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The tetraspanin superfamily member NET-6 is a new tumor suppressor gene

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Abstract

Purpose

NET-6 is a largely uncharacterized member of the tetraspanin superfamily. We have recently shown that its expression level was lowest in breast carcinomas with aggressive characteristics. We now describe the phenotypic and molecular changes induced in MDA-MB-231 breast carcinoma cells by ectopic NET-6 expression.

Methods

A GFP-NET-6 construct was transfected into very low expressing MDA-MB-231 cells. The subcellular distribution was studied by confocal microscopy. Cell proliferation in vitro was measured by MTT assay. Subcutaneous tumor formation in SCID mice was also studied. Other phenotypic parameters measured included growth in soft agar and extracellular matrix invasion. The effect of NET-6 transfection on the cell cycle was interrogated by flow cytometry. Deregulation of metalloproteinase expression was investigated by RT-PCR. Deregulation of proteins involved in apoptosis and cell cycle control was studied by Western blotting.

Results

Ectopic expression of NET-6 inhibited anchorage independent growth and invasion in a Boyden chamber assay. These effects were associated with downregulation of the matrix metalloproteinases MMP-1 and MMP-3. NET-6 had marked antiproliferative activity, both in vitro and in SCID mice. This effect was largely due to increased apoptosis. We identified upregulation of the pro-apoptotic molecules p53, bax, bak and caspase 3.

Conclusions

Our data provide novel and compelling evidence that NET-6 is a potent new breast cancer suppressor gene.

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References

  • Berditchevski F, Odintsova E (1999) Characterization of integrin-tetraspanin adhesion complexes: role of tetraspanins in integrin signaling. J Cell Biol 146:477–492

    Article  PubMed  CAS  Google Scholar 

  • Berditchevski F, Odintsova E, Sawada S, Gilbert E (2002) Expression of the palmitoylation-deficient CD151 weakens the association of alpha 3 beta 1 integrin with the tetraspanin-enriched microdomains and affects integrin-dependent signaling. J Biol Chem 277:36991–37000

    Article  PubMed  CAS  Google Scholar 

  • Carloni V, Mazzocca A, Ravichandran KS (2004) Tetraspanin CD81 is linked to ERK/MAPKinase signaling by Shc in liver tumor cells. Oncogene 23:1566–1574

    Article  PubMed  CAS  Google Scholar 

  • Chattopadhyay N, Wang Z, Ashman LK, Brady-Kalnay SM, Kreidberg JA (2003) Alpha 3 beta1 integrin-CD151, a component of the cadherin-catenin complex, regulates PTPmu expression and cell–cell adhesion. J Cell Biol 163:1351–1362

    Article  PubMed  CAS  Google Scholar 

  • Cherukuri A, Shoham T, Sohn HW, Levy S, Brooks S, Carter R, Pierce SK (2004) The tetraspanin CD81 is necessary for partitioning of coligated CD19/CD21-B cell antigen receptor complexes into signaling-active lipid rafts. J Immunol 172:370–380

    PubMed  CAS  Google Scholar 

  • Dong JT, Lamb PW, Rinker-Schaeffer CW, Vukanovic J, Ichikawa T, Isaacs JT, Barrett JC (1995) KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2. Science 268:884–886

    Article  PubMed  CAS  Google Scholar 

  • Furuya M, Kato H, Nishimura N, Ishiwata I, Ikeda H, Ito R, Yoshiki T, Ishikura H (2005) Down-regulation of CD9 in human ovarian carcinoma cell might contribute to peritoneal dissemination: morphologic alteration and reduced expression of beta1 integrin subsets. Cancer Res 65:2617–2625

    Article  PubMed  CAS  Google Scholar 

  • Geradts J, Maynard R, Birrer MJ, Hendricks D, Abbondanzo SL, Fong KM, Barrett JC, Lombardi DP (1999) Frequent loss of KAI1 expression in squamous and lymphoid neoplasms. An immunohistochemical study of archival tissues. Am J Pathol 154:1665–1671

    PubMed  CAS  Google Scholar 

  • Huang CI, Kohno N, Ogawa E, Adachi M, Taki T, Miyake M (1998) Correlation of reduction in MRP-1/CD9 and KAI1/CD82 expression with recurrences in breast cancer patients. Am J Pathol 153:973–983

    PubMed  CAS  Google Scholar 

  • Huang CL, Liu D, Masuya D, Kameyama K, Nakashima T, Yokomise H, Ueno M, Miyake M (2004) MRP-1/CD9 gene transduction downregulates Wnt signal pathways. Oncogene 23:7475–7483

    Article  PubMed  CAS  Google Scholar 

  • Huang H, Groth J, Sossey-Alaoui K, Hawthorn L, Beall S, Geradts J (2005) Aberrant expression of novel and previously described cell membrane markers in human breast cancer cell lines and tumors. Clin Cancer Res 11:4357–4364

    Article  PubMed  CAS  Google Scholar 

  • Itahana Y, Singh J, Sumida T, Coppe JP, Parrinello S, Bennington JL, Desprez PY (2003) Role of Id-2 in the maintenance of a differentiated and noninvasive phenotype in breast cancer cells. Cancer Res 63:7098–7105

    PubMed  CAS  Google Scholar 

  • Jang HI, Lee H (2003) A decrease in the expression of CD63 tetraspanin protein elevates invasive potential of human melanoma cells. Exp Mol Med 35:317–323

    PubMed  CAS  Google Scholar 

  • Johnson MR, Wand K, Smith JB, Heslin MJ, Diasio RB (2000) Quantitation of dihydropyrimidine dehydrogenase expression by real-time reverse transcription polymerase chain reaction. Anal Biochem 278:175–184

    Article  PubMed  CAS  Google Scholar 

  • Lee M, Hadi M, Hallden G, Aponte GW (2005) Peptide YY and neuropeptide Y induce willin expression, reduce adhesion, and enhance migration in small intestinal cells through the regulation of CD63, matrix metalloproteinase-3, and Cdc42 activity. J Biol Chem 280:125–136

    PubMed  CAS  Google Scholar 

  • Lombardi DP, Geradts J, Foley JF, Chiao C, Lamb PW, Barrett JC (1999) Loss of KAI1 expression in the progression of colorectal cancer. Cancer Res 59:5724–5731

    PubMed  CAS  Google Scholar 

  • Mazzocca A, Sciammetta SC, Carloni V, Cosmi L, Annunziato F, Harada T, Abrignani S, Pinzani M (2005) Binding of hepatitis C virus envelope protein E2 to CD81 up-regulates matrix metalloproteinase-2 in human hepatic stellate cells. J Biol Chem 280:11329–11339

    Article  PubMed  CAS  Google Scholar 

  • Murayama Y, Miyagawa J, Oritani K, Yoshida H, Yamamoto K, Kishida O, Miyazaki T, Tsutsui S, Kiyohara T, Miyazaki Y, Higashiyama S, Matsuzawa Y, Shinomura Y (2004) CD9-mediated activation of the p46 Shc isoform leads to apoptosis in cancer cells. J Cell Sci 117:3379–3388

    Article  PubMed  CAS  Google Scholar 

  • Sauer G, Kurzeder C, Grundmann R, Kreienberg R, Zeillinger R, Deissler H (2003) Expression of tetraspanin adaptor proteins below defined threshold values is associated with in vitro invasiveness of mammary carcinoma cells. Oncol Rep 10:405–410

    PubMed  CAS  Google Scholar 

  • Serru V, Dessen P, Boucheix C, Rubinstein E (2000) Sequence and expression of seven new tetraspans. Biochim Biophys Acta 1478:159–163

    PubMed  CAS  Google Scholar 

  • Shinohara T, Miki T, Nishimura N, Nokihara H, Hamada H, Mukaida N, Sone S (2001) Nuclear factor-κB-dependent expression of metastasis suppressor KAI1/CD82 gene in lung cancer cell lines expressing p53. Cancer Res 61:673–678

    PubMed  CAS  Google Scholar 

  • Sossey-Alaoui K, Vieira L, David D, Boavida MG, Cowell JK (2003) Molecular characterization of a 7p15-21 homozygous deletion in a Wilms tumor. Genes Chromosomes Cancer 36:1–6

    Article  PubMed  CAS  Google Scholar 

  • Sugiura T, Berditchevski F (1999) Function of alpha3beta1-tetraspanin protein complexes in tumor cell invasion. Evidence for the role of the complexes in production of matrix metalloproteinase 2 (MMP-2). J Cell Biol 146:1375–1389

    Article  PubMed  CAS  Google Scholar 

  • Takino T, Miyamori H, Kawaguchi N, Uekita T, Seiki M, Sato H (2003) Tetraspanin CD63 promotes targeting and lysosomal proteolysis of membrane-type 1 matrix metalloproteinase. Biochem Biophys Res Commun 304:160–166

    Article  PubMed  CAS  Google Scholar 

  • Todd SC, Doctor VS, Levy S (1998) Sequences and expression of six new members of the tetraspanin/TM4SF family. Biochim Biophys Acta 1399:101–104

    PubMed  CAS  Google Scholar 

  • van de Loosdrecht AA, Beelen RH, Ossenkoppele GJ, Broekhoven MG, Langenhuijsen MM (1994) A tetrazolium-based colorimetric MTT assay to quantitate human monocyte mediated cytotoxicity against leukemic cells from cell lines and patients with acute myeloid leukemia. J Immunol Methods 174:311–320

    Article  PubMed  Google Scholar 

  • Wilson KS, Roberts H, Leek R, Harris AL, Geradts J (2002) Differential gene expression patterns in HER2/neu-positive and -negative breast cancer cell lines and tissues. Am J Pathol 161:1171–1185

    PubMed  CAS  Google Scholar 

  • Yang X, Wei LL, Tang C, Slack R, Mueller S, Lippman ME (2001) Overexpression of KAI1 suppresses in vitro invasiveness and in vivo metastasis in breast cancer cells. Cancer Res 61:5284–5288

    PubMed  CAS  Google Scholar 

  • Zhang XA, Bontrager AL, Hemler ME (2001) Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta (1) integrins. J Biol Chem 276:25005–25013

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was supported by NCI grant 5R21CA098842 to KSA, and by the NCI-funded Cancer Center Support Grant P30CA16056. We thank Dr. Fengzhi Li who generously provided to us the anti-survivin polyclonal antibody and Dr. Candace Johnson for providing us with antibodies against the apoptotic pathway proteins. We also thank Earl Timm and Joseph Tario for assisting with the flow cytometry experiments and Edward Hurley for his help with confocal microscopy.

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Authors and Affiliations

  1. Department of Pathology, Roswell Park Cancer Institute, Buffalo, NY, 14263, USA

    Huayi Huang

  2. Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, 14263, USA

    Khalid Sossey-Alaoui

  3. Department of Molecular and Cellular Biophysics and Biochemistry, Roswell Park Cancer Institute, Buffalo, NY, 14263, USA

    Sarah H. Beachy

  4. Department of Pathology, Duke University Medical Center, DUMC 3712, Durham, NC, 27710, USA

    Joseph Geradts

Authors
  1. Huayi Huang
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  2. Khalid Sossey-Alaoui
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  3. Sarah H. Beachy
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  4. Joseph Geradts
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Corresponding author

Correspondence to Joseph Geradts.

Additional information

Huayi Huang and Khalid Sossey-Alaoui made comparable contributions to this work.

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Huang, H., Sossey-Alaoui, K., Beachy, S.H. et al. The tetraspanin superfamily member NET-6 is a new tumor suppressor gene. J Cancer Res Clin Oncol 133, 761–769 (2007). https://doi.org/10.1007/s00432-007-0221-1

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  • DOI: https://doi.org/10.1007/s00432-007-0221-1

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