Genomic amplification and oncogenic properties of the GASC1 histone demethylase gene in breast cancer

G Liu, A Bollig-Fischer, B Kreike, M J van de Vijver, J Abrams, S P Ethier, Z-Q Yang, G Liu, A Bollig-Fischer, B Kreike, M J van de Vijver, J Abrams, S P Ethier, Z-Q Yang

Abstract

Earlier, mapping of the 9p23-24 amplicon in esophageal cancer cell lines led us to the positional cloning of gene amplified in squamous cell carcinoma 1 (GASC1), which encodes a nuclear protein with a Jumonji C domain that catalyzes lysine (K) demethylation of histones. However, the transforming roles of GASC1 in breast cancer remain to be determined. In this study, we identified GASC1 as one of the amplified genes for the 9p23-24 region in breast cancer, particularly in basal-like subtypes. The levels of GASC1 transcript expression were significantly higher in aggressive, basal-like breast cancers compared with nonbasal-like breast cancers. Our in vitro assays demonstrated that GASC1 induces transformed phenotypes, including growth factor-independent proliferation, anchorage-independent growth, altered morphogenesis in Matrigel, and mammosphere forming ability, when overexpressed in immortalized, nontransformed mammary epithelial MCF10A cells. Additionally, GASC1 demethylase activity regulates the expression of genes critical for stem cell self-renewal, including NOTCH1, and may be linked to the stem cell phenotypes in breast cancer. Thus, GASC1 is a driving oncogene in the 9p23-24 amplicon in human breast cancer and targeted inhibition of GASC1 histone demethylase in cancer could provide potential new avenues for therapeutic development.

Figures

Figure 1
Figure 1
Genomic analysis of the 9p23-24 region in breast cancer cell lines. (A) Genome view of chromosome 9 (left panel) and 9p23-24 region (right panel) analyzed on the Agilent oligonucleotide array (Agilent Technologies) in the SUM-149 breast cancer cell line. (B) FISH image of a GASC1 probe (RP11-46L22) hybridized to interphase nuclei of SUM-149 and HCC 1954 cells. (C) PCR analyses of genomic DNA obtained from SUM-149 cells and normal human DNA, with p16 and GAPDH primer controls.
Figure 2
Figure 2
Over expression of GASC1 at the RNA and protein levels in the breast cancer cells. (A) GASC1 mRNA expression was measured by semiquantitative real-time PCR in five breast cancer cell lines with or without 9p23-24 amplification compared to MCF10A cells. For the MCF10A cells, the baseline was set to 1. (B) GASC1 protein levels were analyzed by western blot in five breast cancer cell lines and MCF10A line. (C) Expression levels of GASC1 obtained from the gene expression dataset in basal-like and non basal-like breast cancer (Kreike et al., 2007). Bars indicate medians; P-values of Kruskal-Wallis test are provided.
Figure 3
Figure 3
Stable over expresses GASC1 in MCF10A cells with the pLenti6/V5-GASC1 construct (MCF10A-GASC1). Over expression of GASC1 mRNA and protein in this cell line was confirmed with (A) semiquantitative RT-PCR and (B) western blot assays. (C) In vitro growth rate of the MCF10A cells that stably over express GASC1 relative to MCF10A control cells in insulin deficient media. Cells were seeded into 35-mm culture wells and grown in the absence of insulin-like growth factors.
Figure 4
Figure 4
(A) Number and representative pictures of MCF10A-GASC1 and control cell soft-agar colonies. Cells were grown for 3–4 weeks in soft agar and stained with the vital dye p-iodonitrotetrazolium violet. (B) Effects of GASC1 on mammary acinar morphogenesis. MCF-10A-GASC1 and control cells were cultured on a bed of Matrigel as described in Materials and Methods. Top row: Representative bright-field images of acini taken on day 10. Bottom row: Representative images of structures with staining for actin with phalloidin conjugated to Alexa Fluor-568 (red), and DAPI as a marker of nuclei (blue).
Figure 5
Figure 5
(A) Mammosphere formation assay of MCF10A-GASC1 and MCF10A control cells. The top- panel shows representative images of mammospheres formed from MCF10A-GASC1 cells and MCF10A control cells on day 12. The bottom panel shows viable cells in the mammospheres adhered to the culture dish after replanting mammosphere culture into the attachment plate. (B) shRNA-mediated knock down of GASC1 inhibits colony formation in breast cancer cells with GASC1 amplification. The left row showed the TurboGFP fluorescence of pGIPZ shRNA in HCC1954 cells after 2 weeks. (C) Knock down of GASC1 with pLKO GASC1 shRNA inhibits cell growth in HCC1954 and Colo824 lines. HCC1954 and Colo824 cells for GASC1 knock down by pLKO shRNA and No-silencing control vector were seeded at 3×105 cells/well in six-well plates. After 10 days, cell counts were determined using a Coulter counter.
Figure 6
Figure 6
(A) Notch1 expression level in MCF10A-GASC1 and control cells was measured by semiquantitative RT-PCR in normal culture and mammosphere (Mamm.) culture conditions. The baseline for the MCF10A control cells was set to 1. (B) Notch1 in MCF10A and MCF10A-GASC1 cells was analyzed by Western blot. (C) Notch1 expression level was measured by semiquantitative RT-PCR in GASC1 knock down MCF10A and Colo824 cells. The baseline for the MCF10A cells with Non-silencing shRNA control was set to 1.

References

    1. Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW, Regev A, et al. An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors. Nat Genet. 2008;40:499–507.
    1. Cloos PA, Christensen J, Agger K, Maiolica A, Rappsilber J, Antal T, et al. The putative oncogene GASC1 demethylates tri- and dimethylated lysine 9 on histone H3. Nature. 2006;442:307–11.
    1. Debnath J, Muthuswamy SK, Brugge JS. Morphogenesis and oncogenesis of MCF-10A mammary epithelial acini grown in three-dimensional basement membrane cultures. Methods. 2003;30:256–68.
    1. Dontu G, Jackson KW, McNicholas E, Kawamura MJ, Abdallah WM, Wicha MS. Role of Notch signaling in cell-fate determination of human mammary stem/progenitor cells. Breast Cancer Res. 2004;6:R605–15.
    1. Esteller M. Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet. 2007;8:286–98.
    1. Farnie G, Clarke RB. Mammary stem cells and breast cancer--role of Notch signalling. Stem Cell Rev. 2007;3:169–75.
    1. Forozan F, Veldman R, Ammerman CA, Parsa NZ, Kallioniemi A, Kallioniemi OP, et al. Molecular cytogenetic analysis of 11 new breast cancer cell lines. Br J Cancer. 1999;81:1328–34.
    1. Geli J, Nord B, Frisk T, Edstrom Elder E, Ekstrom TJ, Carling T, et al. Deletions and altered expression of the RIZ1 tumour suppressor gene in 1p36 in pheochromocytomas and abdominal paragangliomas. Int J Oncol. 2005;26:1385–91.
    1. Han W, Jung EM, Cho J, Lee JW, Hwang KT, Yang SJ, et al. DNA copy number alterations and expression of relevant genes in triple-negative breast cancer. Genes Chromosomes Cancer. 2008;47:490–9.
    1. Hess JL. MLL: a histone methyltransferase disrupted in leukemia. Trends Mol Med. 2004;10:500–7.
    1. Hu C, Dievart A, Lupien M, Calvo E, Tremblay G, Jolicoeur P. Overexpression of activated murine Notch1 and Notch3 in transgenic mice blocks mammary gland development and induces mammary tumors. Am J Pathol. 2006;168:973–90.
    1. Ignatoski KM, Lapointe AJ, Radany EH, Ethier SP. erbB-2 overexpression in human mammary epithelial cells confers growth factor independence. Endocrinology. 1999;140:3615–22.
    1. Ignatoski KM, Maehama T, Markwart SM, Dixon JE, Livant DL, Ethier SP. ERBB-2 overexpression confers PI 3′ kinase-dependent invasion capacity on human mammary epithelial cells. Br J Cancer. 2000;82:666–74.
    1. Italiano A, Attias R, Aurias A, Perot G, Burel-Vandenbos F, Otto J, et al. Molecular cytogenetic characterization of a metastatic lung sarcomatoid carcinoma: 9p23 neocentromere and 9p23-p24 amplification including JAK2 and JMJD2C. Cancer Genet Cytogenet. 2006;167:122–30.
    1. Jones PA, Baylin SB. The epigenomics of cancer. Cell. 2007;128:683–92.
    1. Katoh Y, Katoh M. Comparative integromics on JMJD2A, JMJD2B and JMJD2C: Preferential expression of JMJD2C in undifferentiated ES cells. Int J Mol Med. 2007;20:269–73.
    1. Klose RJ, Kallin EM, Zhang Y. JmjC-domain-containing proteins and histone demethylation. Nat Rev Genet. 2006;7:715–27.
    1. Klose RJ, Zhang Y. Regulation of histone methylation by demethylimination and demethylation. Nat Rev Mol Cell Biol. 2007;8:307–18.
    1. Knuutila S, Bjorkqvist AM, Autio K, Tarkkanen M, Wolf M, Monni O, et al. DNA copy number amplifications in human neoplasms: review of comparative genomic hybridization studies. Am J Pathol. 1998;152:1107–23.
    1. Kreike B, van Kouwenhove M, Horlings H, Weigelt B, Peterse H, Bartelink H, et al. Gene expression profiling and histopathological characterization of triple-negative/basal-like breast carcinomas. Breast Cancer Res. 2007;9:R65.
    1. Krivtsov AV, Armstrong SA. MLL translocations, histone modifications and leukaemia stem-cell development. Nat Rev Cancer. 2007;7:823–33.
    1. Krivtsov AV, Twomey D, Feng Z, Stubbs MC, Wang Y, Faber J, et al. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature. 2006;442:818–22.
    1. Lal G, Padmanabha L, Smith BJ, Nicholson RM, Howe JR, O’Dorisio MS, et al. RIZ1 is epigenetically inactivated by promoter hypermethylation in thyroid carcinoma. Cancer. 2006;107:2752–9.
    1. Loh YH, Zhang W, Chen X, George J, Ng HH. Jmjd1a and Jmjd2c histone H3 Lys 9 demethylases regulate self-renewal in embryonic stem cells. Genes Dev. 2007;21:2545–57.
    1. Moffa AB, Tannheimer SL, Ethier SP. Transforming potential of alternatively spliced variants of fibroblast growth factor receptor 2 in human mammary epithelial cells. Mol Cancer Res. 2004;2:643–52.
    1. Neve RM, Chin K, Fridlyand J, Yeh J, Baehner FL, Fevr T, et al. A collection of breast cancer cell lines for the study of functionally distinct cancer subtypes. Cancer Cell. 2006;10:515–27.
    1. Northcott PA, Nakahara Y, Wu X, Feuk L, Ellison DW, Croul S, et al. Multiple recurrent genetic events converge on control of histone lysine methylation in medulloblastoma. Nat Genet 2009
    1. Peters AH, O’Carroll D, Scherthan H, Mechtler K, Sauer S, Schofer C, et al. Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell. 2001;107:323–37.
    1. Poetsch M, Dittberner T, Woenckhaus C. Frameshift mutations of RIZ, but no point mutations in RIZ1 exons in malignant melanomas with deletions in 1p36. Oncogene. 2002;21:3038–42.
    1. Politi K, Feirt N, Kitajewski J. Notch in mammary gland development and breast cancer. Semin Cancer Biol. 2004;14:341–7.
    1. Rizzo P, Osipo C, Foreman K, Golde T, Osborne B, Miele L. Rational targeting of Notch signaling in cancer. Oncogene. 2008;27:5124–31.
    1. Sansone P, Storci G, Giovannini C, Pandolfi S, Pianetti S, Taffurelli M, et al. p66Shc/Notch-3 interplay controls self-renewal and hypoxia survival in human stem/progenitor cells of the mammary gland expanded in vitro as mammospheres. Stem Cells. 2007;25:807–15.
    1. Savelyeva L, Claas A, An H, Weber RG, Lichter P, Schwab M. Retention of polysomy at 9p23-24 during karyotypic evolution in human breast cancer cell line COLO 824. Genes Chromosomes Cancer. 1999;24:87–93.
    1. Savelyeva L, Claas A, Matzner I, Schlag P, Hofmann W, Scherneck S, et al. Constitutional genomic instability with inversions, duplications, and amplifications in 9p23-24 in BRCA2 mutation carriers. Cancer Res. 2001;61:5179–85.
    1. Sharpless NE. INK4a/ARF: a multifunctional tumor suppressor locus. Mutat Res. 2005;576:22–38.
    1. Shi Y, Whetstine JR. Dynamic regulation of histone lysine methylation by demethylases. Mol Cell. 2007;25:1–14.
    1. Somervaille TC, Cleary ML. Identification and characterization of leukemia stem cells in murine MLL-AF9 acute myeloid leukemia. Cancer Cell. 2006;10:257–68.
    1. Steele-Perkins G, Fang W, Yang XH, Van Gele M, Carling T, Gu J, et al. Tumor formation and inactivation of RIZ1, an Rb-binding member of a nuclear protein-methyltransferase superfamily. Genes Dev. 2001;15:2250–62.
    1. Vinatzer U, Gollinger M, Mullauer L, Raderer M, Chott A, Streubel B. Mucosa-associated lymphoid tissue lymphoma: novel translocations including rearrangements of ODZ2, JMJD2C, and CNN3. Clin Cancer Res. 2008;14:6426–31.
    1. Wang GG, Cai L, Pasillas MP, Kamps MP. NUP98-NSD1 links H3K36 methylation to Hox-A gene activation and leukaemogenesis. Nat Cell Biol. 2007;9:804–812.
    1. Whetstine JR, Nottke A, Lan F, Huarte M, Smolikov S, Chen Z, et al. Reversal of histone lysine trimethylation by the JMJD2 family of histone demethylases. Cell. 2006;125:467–81.
    1. Wissmann M, Yin N, Muller JM, Greschik H, Fodor BD, Jenuwein T, et al. Cooperative demethylation by JMJD2C and LSD1 promotes androgen receptor-dependent gene expression. Nat Cell Biol. 2007;9:347–53.
    1. Woods Ignatoski KM, Livant DL, Markwart S, Grewal NK, Ethier SP. The role of phosphatidylinositol 3′-kinase and its downstream signals in erbB-2-mediated transformation. Mol Cancer Res. 2003;1:551–60.
    1. Yang ZQ, Imoto I, Fukuda Y, Pimkhaokham A, Shimada Y, Imamura M, et al. Identification of a novel gene, GASC1, within an amplicon at 9p23–24 frequently detected in esophageal cancer cell lines. Cancer Res. 2000;60:4735–9.
    1. Yang ZQ, Imoto I, Pimkhaokham A, Shimada Y, Sasaki K, Oka M, et al. A novel amplicon at 9p23 - 24 in squamous cell carcinoma of the esophagus that lies proximal to GASC1 and harbors NFIB. Jpn J Cancer Res. 2001;92:423–8.
    1. Yang ZQ, Streicher KL, Ray ME, Abrams J, Ethier SP. Multiple interacting oncogenes on the 8p11-p12 amplicon in human breast cancer. Cancer Research. 2006;66:11632–11643.

Source: PubMed

Sponsorer og samarbeidspartnere

Medisinsk tilstand

Legemiddelintervensjoner

3
Abonnere