Frontiers of Medicine

, Volume 10, Issue 1, pp 41–51 | Cite as

Midline2 is overexpressed and a prognostic indicator in human breast cancer and promotes breast cancer cell proliferation in vitro and in vivo

  • Lan Wang
  • Jueheng Wu
  • Jie Yuan
  • Xun Zhu
  • Hongmei Wu
  • Mengfeng LiEmail author
Research Article


Midline2 (MID2) is an ubiquitin-conjugating E2 enzyme linked to tumor progression and a novel interacting partner of breast cancer 1, early-onset (BRCA1). However, the role of MID2 in breast cancer remains unknown. This study investigated the expression, prognostic value, and role of MID2 in breast cancer. The expression of MID2 mRNA and protein was significantly upregulated in breast cancer tissue and established cell lines compared with that in normal breast epithelial cells and paired adjacent non-tumor tissue (P < 0.001). Immunohistochemical analysis demonstrated that MID2 was overexpressed in 272 of 284 (95.8%) paraffinembedded, archived breast cancer tissue. Moreover, MID2 expression increased with advanced clinical stage (P < 0.001). High MID2 expression was significantly associated with advanced clinical stages and T, N, and M staging (all P < 0.05). Univariate and multivariate analyses indicated that high MID2 expression was an independent prognostic factor for poor overall survival in the entire cohort (93.73 vs. 172.1 months; P < 0.001, logrank test) and in subgroups with stages Tis + I + II and III + IV. Furthermore, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide colony formation, and anchorage-independent growth ability assays were conducted. Results showed that siRNA silencing of MID2 expression significantly reduced MCF-7 and MDA-MB-231 cell proliferation in vitro and blocked the growth of MDA-MB-231 cell xenograft tumors in vivo (P < 0.05). This study indicated that MID2 may be a novel prognostic marker and interventional target in breast cancer.


breast cancer MID2 proliferation overall survival xenograft 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. 1.
    Abrams J, Kramer B, Doroshow JH, Varmus H. National Cancer Institute-supported clinical trials networks. J Clin Oncol 2015; 33(3): 293CrossRefPubMedGoogle Scholar
  2. 2.
    Kulkarni KP. In the realms of future: new frontiers of ‘technooncology’ as a platform for global improvement in the outcomes of childhood cancer. Expert Rev Hematol 2015; 8(1): 1–4CrossRefPubMedGoogle Scholar
  3. 3.
    Banday AH, Jeelani S, Hruby VJ. Cancer vaccine adjuvants—recent clinical progress and future perspectives. Immunopharmacol Immunotoxicol 2015; 37(1): 1–11CrossRefPubMedGoogle Scholar
  4. 4.
    Milanezi F, Carvalho S, Schmitt FC. EGFR/HER2 in breast cancer: a biological approach for molecular diagnosis and therapy. Expert Rev Mol Diagn 2008; 8(4): 417–434CrossRefPubMedGoogle Scholar
  5. 5.
    Pellisé M, Castells A, Ginès A, Solé M, Mora J, Castellví-Bel S, Rodríguez-Moranta F, Fernàndez-Esparrach G, Llach J, Bordas JM, Navarro S, Piqué JM. Clinical usefulness of KRAS mutational analysis in the diagnosis of pancreatic adenocarcinoma by means of endosonography-guided fine-needle aspiration biopsy. Aliment Pharmacol Ther 2003; 17(10): 1299–1307CrossRefPubMedGoogle Scholar
  6. 6.
    Colombo M, Corsi F, Foschi D, Mazzantini E, Mazzucchelli S, Morasso C, Occhipinti E, Polito L, Prosperi D, Ronchi S, Verderio P. HER2 targeting as a two-sided strategy for breast cancer diagnosis and treatment: outlook and recent implications in nanomedical approaches. Pharmacol Res 2010; 62(2): 150–165CrossRefPubMedGoogle Scholar
  7. 7.
    American College of Chest Physicians. Diagnosis and management of lung cancer: ACCP evidence-based guidelines. Chest 2003; 123: D-G, 1S–337SGoogle Scholar
  8. 8.
    Grabiec M, Nowicki P, Walentowicz M, Grezlikowska U, Mierzwa T, Chmielewska W. Role of Ca-125 in the differential diagnosis of adnexal mass in breast cancer patients. Ginekol Pol 2005; 76(5): 371–376 (in Polish)PubMedGoogle Scholar
  9. 9.
    Katai H, Sano T. Early gastric cancer: concepts, diagnosis, and management. Int J Clin Oncol 2005; 10(6): 375–383CrossRefPubMedGoogle Scholar
  10. 10.
    Li J, Yang L, Song L, Xiong H, Wang L, Yan X, Yuan J, Wu J, Li M. Astrocyte elevated gene-1 is a proliferation promoter in breast cancer via suppressing transcriptional factor FOXO1. Oncogene 2009; 28(36): 3188–3196CrossRefPubMedGoogle Scholar
  11. 11.
    Li J, Guan HY, Gong LY, Song LB, Zhang N, Wu J, Yuan J, Zheng YJ, Huang ZS, Li M. Clinical significance of sphingosine kinase-1 expression in human astrocytomas progression and overall patient survival. Clin Cancer Res 2008; 14(21): 6996–7003CrossRefPubMedGoogle Scholar
  12. 12.
    Joseph P, Lei YX, Whong WZ, Ong TM. Oncogenic potential of mouse translation elongation factor-1 d, a novel cadmiumresponsive proto-oncogene. J Biol Chem 2002; 277(8): 6131–6136CrossRefPubMedGoogle Scholar
  13. 13.
    Li LB, Louie MC, Chen HW, Zou JX. Proto-oncogene ACTR/AIB1 promotes cancer cell invasion by up-regulating specific matrix metalloproteinase expression. Cancer Lett 2008; 261(1): 64–73CrossRefPubMedGoogle Scholar
  14. 14.
    Liu Z, Lu H, Shi H, Du Y, Yu J, Gu S, Chen X, Liu KJ, Hu CA. PUMA overexpression induces reactive oxygen species generation and proteasome-mediated stathmin degradation in colorectal cancer cells. Cancer Res 2005; 65(5): 1647–1654CrossRefPubMedGoogle Scholar
  15. 15.
    Peng HM, Morishima Y, Jenkins GJ, Dunbar AY, Lau M, Patterson C, Pratt WB, Osawa Y. Ubiquitylation of neuronal nitric-oxide synthase by CHIP, a chaperone-dependent E3 ligase. J Biol Chem 2004; 279(51): 52970–52977CrossRefPubMedGoogle Scholar
  16. 16.
    van Wijk SJ, de Vries SJ, Kemmeren P, Huang A, Boelens R, Bonvin AM, Timmers HT. A comprehensive framework of E2- RING E3 interactions of the human ubiquitin-proteasome system. Mol Syst Biol 2009; 5: 295PubMedPubMedCentralGoogle Scholar
  17. 17.
    Wang Y, Ren F, Wang Y, Feng Y, Wang D, Jia B, Qiu Y, Wang S, Yu J, Sung JJ, Xu J, Zeps N, Chang Z. CHIP/Stub1 functions as a tumor suppressor and represses NF-kB-mediated signaling in colorectal cancer. Carcinogenesis 2014; 35(5): 983–991CrossRefPubMedGoogle Scholar
  18. 18.
    Wosnitzer MS, Mielnik A, Dabaja A, Robinson B, Schlegel PN, Paduch DA. Ubiquitin Specific Protease 26 (USP26) expression analysis in human testicular and extragonadal tissues indicates diverse action of USP26 in cell differentiation and tumorigenesis. PLoS ONE 2014; 9(6): e98638CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Perez EA, Dueck AC, McCullough AE, Chen B, Geiger XJ, Jenkins RB, Lingle WL, Davidson NE, Martino S, Kaufman PA, Kutteh LA, Sledge GW, Harris LN, Gralow JR, Reinholz MM. Impact of PTEN protein expression on benefit from adjuvant trastuzumab in earlystage human epidermal growth factor receptor 2-positive breast cancer in the North Central Cancer Treatment Group N9831 trial. J Clin Oncol 2013; 31(17): 2115–2122CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Napolitano LM, Jaffray EG, Hay RT, Meroni G. Functional interactions between ubiquitin E2 enzymes and TRIM proteins. Biochem J 2011; 434(2): 309–319CrossRefPubMedGoogle Scholar
  21. 21.
    Vichi A, Payne DM, Pacheco-Rodriguez G, Moss J, Vaughan M. E3 ubiquitin ligase activity of the trifunctional ARD1 (ADP-ribosylation factor domain protein 1). Proc Natl Acad Sci USA 2005; 102(6): 1945–1950CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Trockenbacher A, Suckow V, Foerster J, Winter J, Krauss S, Ropers HH, Schneider R, Schweiger S. MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation. Nat Genet 2001; 29(3): 287–294CrossRefPubMedGoogle Scholar
  23. 23.
    Urano T, Saito T, Tsukui T, Fujita M, Hosoi T, Muramatsu M, Ouchi Y, Inoue S. Efp targets 14-3-3 sigma for proteolysis and promotes breast tumour growth. Nature 2002; 417(6891): 871–875CrossRefPubMedGoogle Scholar
  24. 24.
    Niikura T, Hashimoto Y, Tajima H, Ishizaka M, Yamagishi Y, Kawasumi M, Nawa M, Terashita K, Aiso S, Nishimoto I. A tripartite motif protein TRIM11 binds and destabilizes Humanin, a neuroprotective peptide against Alzheimer’s disease-relevant insults. Eur J Neurosci 2003; 17(6): 1150–1158CrossRefPubMedGoogle Scholar
  25. 25.
    Horn EJ, Albor A, Liu Y, El-Hizawi S, Vanderbeek GE, Babcock M, Bowden GT, Hennings H, Lozano G, Weinberg WC, Kulesz-Martin M. RING protein Trim32 associated with skin carcinogenesis has anti-apoptotic and E3-ubiquitin ligase properties. Carcinogenesis 2004; 25(2): 157–167CrossRefPubMedGoogle Scholar
  26. 26.
    Wada K, Kamitani T. Autoantigen Ro52 is an E3 ubiquitin ligase. Biochem Biophys Res Commun 2006; 339(1): 415–421CrossRefPubMedGoogle Scholar
  27. 27.
    Reymond A, Meroni G, Fantozzi A, Merla G, Cairo S, Luzi L, Riganelli D, Zanaria E, Messali S, Cainarca S, Guffanti A, Minucci S, Pelicci PG, Ballabio A. The tripartite motif family identifies cell compartments. EMBO J 2001; 20(9): 2140–2151CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Liu C, Huang X, Hou S, Hu B, Li H. Silencing of tripartite motif (TRIM) 29 inhibits proliferation and invasion and increases chemosensitivity to cisplatin in human lung squamous cancer NCI-H520 cells. Thorac Cancer 2015; 6(1): 31–37CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Short KM, Cox TC. Subclassification of the RBCC/TRIM superfamily reveals a novel motif necessary for microtubule binding. J Biol Chem 2006; 281(13): 8970–8980CrossRefPubMedGoogle Scholar
  30. 30.
    Yap MW, Nisole S, Lynch C, Stoye JP. Trim5a protein restricts both HIV-1 and murine leukemia virus. Proc Natl Acad Sci USA 2004; 101(29): 10786–10791CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Jehee FS, Rosenberg C, Krepischi-Santos AC, Kok F, Knijnenburg J, Froyen G, Vianna-Morgante AM, Opitz JM, Passos-Bueno MR. An Xq22.3 duplication detected by comparative genomic hybridization microarray (Array-CGH) defines a new locus (FGS5) for FG syndrome. Am J Med Genet A 2005; 139(3): 221–226CrossRefPubMedGoogle Scholar
  32. 32.
    Uchil PD, Hinz A, Siegel S, Coenen-Stass A, Pertel T, Luban J, Mothes W. TRIM protein-mediated regulation of inflammatory and innate immune signaling and its association with antiretroviral activity. J Virol 2013; 87(1): 257–272CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Corominas R, Yang X, Lin GN, Kang S, Shen Y, Ghamsari L, Broly M, Rodriguez M, Tam S, Trigg SA, Fan C, Yi S, Tasan M, Lemmens I, Kuang X, Zhao N, Malhotra D, Michaelson JJ, Vacic V, Calderwood MA, Roth FP, Tavernier J, Horvath S, Salehi-Ashtiani K, Korkin D, Sebat J, Hill DE, Hao T, Vidal M, Iakoucheva LM. Protein interaction network of alternatively spliced isoforms from brain links genetic risk factors for autism. Nat Commun 2014; 5: 3650CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Song L, Wang L, Li Y, Xiong H, Wu J, Li J, Li M. Sam68 upregulation correlates with, and its down-regulation inhibits, proliferation and tumourigenicity of breast cancer cells. J Pathol 2010; 222(3): 227–237CrossRefPubMedGoogle Scholar
  35. 35.
    Li J, Zhang N, Song LB, Liao WT, Jiang LL, Gong LY, Wu J, Yuan J, Zhang HZ, Zeng MS, Li M. Astrocyte elevated gene-1 is a novel prognostic marker for breast cancer progression and overall patient survival. Clin Cancer Res 2008; 14(11): 3319–3326CrossRefPubMedGoogle Scholar
  36. 36.
    Carey LA, Metzger R, Dees EC, Collichio F, Sartor CI, Ollila DW, Klauber-DeMore N, Halle J, Sawyer L, Moore DT, Graham ML. American Joint Committee on Cancer tumor-node-metastasis stage after neoadjuvant chemotherapy and breast cancer outcome. J Natl Cancer Inst 2005; 97(15): 1137–1142CrossRefPubMedGoogle Scholar
  37. 37.
    Zhang Z, Li J, Zheng H, Yu C, Chen J, Liu Z, Li M, Zeng M, Zhou F, Song L. Expression and cytoplasmic localization of SAM68 is a significant and independent prognostic marker for renal cell carcinoma. Cancer Epidemiol Biomarkers Prev 2009; 18(10): 2685–2693CrossRefPubMedGoogle Scholar
  38. 38.
    Wei P, Zhang N, Xu Y, Li X, Shi D, Wang Y, Li D, Cai S. TPX2 is a novel prognostic marker for the growth and metastasis of colon cancer. J Transl Med 2013; 11(1): 313CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Takata M, Yamanaka N, Tanaka T, Yamanaka J, Maeda S, Okamoto E, Yasojima H, Uematsu K, Watanabe H, Uragari Y. What patients can survive disease free after complete resection for hepatocellular carcinoma? A multivariate analysis. Jpn J Clin Oncol 2000; 30(2): 75–81CrossRefPubMedGoogle Scholar
  40. 40.
    Stuckey AR, Onstad MA. Hereditary breast cancer: an update on risk assessment and genetic testing in 2015. Am J Obstet Gynecol 2015; 213(2): 161–165CrossRefPubMedGoogle Scholar
  41. 41.
    Cainarca S, Messali S, Ballabio A, Meroni G. Functional characterization of the Opitz syndrome gene product (midin): evidence for homodimerization and association with microtubules throughout the cell cycle. Hum Mol Genet 1999; 8(8): 1387–1396CrossRefPubMedGoogle Scholar
  42. 42.
    Short KM, Hopwood B, Yi Z, Cox TC. MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders. BMC Cell Biol 2002; 3(1): 1CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Seshacharyulu P, Pandey P, Datta K, Batra SK. Phosphatase: PP2A structural importance, regulation and its aberrant expression in cancer. Cancer Lett 2013; 335(1): 9–18CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Colella S, Ohgaki H, Ruediger R, Yang F, Nakamura M, Fujisawa H, Kleihues P, Walter G. Reduced expression of the a subunit of protein phosphatase 2A in human gliomas in the absence of mutations in the Aalpha and Abeta subunit genes. Int J Cancer 2001; 93(6): 798–804CrossRefPubMedGoogle Scholar
  45. 45.
    Liu W, Bagaitkar J, Watabe K. Roles of AKT signal in breast cancer. Front Biosci 2007; 12(8–12): 4011–4019CrossRefPubMedGoogle Scholar
  46. 46.
    Ostrakhovitch EA, Cherian MG. Role of p53 and reactive oxygen species in apoptotic response to copper and zinc in epithelial breast cancer cells. Apoptosis 2005; 10(1): 111–121CrossRefPubMedGoogle Scholar
  47. 47.
    Dueck AC, Reinholz MM, Geiger XJ, Tenner K, Ballman K, Jenkins RB, Riehle D, Chen B, McCullough AE, Davidson NE, Martino S, Sledge GW, Kaufman PA, Kutteh LA, Gralow J, Harris LN, Ingle JN, Lingle WL, Perez EA. Impact of c-MYC protein expression on outcome of patients with early-stage HER2+ breast cancer treated with adjuvant trastuzumab NCCTG (alliance) N9831. Clin Cancer Res 2013; 19(20): 5798–5807CrossRefPubMedPubMedCentralGoogle Scholar
  48. 48.
    Jiang G, Xiao X, Zeng Y, Nagabhushanam K, Majeed M, Xiao D. Targeting beta-catenin signaling to induce apoptosis in human breast cancer cells by z-guggulsterone and Gugulipid extract of Ayurvedic medicine plant Commiphora mukul. BMC Complement Altern Med 2013; 13(1): 203CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Nakshatri H, Goulet RJJr. NF-?B and breast cancer. Curr Probl Cancer 2002; 26(5): 282–309CrossRefPubMedGoogle Scholar
  50. 50.
    Shyamsunder P, Verma RS, Lyakhovich A. ROMO1 regulates RedOx states and serves as an inducer of NF-kB-driven EMT factors in Fanconi anemia. Cancer Lett 2015; 361(1): 33–38CrossRefPubMedGoogle Scholar
  51. 51.
    Hill SJ, Rolland T, Adelmant G, Xia X, Owen MS, Dricot A, Zack TI, Sahni N, Jacob Y, Hao T, McKinney KM, Clark AP, Reyon D, Tsai SQ, Joung JK, Beroukhim R, Marto JA, Vidal M, Gaudet S, Hill DE, Livingston DM. Systematic screening reveals a role for BRCA1 in the response to transcription-associated DNA damage. Genes Dev 2014; 28(17): 1957–1975CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Tu Z, Aird KM, Bitler BG, Nicodemus JP, Beeharry N, Xia B, Yen TJ, Zhang R. Oncogenic RAS regulates BRIP1 expression to induce dissociation of BRCA1 from chromatin, inhibit DNA repair, and promote senescence. Dev Cell 2011; 21(6): 1077–1091CrossRefPubMedPubMedCentralGoogle Scholar
  53. 53.
    Rytelewski M, Tong JG, Buensuceso A, Leong HS, Maleki Vareki S, Figueredo R, Di Cresce C, Wu SY, Herbrich SM, Baggerly KA, Romanow L, Shepherd T, Deroo BJ, Sood AK, Chambers AF, Vincent M, Ferguson PJ, Koropatnick J. BRCA2 inhibition enhances cisplatin-mediated alterations in tumor cell proliferation, metabolism, and metastasis. Mol Oncol 2014; 8(8): 1429–1440CrossRefPubMedGoogle Scholar
  54. 54.
    Pathania R, Ramachandran S, Elangovan S, Padia R, Yang P, Cinghu S, Veeranan-Karmegam R, Arjunan P, Gnana-Prakasam JP, Sadanand F, Pei L, Chang CS, Choi JH, Shi H, Manicassamy S, Prasad PD, Sharma S, Ganapathy V, Jothi R, Thangaraju M. DNMT1 is essential for mammary and cancer stem cell maintenance and tumorigenesis. Nat Commun 2015; 6: 6910CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Higher Education Press and Springer-Verlag Berlin Heidelberg 2016

Authors and Affiliations

  • Lan Wang
    • 1
  • Jueheng Wu
    • 3
  • Jie Yuan
    • 3
  • Xun Zhu
    • 2
    • 3
  • Hongmei Wu
    • 1
  • Mengfeng Li
    • 2
    • 3
    Email author
  1. 1.Department of Microbiology and Immunology, School of Basic CoursesGuangdong Pharmaceutical UniversityGuangzhouChina
  2. 2.Department of Microbiology, Zhongshan School of MedicineSun Yat-sen UniversityGuangzhouChina
  3. 3.Key Laboratory of Tropical Disease Control (Sun Yat-sen University), Ministry of EducationGuangzhouChina

Personalised recommendations