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The Role of Multicellular Aggregation in the Survival of ErbB2-positive Breast Cancer Cells during Extracellular Matrix Detachment*

  • Raju R. Rayavarapu
    Affiliations
    Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
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  • Author Footnotes
    1 Both authors contributed equally to this work.
    Brendan Heiden
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
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  • Author Footnotes
    1 Both authors contributed equally to this work.
    Nicholas Pagani
    Footnotes
    1 Both authors contributed equally to this work.
    Affiliations
    Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
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  • Melissa M. Shaw
    Affiliations
    Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
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  • Sydney Shuff
    Affiliations
    Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
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  • Author Footnotes
    2 Recipient of a Pathway to Independence Award (5R00CA158066) from the NIH.
    Siyuan Zhang
    Footnotes
    2 Recipient of a Pathway to Independence Award (5R00CA158066) from the NIH.
    Affiliations
    Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
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  • Zachary T. Schafer
    Correspondence
    Recipient of a Lee National Denim Day Research Scholar Grant (RSG-14-145-01-CSM) from the American Cancer Society and a Career Catalyst Grant (CCR14302768) from Susan G. Komen. To whom correspondence should be addressed: Dept. of Biological Sciences, University of Notre Dame, 222 Galvin Life Science Center, Notre Dame, IN 46556. Tel.: 574-631-0875; Fax: 574-631-7413; E-mail: [email protected]
    Affiliations
    Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556
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  • Author Footnotes
    * This work was supported by the Glynn Family Honors Program and the Notre Dame NSF-REU program in biology (to N. P.).
    1 Both authors contributed equally to this work.
    2 Recipient of a Pathway to Independence Award (5R00CA158066) from the NIH.
    4 The abbreviations used are: ECMextracellular matrixDAPI4,6-diamidino-2-phenylindoleDNECADdominant-negative E-cadherinEMTepithelial-mesenchymal transitionMETmesenchymal to epithelial transitionCTCcirculating tumor cellMCmethylcelluloseEGFRepidermal growth factor receptor.
Open AccessPublished:February 13, 2015DOI:https://doi.org/10.1074/jbc.M114.612754
      The metastasis of cancer cells from the site of the primary tumor to distant sites in the body represents the most deadly manifestation of cancer. In order for metastasis to occur, cancer cells need to evade anoikis, which is defined as apoptosis caused by loss of attachment to extracellular matrix (ECM). Signaling from ErbB2 has previously been linked to the evasion of anoikis in breast cancer cells but the precise molecular mechanisms by which ErbB2 blocks anoikis have yet to be unveiled. In this study, we have identified a novel mechanism by which anoikis is inhibited in ErbB2-expressing cells: multicellular aggregation during ECM-detachment. Our data demonstrate that disruption of aggregation in ErbB2-positive cells is sufficient to induce anoikis and that this anoikis inhibition is a result of aggregation-induced stabilization of EGFR and consequent ERK/MAPK survival signaling. Furthermore, these data suggest that ECM-detached ErbB2-expressing cells may be uniquely susceptible to targeted therapy against EGFR and that this sensitivity could be exploited for specific elimination of ECM-detached cancer cells.

      Introduction

      The overwhelming majority of cancer-related deaths (90%) are a direct result of the metastasis of cancer cells from the primary tumor to distant sites (
      • Nguyen D.X.
      • Massagué J.
      Genetic determinants of cancer metastasis.
      ,
      • Hanahan D.
      • Weinberg R.A.
      Hallmarks of cancer: the next generation.
      • Wan L.
      • Pantel K.
      • Kang Y.
      Tumor metastasis: moving new biological insights into the clinic.
      ). Metastasis is an inherently inefficient process as a significant percentage of cells that escape the primary tumor are not successful in colonizing secondary sites. It is currently understood that a significant contributor to this inefficiency is the induction of cell death, particularly in cells that lack attachment to the extracellular matrix (ECM)
      The abbreviations used are: ECM
      extracellular matrix
      DAPI
      4,6-diamidino-2-phenylindole
      DNECAD
      dominant-negative E-cadherin
      EMT
      epithelial-mesenchymal transition
      MET
      mesenchymal to epithelial transition
      CTC
      circulating tumor cell
      MC
      methylcellulose
      EGFR
      epidermal growth factor receptor.
      (
      • Mehlen P.
      • Puisieux A.
      Metastasis: a question of life or death.
      ). Caspase-dependent programmed cell death that is caused by ECM-detachment is known as anoikis, and anoikis resistance is an important factor in determining the success of cancer cells in navigating the metastatic cascade (
      • Frisch S.M.
      • Francis H.
      Disruption of epithelial cell-matrix interactions induces apoptosis.
      ,
      • Frisch S.M.
      • Screaton R.A.
      Anoikis mechanisms.
      ). In addition to anoikis, recent studies have discovered multiple, distinct cellular alterations that can impact the survival of ECM-detached cancer cells in an anoikis-independent fashion, suggesting that cancer cells may need to utilize a multifaceted approach to survive in the absence of ECM-attachment (
      • Avivar-Valderas A.
      • Bobrovnikova-Marjon E.
      • Alan Diehl J.
      • Bardeesy N.
      • Debnath J.
      • Aguirre-Ghiso J.A.
      Regulation of autophagy during ECM detachment is linked to a selective inhibition of mTORC1 by PERK.
      • Avivar-Valderas A.
      • Salas E.
      • Bobrovnikova-Marjon E.
      • Diehl J.A.
      • Nagi C.
      • Debnath J.
      • Aguirre-Ghiso J.A.
      PERK integrates autophagy and oxidative stress responses to promote survival during extracellular matrix detachment.
      ,
      • Buchheit C.L.
      • Rayavarapu R.R.
      • Schafer Z.T.
      The regulation of cancer cell death and metabolism by extracellular matrix attachment.
      ,
      • Davison C.A.
      • Durbin S.M.
      • Thau M.R.
      • Zellmer V.R.
      • Chapman S.E.
      • Diener J.
      • Wathen C.
      • Leevy W.M.
      • Schafer Z.T.
      Antioxidant enzymes mediate survival of breast cancer cells deprived of extracellular matrix.
      ,
      • Fung C.
      • Lock R.
      • Gao S.
      • Salas E.
      • Debnath J.
      Induction of autophagy during extracellular matrix detachment promotes cell survival.
      ,
      • Schafer Z.T.
      • Grassian A.R.
      • Song L.
      • Jiang Z.
      • Gerhart-Hines Z.
      • Irie H.Y.
      • Gao S.
      • Puigserver P.
      • Brugge J.S.
      Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment.
      ,
      • Whelan K.A.
      • Caldwell S.A.
      • Shahriari K.S.
      • Jackson S.R.
      • Franchetti L.D.
      • Johannes G.J.
      • Reginato M.J.
      Hypoxia suppression of Bim and Bmf blocks anoikis and luminal clearing during mammary morphogenesis.
      • Buchheit C.L.
      • Weigel K.J.
      • Schafer Z.T.
      Cancer cell survival during detachment from the ECM: multiple barriers to tumour progression.
      ).
      The activation of oncogenic signaling in cancer cells is of paramount importance to developing anoikis resistance and to rectifying other cellular alterations that compromise cell viability in absence of ECM-attachment (
      • Buchheit C.L.
      • Rayavarapu R.R.
      • Schafer Z.T.
      The regulation of cancer cell death and metabolism by extracellular matrix attachment.
      ). In particular, overexpression of the ErbB2 oncogene has been linked to the survival of ECM-detached cells in a number of different contexts (
      • Schafer Z.T.
      • Grassian A.R.
      • Song L.
      • Jiang Z.
      • Gerhart-Hines Z.
      • Irie H.Y.
      • Gao S.
      • Puigserver P.
      • Brugge J.S.
      Antioxidant and oncogene rescue of metabolic defects caused by loss of matrix attachment.
      ,
      • Whelan K.A.
      • Caldwell S.A.
      • Shahriari K.S.
      • Jackson S.R.
      • Franchetti L.D.
      • Johannes G.J.
      • Reginato M.J.
      Hypoxia suppression of Bim and Bmf blocks anoikis and luminal clearing during mammary morphogenesis.
      ,
      • Grassian A.R.
      • Schafer Z.T.
      • Brugge J.S.
      ErbB2 stabilizes epidermal growth factor receptor (EGFR) expression via Erk and Sprouty2 in extracellular matrix-detached cells.
      ,
      • Haenssen K.K.
      • Caldwell S.A.
      • Shahriari K.S.
      • Jackson S.R.
      • Whelan K.A.
      • Klein-Szanto A.J.
      • Reginato M.J.
      ErbB2 requires integrin α5 for anoikis resistance via Src regulation of receptor activity in human mammary epithelial cells.
      • Buchheit C.L.
      • Angarola B.L.
      • Steiner A.
      • Weigel K.J.
      • Schafer Z.T.
      Anoikis evasion in inflammatory breast cancer cells is mediated by Bim-EL sequestration.
      ). While these studies have unveiled distinct mechanisms by which ErbB2 can promote anchorage-independent survival, it remains unclear if ErbB2 can promote survival through additional molecular mechanisms. Given the tremendous biological heterogeneity in ErbB2-positive breast tumors, it seems likely that the ability of ErbB2 to promote the survival of ECM-detached cells is not solely limited to the aforementioned studies (
      • Arteaga C.L.
      • Engelman J.A.
      ERBB receptors: from oncogene discovery to basic science to mechanism-based cancer therapeutics.
      ).
      Among the signaling pathways associated with ErbB2 that have yet to be investigated during the survival of ECM-detached cancer cells are those regulating cell-cell adhesion. Signaling from the ErbB2 receptor has been shown to impinge upon key molecules that determine the nature and efficacy of cell-cell contacts in a number of settings (
      • Higgins M.J.
      • Baselga J.
      Targeted therapies for breast cancer.
      ). In addition, circulating tumor cells (which lack normal attachment to ECM) have often been discovered as multicellular aggregates. This is particularly true in malignancies like inflammatory breast cancer and epithelial ovarian cancer (
      • Moss N.M.
      • Barbolina M.V.
      • Liu Y.
      • Sun L.
      • Munshi H.G.
      • Stack M.S.
      Ovarian cancer cell detachment and multicellular aggregate formation are regulated by membrane type 1 matrix metalloproteinase: a potential role in I.p. metastatic dissemination.
      ,
      • Robertson F.M.
      • Bondy M.
      • Yang W.
      • Yamauchi H.
      • Wiggins S.
      • Kamrudin S.
      • Krishnamurthy S.
      • Le-Petross H.
      • Bidaut L.
      • Player A.N.
      • Barsky S.H.
      • Woodward W.A.
      • Buchholz T.
      • Lucci A.
      • Ueno N.T.
      • Cristofanilli M.
      Inflammatory breast cancer: the disease, the biology, the treatment.
      ), which are cancers that are oftentimes driven by ErbB2-mediated signaling. Furthermore, the oncogene TrkB, which has been shown to stimulate survival signaling pathways (including those also downstream of ErbB2) in a fashion that blocks anoikis and promotes metastasis (
      • Douma S.
      • Van Laar T.
      • Zevenhoven J.
      • Meuwissen R.
      • Van Garderen E.
      • Peeper D.S.
      Suppression of anoikis and induction of metastasis by the neurotrophic receptor TrkB.
      ), can enhance multicellular aggregation during ECM-detachment (
      • Geiger T.R.
      • Peeper D.S.
      Critical role for TrkB kinase function in anoikis suppression, tumorigenesis, and metastasis.
      ).
      These data have motivated us to examine the relationship between ErbB2 and multicellular aggregation during ECM-detached conditions. In this study, we have discovered that ErbB2-induced multicellular aggregation is critical to the inhibition of anoikis. Interestingly, we have found that this multicellular aggregation during ECM-detachment promotes ErbB2/EGFR-mediated activation of ERK/MAPK by preventing EGFR from being internalized and trafficked to the lysosome. Furthermore, our data suggest that ErbB2 and E-cadherin-expressing cancer cells may be uniquely susceptible to therapies antagonizing EGFR activity during ECM-detachment.

      DISCUSSION

      The evasion of anoikis by cancer cells is of paramount importance to their ability to successfully metastasize to distant sites. A better understanding of the molecular mechanisms and signal transduction pathways underlying anoikis evasion in cancer cells could potentially lead to the development of novel therapeutics that eliminate ECM-detached cancer cells. Here, we demonstrate that ErbB2-expressing breast cancer cells form large, multicellular aggregates when detached from the ECM and that these aggregates play a critical role in anoikis evasion (see model in Fig. 8). While this study focuses specifically on aggregate formation during ECM-detachment in ErbB2-positive cells, our data raise the possibility that signaling that resonates from other oncogenes or in other cancers may also promote aggregate formation and anoikis evasion during ECM-detachment. Indeed, previous research in squamous cell carcinoma cells characterized a process known as “synoikis” whereby cell-cell contacts facilitate survival through the elevation of Bcl-2 protein (
      • Shen X.
      • Kramer R.H.
      Adhesion-mediated squamous cell carcinoma survival through ligand-independent activation of epidermal growth factor receptor.
      ). That being said, evidence seems to suggest that the mechanism described in our study is specific to ErbB2-expressing cells. For example, in Fig. 4D, we show that cells engineered to have constitutive activation of ERK (via the expression of MEKDD) do not induce anoikis upon disruption of aggregation, suggesting that when ERK signaling is sustained by means other than ErbB2 overexpression, multicellular aggregation is not protective against anoikis. Given that our study suggests that stabilization of EGFR (potentially due to complex formation with ErbB2 and E-cadherin) is necessary for aggregation-induced anoikis evasion, it might be the case that ErbB2 expression is necessary for aggregation to be cytoprotective during ECM-detachment in breast cancer cells. Alternatively, other types of oncogenic signaling may stabilize EGFR through other mechanisms.
      Figure thumbnail gr8
      FIGURE 8Model for aggregate-mediated EGFR stabilization and anoikis suppression in ECM-detached ErbB2-overexpressing cells. A, model for aggregate-mediated ECM-detached survival in ErbB2-overexpressing cells. B, model for anoikis induction upon aggregate disruption in ECM-detached ErbB2-overexpressing cells.
      The results presented here in our study also clearly delineate a role for E-cadherin-mediated cell-cell adhesion in protection from anoikis in ErbB2-expressing cells. One aspect of these data that we find particularly intriguing is that multiple types of cancer cells are well known to lose E-cadherin during an epithelial-mesenchymal transition (EMT), which facilitates migration and metastasis (
      • Lamouille S.
      • Xu J.
      • Derynck R.
      Molecular mechanisms of epithelial-mesenchymal transition.
      ). In fact, EMT has itself been directly linked to the evasion of anoikis through eloquent studies involving the protein NRAGE (
      • Kumar S.
      • Park S.H.
      • Cieply B.
      • Schupp J.
      • Killiam E.
      • Zhang F.
      • Rimm D.L.
      • Frisch S.M.
      A pathway for the control of anoikis sensitivity by E-cadherin and epithelial-to-mesenchymal transition.
      ). Therefore, how do we reconcile the data from this study implicating E-cadherin in anoikis protection with the fact that E-cadherin is often lost during tumor progression? Recent studies have revealed that while EMT does often occur during the early stages of metastasis, a re-acquisition of E-cadherin through a mesenchymal to epithelial transition (MET) can be critical for successful metastasis (
      • Ocaña O.H.
      • Córcoles R.
      • Fabra A.
      • Moreno-Bueno G.
      • Acloque H.
      • Vega S.
      • Barrallo-Gimeno A.
      • Cano A.
      • Nieto M.A.
      Metastatic colonization requires the repression of the epithelial-mesenchymal transition inducer Prrx1.
      ,
      • Tsai J.H.
      • Donaher J.L.
      • Murphy D.A.
      • Chau S.
      • Yang J.
      Spatiotemporal regulation of epithelial-mesenchymal transition is essential for squamous cell carcinoma metastasis.
      • Chao Y.L.
      • Shepard C.R.
      • Wells A.
      Breast carcinoma cells re-express E-cadherin during mesenchymal to epithelial reverting transition.
      ). These data raise the possibility that an MET (following an EMT) could facilitate aggregation-induced anoikis evasion at later stages of the metastatic cascade when disseminated cancer cells need to survive in foreign matrix environments. Furthermore, other studies have unequivocally demonstrated the presence of E-cadherin in a percentage of both primary breast tumors and distant metastases (
      • Kowalski P.J.
      • Rubin M.A.
      • Kleer C.G.
      E-cadherin expression in primary carcinomas of the breast and its distant metastases.
      ) and a correlation between cellular aggregation and successful metastasis was first published over 50 years ago (
      • Fidler I.J.
      The relationship of embolic homogeneity, number, size and viability to the incidence of experimental metastasis.
      ,
      • Watanabe S.
      The metastasizability of tumor cells.
      ). More recent studies examining circulating tumor cells (CTCs) have provided evidence that these cells often exist in the bloodstream as multicellular aggregates (
      • Stott S.L.
      • Hsu C.H.
      • Tsukrov D.I.
      • Yu M.
      • Miyamoto D.T.
      • Waltman B.A.
      • Rothenberg S.M.
      • Shah A.M.
      • Smas M.E.
      • Korir G.K.
      • Floyd Jr., F.P.
      • Gilman A.J.
      • Lord J.B.
      • Winokur D.
      • Springer S.
      • Irimia D.
      • Nagrath S.
      • Sequist L.V.
      • Lee R.J.
      • Isselbacher K.J.
      • Maheswaran S.
      • Haber D.A.
      • Toner M.
      Isolation of circulating tumor cells using a microvortex-generating herringbone-chip.
      ,
      • Fernandez S.V.
      • Robertson F.M.
      • Pei J.
      • Aburto-Chumpitaz L.
      • Mu Z.
      • Chu K.
      • Alpaugh R.K.
      • Huang Y.
      • Cao Y.
      • Ye Z.
      • Cai K.Q.
      • Boley K.M.
      • Klein-Szanto A.J.
      • Devarajan K.
      • Addya S.
      • Cristofanilli M.
      Inflammatory breast cancer (IBC): clues for targeted therapies.
      ,
      • Yu M.
      • Stott S.
      • Toner M.
      • Maheswaran S.
      • Haber D.A.
      Circulating tumor cells: approaches to isolation and characterization.
      ). Our data may help explain the significance of these findings in that aggregation may occur in CTCs and may facilitate metastasis through the inhibition of anoikis.
      In addition to our data demonstrating a role for multicellular aggregation in blocking anoikis, our study has also uncovered a unique vulnerability of ErbB2-expressing cells: the inhibition of EGFR during ECM-detachment (see Fig. 7). Numerous studies have demonstrated that EGFR inhibition is not an effective strategy for patients with ErbB2-positive breast cancer (
      • Yonesaka K.
      • Zejnullahu K.
      • Okamoto I.
      • Satoh T.
      • Cappuzzo F.
      • Souglakos J.
      • Ercan D.
      • Rogers A.
      • Roncalli M.
      • Takeda M.
      • Fujisaka Y.
      • Philips J.
      • Shimizu T.
      • Maenishi O.
      • Cho Y.
      • Sun J.
      • Destro A.
      • Taira K.
      • Takeda K.
      • Okabe T.
      • Swanson J.
      • Itoh H.
      • Takada M.
      • Lifshits E.
      • Okuno K.
      • Engelman J.A.
      • Shivdasani R.A.
      • Nishio K.
      • Fukuoka M.
      • Varella-Garcia M.
      • Nakagawa K.
      • Jänne P.A.
      Activation of ERBB2 signaling causes resistance to the EGFR-directed therapeutic antibody cetuximab.
      ,
      • Wang S.E.
      • Narasanna A.
      • Perez-Torres M.
      • Xiang B.
      • Wu F.Y.
      • Yang S.
      • Carpenter G.
      • Gazdar A.F.
      • Muthuswamy S.K.
      • Arteaga C.L.
      HER2 kinase domain mutation results in constitutive phosphorylation and activation of HER2 and EGFR and resistance to EGFR tyrosine kinase inhibitors.
      ). Our data suggest that while EGFR inhibition is not effective in ECM-attached cells, ECM-detached cells are sensitive to EGFR inhibitors. We postulate that this sensitivity is due to the reliance of ECM-detached cells on aggregation/E-cadherin-induced activation of EGFR for sustained ERK signaling. Indeed, in other tumors that rely heavily on EGFR signaling (e.g. NSCLC), E-cadherin expression can enhance the sensitivity of cancer cells to EGFR inhibitors (
      • Witta S.E.
      • Gemmill R.M.
      • Hirsch F.R.
      • Coldren C.D.
      • Hedman K.
      • Ravdel L.
      • Helfrich B.
      • Dziadziuszko R.
      • Chan D.C.
      • Sugita M.
      • Chan Z.
      • Baron A.
      • Franklin W.
      • Drabkin H.A.
      • Girard L.
      • Gazdar A.F.
      • Minna J.D.
      • Bunn Jr., P.A.
      Restoring E-cadherin expression increases sensitivity to epidermal growth factor receptor inhibitors in lung cancer cell lines.
      ). Therefore, it seems possible that EGFR inhibition may be helpful in eliminating ECM-detached, ErbB2-positive breast cancer cells, while alternative approaches to antagonize ERK signaling may be necessary to eliminate ErbB2-positive, ECM-attached cells.

      Acknowledgments

      We thank Cassandra Buchheit, Kelsey Weigel, Joshua Mason, Amy Leliaert, Kelsea Hosoda, Veronica Schafer, and the rest of the Schafer laboratory for helpful comments and discussion. We thank Lisa Checkley and Michael Ferdig at Notre Dame for providing chloroquine. Additionally, we thank Mary Ann McDowell at Notre Dame for the use of the Shandon cytospin3 and Michael Overholtzer at Memorial Sloan-Kettering Cancer Center for the E-cadherin-positive SKBR3 cells. We thank Kimbra Turner at the University of Notre Dame for help with figure design, layout, and formatting.

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