnification per group and averaged. *P < 0.001 between Cav-1 KO (KO) and WT; P < 0.001 between Cav-1 KO + placebo (PLAC) and Cav-1 KO + RAPA.

      Mammary Tumor Growth in Cav-1 KO Mice Is Hormone Independent

      To exclude the possibility that mammary tumors grew faster in Cav-1 KO fat pads because of differential hormonal sensitivity of the host's mammary fat pad, we performed a bilateral ovariectomy (OVX). Figure 11 shows that tumor formation was not affected by depletion of ovarian hormones in both WT and Cav-1 KO mice. The success of the ovariectomy procedure was confirmed by measuring uterine atrophy, as previously described.
      • Mercier I.
      • Casimiro M.C.
      • Zhou J.
      • Wang C.
      • Plymire C.
      • Bryant K.G.
      • Daumer K.M.
      • Sotgia F.
      • Bonuccelli G.
      • Witkiewicz A.K.
      • Lin J.
      • Tran T.H.
      • Milliman J.
      • Frank P.G.
      • Jasmin J.F.
      • Rui H.
      • Pestell R.G.
      • Lisanti M.P.
      Genetic ablation of caveolin-1 drives estrogen-hypersensitivity and the development of DCIS-like mammary lesions.
      Interestingly, tumors still grew approximately 4.5-fold bigger in ovariectomized Cav-1 KO mice when compared with their WT counterparts (P < 0.01; n = 9 and n = 12, respectively). Most important, even in the absence of ovarian hormones, rapamycin successfully prevented tumor growth in Cav-1 KO mice (4.2-fold; P < 0.01 compared with Cav-1 KO/OVX-placebo). Also important, there was no significant difference between Cav-1 KO/Sham-RAPA versus Cav-1 KO/OVX-RAPA (P = 0.486).
      Figure thumbnail gr11
      Figure 11Accelerated mammary tumor growth in Cav-1 KO mice is hormone independent. Tumor weight after tumor cell injection and rapamycin treatment in OVX WT and Cav-1 KO mice is depicted. Cav-1 KO mice develop larger tumors and remain rapamycin sensitive in the absence of ovarian hormones. *P < 0.001 versus WT sham; P < 0.001 versus WT-OVX-PLACEBO; P < 0.01 versus Cav-1 KO-OVX-PLACEBO; §P < 0.05 versus Cav-1 KO sham.

      Transcriptional Evidence that mTOR/S6-Kinase Signaling in the Tumor Microenvironment Is Increased in Human Breast Cancer Patients

      To further assess the clinical relevance of our current findings, we next re-examined the transcriptional profiles of human tumor stroma that was isolated from a series of breast cancer patients, via laser-capture microdissection.
      • Finak G.
      • Bertos N.
      • Pepin F.
      • Sadekova S.
      • Souleimanova M.
      • Zhao H.
      • Chen H.
      • Omeroglu G.
      • Meterissian S.
      • Omeroglu A.
      • Hallett M.
      • Park M.
      Stromal gene expression predicts clinical outcome in breast cancer.
      These data included three complementary gene sets that were also associated with clinical outcome.
      • Pavlides S.
      • Tsirigos A.
      • Vera I.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      Transcriptional evidence for the “Reverse Warburg Effect” in human breast cancer tumor stroma and metastasis: similarities with oxidative stress, inflammation, Alzheimer's disease, and “Neuron-Glia Metabolic Coupling.”.
      i) The Tumor Stroma versus Normal Stroma List evaluated the transcriptional profiles of tumor stroma obtained from 53 patients with normal stroma obtained from 38 patients (containing 6777 genes).
      • Pavlides S.
      • Tsirigos A.
      • Vera I.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      Transcriptional evidence for the “Reverse Warburg Effect” in human breast cancer tumor stroma and metastasis: similarities with oxidative stress, inflammation, Alzheimer's disease, and “Neuron-Glia Metabolic Coupling.”.
      ii) The Recurrence Stroma List evaluated the transcriptional profiles of tumor stroma obtained from 11 patients (with tumor recurrence) with the tumor stroma of 42 patients (without tumor recurrence) (containing 3354 genes).
      • Pavlides S.
      • Tsirigos A.
      • Vera I.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      Transcriptional evidence for the “Reverse Warburg Effect” in human breast cancer tumor stroma and metastasis: similarities with oxidative stress, inflammation, Alzheimer's disease, and “Neuron-Glia Metabolic Coupling.”.
      iii) The Lymph-Node (LN) Metastasis Stroma List evaluated the transcriptional profiles of tumor stroma (obtained from 25 patients with LN metastasis) with the tumor stroma of 25 patients (without LN metastasis) (containing 1182 genes).
      • Pavlides S.
      • Tsirigos A.
      • Vera I.
      • Flomenberg N.
      • Frank P.G.
      • Casimiro M.C.
      • Wang C.
      • Pestell R.G.
      • Martinez-Outschoorn U.E.
      • Howell A.
      • Sotgia F.
      • Lisanti M.P.
      Transcriptional evidence for the “Reverse Warburg Effect” in human breast cancer tumor stroma and metastasis: similarities with oxidative stress, inflammation, Alzheimer's disease, and “Neuron-Glia Metabolic Coupling.”.
      All gene transcripts that were up-regulated in the tumor stroma of patients were all selected and assigned a P value, with a cutoff of P < 0.05.
      The results of this analysis are summarized in Table 1. Many gene transcripts associated with mTOR/S6-kinase signaling, and S6-kinase itself, and other ribosomal proteins were all specifically up-regulated in the tumor stroma of human breast cancer patients. Interestingly, many mitochondrial ribosomal proteins were also up-regulated. Such a large increase in the anabolic protein synthesis machinery may be a necessary stress response to compensate for the onset of catabolic protein degradation, via autophagy and mitophagy, in the tumor stroma. Many of these overexpressed transcripts were also specifically associated with tumor recurrence and LN metastasis.
      To further assess the association of mTOR/S6-kinase signaling with a Cav-1–deficient tumor stroma, we next re-examined the gene profiles obtained from human tumor stroma isolated from breast cancer patients that were separated based on the status of stromal Cav-1.
      • Witkiewicz A.K.
      • Kline J.
      • Queenan M.
      • Brody J.R.
      • Tsirigos A.
      • Bilal E.
      • Pavlides S.
      • Ertel A.
      • Sotgia F.
      • Lisanti M.P.
      Molecular profiling of a lethal tumor microenvironment, as defined by stromal caveolin-1 status in breast cancers.
      The results of this analysis are summarized in Table 2. Importantly, many of the same mTOR/S6-kinase–related gene transcripts were specifically elevated in patients with a loss of stromal Cav-1 (highlighted in bold), as predicted. In this regard, Lactb (a mitochondrial ribosomal protein) is one of the gene transcripts that was most highly up-regulated in the stroma of patients with a loss of Cav-1. In light of these clinical data, our current results using Cav-1–deficient mice as a preclinical model may have important translational significance for human breast cancer patients.

      Discussion

      New evidence suggests a dynamic function of the surrounding tumor stroma in breast cancer pathogenesis.
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      Stromal myofibroblasts in breast cancer: relations between their occurrence, tumor grade and expression of some tumour markers.
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      Cancer-associated stromal fibroblasts promote pancreatic tumor progression.
      Cav-1, an important tumor suppressor, was recently shown to be expressed in the tumor-associated stroma.
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      An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor clinical outcome in human breast cancers.
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      Stromal cell expression of caveolin-1 predicts outcome in breast cancer.
      Importantly, a loss of stromal Cav-1 is a powerful predictive biomarker that is associated with early tumor recurrence, LN metastasis, and tamoxifen resistance, driving poor clinical outcome in breast cancer patients.
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      • Brody J.R.
      • Lisanti M.P.
      An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor clinical outcome in human breast cancers.
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      • Henderson M.A.
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      • Cuello-Carrion F.D.
      • Gago F.E.
      • Anderson R.L.
      Stromal cell expression of caveolin-1 predicts outcome in breast cancer.
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      Quis custodiet ipsos custodies: who watches the watchmen?.
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      • Yoshida N.
      • Mikami Y.
      • Wakasa T.
      • Shintaku M.
      • Tsuyuki S.
      • Inamoto T.
      • Toi M.
      Prognostic significance of tumor/stromal caveolin-1 expression in breast cancer patients.
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      • Park S.
      • Kim S.I.
      • Lee S.
      • Park B.W.
      The impact of caveolin protein expression in tumor stroma on prognosis of breast cancer.
      Although these clinical data suggest that Cav-1 can serve as a predictive biomarker of disease outcome, more studies were warranted to understand the mechanisms involved.
      To gain a better understanding of the prognostic value of a loss of stromal Cav-1, mammary tumor cells isolated from the MMTV-PyMT tumor model (Met-1) were injected into the mammary fat pads of WT and Cav-1 KO mice. Interestingly, a lack of Cav-1 expression in the mammary fat pad greatly accelerated tumor formation. The present results are in accordance with previous tumor transplantation experiments in Cav-1 KO fat pads, which also suggested a growth-inhibiting property of stromal Cav-1.
      • Williams T.M.
      • Sotgia F.
      • Lee H.
      • Hassan G.
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      • Mercier I.
      • Rui H.
      • Pestell R.G.
      • Lisanti M.P.
      Stromal and epithelial caveolin-1 both confer a protective effect against mammary hyperplasia and tumorigenesis: caveolin-1 antagonizes cyclin D1 function in mammary epithelial cells.
      Furthermore, recent xenografts and co-injection experiments showed that knockdown of Cav-1 expression in human immortalized fibroblasts was sufficient to accelerate the growth of MDA-MB-231 breast cancer cells.
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      • Sotgia F.
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      • Balliet R.
      • Eaton G.
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      • Pavlides S.
      • Howell A.
      • Iozzo R.V.
      • Pestell R.G.
      • Scherer P.E.
      • Capozza F.
      • Lisanti M.P.
      Caveolin-1 and mitochondrial SOD2 (MnSOD) function as tumor suppressors in the stromal microenvironment: a new genetically tractable model for human cancer associated fibroblasts.
      A striking morphological feature of the mammary tumors grown in Cav-1 KO fat pads was the increased amount of collagen deposition and the presence of spindle-shaped fibroblasts embedded within the tumor mass (Figure 3). In contrast, tumors grown in WT mammary fat pads were almost entirely epithelial. Previous studies described the prognostic value of an abundant stroma (stroma rich) versus an epithelial-dominant tumor mass (stroma poor). For example, colon and breast tumors with abundant stroma showed an increased risk of relapse and a poor prognosis.
      • Mesker W.E.
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      • Tanke H.J.
      Presence of a high amount of stroma and downregulation of SMAD4 predict for worse survival for stage I-II colon cancer patients.
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      • Mesker W.E.
      Tumor-stroma ratio in the primary tumor is a prognostic factor in early breast cancer patients, especially in triple-negative carcinoma patients.
      More specifically, TN breast tumors with less stroma have a 5-year relapse-free rate of 81% compared with 56% for those with abundant stroma. Mechanistically, the prominence of collagen deposition and an increased number of fibroblasts found in the Cav-1 KO tumor stroma could be explained by increased stromal proliferation. In fact, Cav-1 KO CAFs showed increased nuclear expression of the proliferative marker MCM7 (Figure 4). These results suggest that a lack of stromal Cav-1 in tumor could promote the proliferation of fibroblasts.
      Increased nucleolar prominence has been used to predict cellular transformation for years, and abnormalities in nucleolar shape have been reported in cancer as early as the 19th century.
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      Nucleolus, ribosomes, and cancer.
      The nucleolus is an important cellular component involved in ribosome production. Most often, increased cellular proliferation results in an increased demand for cellular constituents, leading to increased protein synthesis, ribosome production, and nucleolar hypertrophy.
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      An encore for ribosome biogenesis in the control of cell proliferation.
      In fact, we noticed that Cav-1 KO CAFs have more prominent nucleoli and hypothesized that they required increased protein synthesis to sustain their greater proliferative rates. Accordingly, Cav-1 KO CAFs expressed significantly more B23/nucleophosmin, a nucleolar protein necessary for ribosomal RNA transcription and processing (Figure 5).
      mTOR, the mammalian target of rapamycin, is a serine/threonine kinase that is often overexpressed or activated in cancer cells and that regulates protein synthesis, cell growth, and survival. Few studies have examined the role of the mTOR pathway in CAFs and tumor growth.
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      • Hidalgo M.
      New targets for therapy in breast cancer: mammalian target of rapamycin (mTOR) antagonists.
      Consistent with B23/nucleophosmin overexpression, a remarkable increase in mTOR activity was observed in Cav-1 KO CAFs, as reflected by increased pS6 protein levels (Figure 6, Figure 7). Consistent with an activated mTOR pathway in the stroma of Cav-1–depleted mammary tumors, these tumors were highly responsive to mTOR inhibitors, such as rapamycin, as seen by the prevention of tumor growth (Figure 7). To our knowledge, although this is the first report of mTOR activation in Cav-1 KO CAFs. A recent study reported that keloids, a dermal fibroproliferative disorder of scar fibroblasts, also display hyperactivation of the mTOR pathway.
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      mTOR as a potential therapeutic target for treatment of keloids and excessive scars.
      In fact, the proliferation of keloids can be inhibited by rapamycin.
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      • Do D.V.
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      • Aalami O.
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      mTOR as a potential therapeutic target for treatment of keloids and excessive scars.
      The hyperactivation of mTOR in Cav-1 KO CAFs is not surprising because they share several similarities with keloids, such as increased proliferation, aberrant growth factor secretion, and increased extracellular matrix production and contractile proteins.
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      • Lisanti M.P.
      Human breast cancer-associated fibroblasts (CAFs) show caveolin-1 downregulation and RB tumor suppressor functional inactivation: implications for the response to hormonal therapy.
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      • Lisanti M.P.
      Caveolin-1-/- null mammary stromal fibroblasts share characteristics with human breast cancer-associated fibroblasts.
      Interestingly, recent reports have shown that stromal phosphatase and tensin homolog (PTEN), an upstream inhibitor of phosphatidylinositol 3-kinase, can inhibit mammary tumor development in an ErB2 breast cancer mouse model.
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      • Leone G.
      Pten in stromal fibroblasts suppresses mammary epithelial tumours.
      Morphologically, rapamycin-treated Cav-1 KO tumors showed less collagen, had decreased tumor-associated fibroblasts, and had significantly less angiogenesis, as reflected by decreased CD31 staining; all of these characteristics predict a good clinical outcome in patients
      • Mesker W.E.
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      • Alberici P.
      • Kuppen P.J.
      • Miranda N.F.
      • van Leeuwen K.A.
      • Morreau H.
      • Szuhai K.
      • Tollenaar R.A.
      • Tanke H.J.
      Presence of a high amount of stroma and downregulation of SMAD4 predict for worse survival for stage I-II colon cancer patients.
      • de Kruijf E.M.
      • van Nes J.G.
      • van de Velde C.J.
      • Putter H.
      • Smit V.T.
      • Liefers G.J.
      • Kuppen P.J.
      • Tollenaar R.A.
      • Mesker W.E.
      Tumor-stroma ratio in the primary tumor is a prognostic factor in early breast cancer patients, especially in triple-negative carcinoma patients.
      • Ellis L.M.
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      Angiogenesis and metastasis.
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      Advances in angiogenesis research: relevance to urological oncology.
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      Angiogenesis as a predictor of long-term survival for patients with node-negative breast cancer.
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      Angiogenesis as a prognostic marker in breast carcinoma with conventional adjuvant chemotherapy: a multiparametric and immunohistochemical analysis.
      (Figure 9). Previous reports have suggested that human breast CAFs can directly promote angiogenesis through the expression of stromal cell–derived factors.
      • Orimo A.
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      Stromal fibroblasts present in invasive human breast carcinomas promote tumor growth and angiogenesis through elevated SDF-1/CXCL12 secretion.
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      • Sheng H.
      Roles of myofibroblasts in prostaglandin E2-stimulated intestinal epithelial proliferation and angiogenesis.
      Furthermore, we previously reported that a lack of Cav-1 expression in mammary fat pads is linked to increased angiogenesis. Indeed, mammary fibroblasts derived from Cav-1 KO mice revealed increased levels of angiogenesis-related gene transcripts.
      • Mercier I.
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      • Lin J.
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      • Frank P.G.
      • Jasmin J.F.
      • Rui H.
      • Pestell R.G.
      • Lisanti M.P.
      Genetic ablation of caveolin-1 drives estrogen-hypersensitivity and the development of DCIS-like mammary lesions.
      • Sotgia F.
      • Del Galdo F.
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      • Bonuccelli G.
      • Mercier I.
      • Whitaker-Menezes D.
      • Daumer K.M.
      • Zhou J.
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      • Xu H.
      • Bosco E.
      • Quong A.A.
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      • Minetti C.
      • Frank P.G.
      • Jimenez S.A.
      • Knudsen E.S.
      • Pestell R.G.
      • Lisanti M.P.
      Caveolin-1-/- null mammary stromal fibroblasts share characteristics with human breast cancer-associated fibroblasts.
      The decrease in angiogenesis observed after rapamycin treatment in tumors grown in Cav-1 KO mice (Figure 10) is interesting and might suggest the involvement of the stromal mTOR pathway on blood vessel formation in these Cav-1–deficient stromal tumors.
      Previous reports have suggested that the anti-tumor effects of rapamycin are estrogen dependent, thus neglecting its potential therapeutic efficacy in estrogen-independent and tamoxifen-resistant breast cancer patients.
      • Chang S.B.
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      Rapamycin inhibits proliferation of estrogen-receptor-positive breast cancer cells.
      To test whether rapamycin would retain its therapeutic potential in breast cancer patients with low stromal Cav-1 in the absence of ovarian hormones, we subjected the mice to bilateral ovariectomies. Interestingly, the accelerated tumor growth observed in Cav-1 KO mice was maintained after the ovariectomy procedure (Figure 11). These results suggest a hormone-independent role for stromal Cav-1 in breast tumor growth. More important, rapamycin also dramatically prevented tumor growth in ovariectomized Cav-1 KO mice. The present results suggest the potential clinical value of mTOR inhibitors in premenopausal and postmenopausal TN breast cancer patients with decreased stromal Cav-1, as well as in estrogen receptor–positive tamoxifen-resistant breast cancer patients.
      Recent mechanistic studies have proposed that Cav-1 might constitute an important link between cancer cells and the tumor stroma.
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      The autophagic tumor stroma model of cancer: role of oxidative stress and ketone production in fueling tumor cell metabolism.
      Metabolomics and gene profiling studies of the Cav-1 KO mammary gland have revealed signatures associated with oxidative stress, mitochondrial dysfunction, and aerobic glycolysis.
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      The autophagic tumor stroma model of cancer: role of oxidative stress and ketone production in fueling tumor cell metabolism.
      This new model proposes that a loss of Cav-1 expression is sufficient to induce oxidative stress in stromal fibroblasts. These events result in lysosome-driven degradation of organelles (autophagy or mitophagy) in CAFs, which release energy-rich nutrients. These recycled stromal nutrients can then be used by the adjacent epithelial cancer cells, resulting in net energy transfer to cancer cells and accelerated tumor growth.
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      The autophagic tumor stroma model of cancer: role of oxidative stress and ketone production in fueling tumor cell metabolism.
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      • Lisanti M.P.
      Oxidative stress in cancer associated fibroblasts drives tumor-stroma co-evolution: a new paradigm for understanding tumor metabolism, the field effect and genomic instability in cancer cells.
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      Autophagy in cancer associated fibroblasts promotes tumor cell survival: role of hypoxia: HIF1 induction and NFkappaB activation in the tumor stromal microenvironment.
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      Ketones and lactate “fuel” tumor growth and metastasis: evidence that epithelial cancer cells use oxidative mitochondrial metabolism.
      The phosphatidylinositol 3-kinase/mTOR/S6-kinase pathway has previously been linked to oxidative stress.
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      TOR kinase and Ran are downstream from PI3K/Akt in H2O2-induced mitosis.
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      Increased Rheb-TOR signaling enhances sensitivity of the whole organism to oxidative stress.
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      Essential role of caveolae in interleukin-6- and insulin-like growth factor I-triggered Akt-1-mediated survival of multiple myeloma cells.
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      Effects of FK506 and rapamycin on generation of reactive oxygen species, nitric oxide production and nuclear factor kappa B activation in rat hepatocytes.
      S6-kinase is the upstream activator of pS6 and was recently required for starvation-induced autophagy.
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      The double-edged sword of autophagy modulation in cancer.
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      High levels of autophagy can actually be detrimental to cells,
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      and mTOR activation protects cells against high levels of autophagy.
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      Nutrient-dependent regulation of autophagy through the target of rapamycin pathway.
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      Hyperactivation of phosphatidylinositol-3 kinase promotes escape from hormone dependence in estrogen receptor-positive human breast cancer.
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      Apoptosis, autophagy, accelerated senescence and reactive oxygen in the response of human breast tumor cells to adriamycin.
      Thus, the increased mTOR/S6-kinase signaling observed in Cav-1 KO CAFs might be a compensatory response to protect fibroblasts from abnormally high levels of autophagy. Interestingly, Narita et al
      • Narita M.
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      Spatial coupling of mTOR and autophagy augments secretory phenotypes.
      have recently come to a similar conclusion.
      • Zoncu R.
      • Sabatini D.M.
      Cell biology: the TASCC of secretion.
      They directly showed that anabolic protein synthesis, via mTOR signaling, is an important stress response in cells undergoing catabolic protein degradation via enhanced autophagy,
      • Narita M.
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      • Nakashima T.
      • Yoshida S.
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      • Reichelt S.
      • Ferreira M.
      • Tavare S.
      • Inoki K.
      • Shimizu S.
      Spatial coupling of mTOR and autophagy augments secretory phenotypes.
      and may lead to an enhanced secretory phenotype, with the increased production of IL-6 and IL-8.
      • Narita M.
      • Young A.R.
      • Arakawa S.
      • Samarajiwa S.A.
      • Nakashima T.
      • Yoshida S.
      • Hong S.
      • Berry L.S.
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      • Ferreira M.
      • Tavare S.
      • Inoki K.
      • Shimizu S.
      Spatial coupling of mTOR and autophagy augments secretory phenotypes.
      However, they did not examine the role of Cav-1 in this process or relate this metabolic phenotype to tumorigenesis.
      The role of the mTOR pathway in oxidative stress–induced proliferation was also previously reported. Hydrogen peroxide can induce the proliferation of lung cells that, in turn, can be inhibited by rapamycin.
      • Radisavljevic Z.M.
      • Gonzalez-Flecha B.
      TOR kinase and Ran are downstream from PI3K/Akt in H2O2-induced mitosis.
      Some reports also suggest that retinoblastoma (RB) is a downstream target of mTOR in adipocytes and prostate and ovarian cancer cells.
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      Retinoblastoma protein phosphorylation via PI 3-kinase and mTOR pathway regulates adipocyte differentiation.
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      G1 cell cycle progression and the expression of G1 cyclins are regulated by PI3K/AKT/mTOR/p70S6K1 signaling in human ovarian cancer cells.
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      Androgens induce prostate cancer cell proliferation through mammalian target of rapamycin activation and post-transcriptional increases in cyclin D proteins.
      Interestingly, we previously reported dysregulation of the RB pathway by Cav-1 in human breast CAFs.
      • Mercier I.
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      • Quong J.
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      Human breast cancer-associated fibroblasts (CAFs) show caveolin-1 downregulation and RB tumor suppressor functional inactivation: implications for the response to hormonal therapy.
      Thus, the increased proliferative potential of Cav-1–negative CAFs surrounding breast tumors might contribute toward a worse prognosis due to mTOR-dependent oxidative stress–induced proliferation, suggesting a new axis in the stroma of breast tumors (Cav-1→mTOR→RB) (Figure 12). Whether breast cancer patients with low stromal Cav-1 expression have more CAFs, increased stromal mTOR, and an inactivated RB pathway remains to be examined.
      Figure thumbnail gr12
      Figure 12Mechanistic diagram summarizing the function of stromal Cav-1 in the mTOR pathway and tumor growth. The levels of Cav-1 expression in the CAFs dictate how tumor cells grow in the mammary fat pad. Fibroblasts lacking Cav-1 expression display significantly more mTOR activation, causing the surrounding tumor cells, through paracrine actions, to grow and develop into significantly larger tumors. This accelerated growth induced by a Cav-1–negative stroma can be specifically prevented by the pharmacological inhibition of the mTOR pathway with rapamycin.
      Apart from its role in autophagy and cancer, mTOR hyperactivation was recently linked to aging.
      • Blagosklonny M.V.
      TOR-driven aging: speeding car without brakes.
      For instance, rapamycin treatment can increase the life span of cancer-prone mice.
      • Anisimov V.N.
      • Zabezhinski M.A.
      • Popovich I.G.
      • Piskunova T.S.
      • Semenchenko A.V.
      • Tyndyk M.L.
      • Yurova M.N.
      • Antoch M.P.
      • Blagosklonny M.V.
      Rapamycin extends maximal lifespan in cancer-prone mice.
      Interestingly, a loss of Cav-1 in mice results in accelerated aging and a decreased life span.
      • Park D.S.
      • Cohen A.W.
      • Frank P.G.
      • Razani B.
      • Lee H.
      • Williams T.M.
      • Chandra M.
      • Shirani J.
      • De Souza A.P.
      • Tang B.
      • Jelicks L.A.
      • Factor S.M.
      • Weiss L.M.
      • Tanowitz H.B.
      • Lisanti M.P.
      Caveolin-1 null (−/−) mice show dramatic reductions in life span.
      • Head B.P.
      • Peart J.N.
      • Panneerselvam M.
      • Yokoyama T.
      • Pearn M.L.
      • Niesman I.R.
      • Bonds J.A.
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      Loss of caveolin-1 accelerates neurodegeneration and aging.
      Whether rapamycin treatment could increase the longevity of Cav-1 KO mice remains to be determined. The current results suggest that a Cav-1–negative tumor stroma possesses all of the characteristics of an aging organ and might contribute to poor disease outcome and shorten the life span of patients because of hyperactivated stromal mTOR signaling.
      In conclusion, the present study investigated the downstream mechanisms involved in the growth-promoting effects of a Cav-1–negative tumor stroma in mammary tumors. We show that Cav-1–negative stroma provides a fertile soil for breast tumor growth. These tumors display a proliferative stroma with hyperactivated mTOR signaling. The treatment of these mice with rapamycin prevented tumor growth, an effect independent of estrogen and progesterone. Thus, our results suggest that the levels of Cav-1 in the microenvironment (Cav-1 rich versus Cav-1 poor) can directly promote the growth of breast tumors, which can be prevented by mTOR inhibitors, such as rapamycin. Breast cancer patients with low stromal Cav-1 levels have a poor prognosis. Whether mTOR inhibitors could prevent tumor growth and increase survival rates in breast cancer patients with decreased stromal Cav-1 remain to be investigated and could have a major clinical impact. In conclusion, we describe a new network between stromal Cav-1 and mTOR signaling, which can be used to design more effective treatments for breast cancer patients.

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