Klinakis et al. 10.1073/pnas.0603371103. |
Supporting Figure 6
Supporting Table 3
Supporting Table 4
Supporting Figure 7
Supporting Materials and Methods
Fig. 6. Hierarchical clustering of genes (rows) and specimens (columns). The dendrogram shown (derived by unsupervised analysis using CYGWIN software) reveals that there is clear discrimination in gene-expression patterns between normal mammary glands of lactating females (L) or parous females after involution (P) and tumor specimens. The latter are carcinomas from MMTV-Myc transgenic mice (M) and regressing (N1) and nonregressing (NR) tumors induced by an MMTV-N1IC transgene. The pattern similarity between the Myc and Notch1IC tumors is striking. A color scale is shown at the bottom.
Fig. 7. Immunophenotyping of mammary glands and tumors for active Erk1/2 and Akt1. We used immunophenotyping to examine whether deregulation of proliferation and inhibition of apoptosis during the development of N1IC-induced nonregressing carcinomas could be correlated with differences between tumors and controls in the status of important markers for these processes, such as the mitogen-activated protein kinases p44 and p42 (Mapk3 and Mapk1, or Erk1/2) and Akt1, respectively. For this analysis, we used antibodies specifically recognizing the active (phosphorylated) forms of Erk1/2 and Akt1 (p-Erk1/2 and p-Akt1). In WT control lactating glands, the glandular epithelial cells are negative for both markers, whereas the myoepithelial cells exhibit nuclear labeling for p-Erk1/2. A strong positive nuclear signal for p-Erk1/2 and p-Akt1 (more widespread for the latter marker) is observed in both regressing and nonregressing tumors from mice with an MMTV-N1IC/Mycfl/fl/Wapcre genotype.
Table 3. Activated Myc-target genes found up-regulated in Myc- and N1IC-induced mouse mammary tumorsa
Symbol | Gene name | Function | Myc/WT | N1IC/WT |
Cdk4b | Cyclin-dependent kinase 4 | Cell cycle | 2.5 | 2.4 |
Ccnb1 | Cyclin B1 | Cell cycle | 10.2 | 5.7 |
Cdc25cc | Cell division cycle 25 homolog C (Saccharomyces cerevisiae) | Cell cycle | 3.6 | 3.0 |
Cks2d | CDC28 protein kinase regulatory subunit 2 | Cell cycle | 9.2 | 6.5 |
Rcc1e | Regulation of chromosome condensation 1 | Cell cycle | 6.6 | 2.5 |
Nme2f | Expressed in nonmetastatic cells 2, protein | Transcriptional regulator | 2.6 | 2.2 |
Bax | Bcl2-associated X protein | Apoptosis | 3.4 | 3.1 |
Ptmag | Prothymosin alpha | Antiapoptotic activity | 2.3 | 3.2 |
Cbx3 | Chromobox homolog 3 (Drosophila HP1 gamma) | Chromatin assembly/disassembly | 4.5 | 2.7 |
Tk1 | Thymidine kinase 1 | DNA metabolism | 5.3 | 3.9 |
Tymsh | Thymidylate synthase | DNA metabolism | 2.1 | 3.8 |
Pold2 | Polymerase (DNA-directed), delta 2, regulatory subunit | DNA replication | 4.7 | 3.0 |
Apex1i | Apurinic/apyrimidinic endonuclease 1 | DNA repair | 3.3 | 2.1 |
Ddx18 | DEAD (Asp-Glu-Ala-Asp) box polypeptide 18 | RNA helicase activity | 5.2 | 5.4 |
Akap1j | A kinase (PRKA) anchor protein 1 | RNA binding | 4.8 | 3.1 |
Sfrs1 | Splicing factor, arginine/serine-rich 1 (ASF/SF2) | RNA binding | 4.6 | 4.5 |
Hnrpa1 | Heterogeneous nuclear ribonucleoprotein A1 | RNA binding | 2.8 | 3.0 |
Snrpd3 | Small nuclear ribonucleoprotein D3 | Ribonucleoprotein complex | 4.4 | 2.6 |
Eif2s1k | Eukaryotic translation initiation factor 2, subunit 1 alpha | Protein biosynthesis | 2.6 | 2.3 |
Jtv1 | JTV1 gene | Protein biosynthesis | 3.4 | 2.3 |
Shmt1l | Serine hydroxymethyl transferase 1 (soluble) | Metabolism | 3.5 | 2.1 |
Srm | Spermidine synthase | Metabolism | 9.9 | 2.8 |
Tfrcm | Transferrin receptor | Endocytosis | 3.5 | 5.8 |
a, Of 1,697 genes currently listed in the "Myc target gene database" (http://www.myc-cancer-gene.org), 501 are considered activated and 1,196 as repressed by Myc (based on microarray analysis or some other criterion using a variety of samples). From these lists, we selected two subsets of 130 activated and 929 repressed genes, which are presumably direct transcriptional targets (assigned, for example, by ChIP analysis). Genes shown by our expression profiling analysis to be commonly up-regulated (656) or down-regualted (790) in MMTV-Myc-induced mouse breast tumors and MMTV-N1IC-induced nonregressing carcinomas and, in addition, were present in the corresponding subsets of Myc direct targets (23/130 and 37/929) are listed in Supplemental Tables 3 and 4. The numbers indicate fold increases or decreases in expression in comparison with WT control values (mRNA levels in normal mammary glands of parous females isolated after involution).
b, Functions downstream of Myc in transformation and its loss inhibits Myc tumorigenic action in epithelial tissues (1).
c, Overexpressed in various carcinomas, including breast cancer (2).
d, Overexpressed in colon (3) and cervical (4) cancer.
e, Overexpressed in lobular breast cancer (5).
f, Associated with various malignancies (for an example, see ref. 6).
g, Nuclear transforming protein (7). Expression is higher in tumors than in normal tissue (8). This gene exerts antiapoptotic action by preventing apoptosome formation, which is consistent with its oncogenic activity (9).
h, Overexpressed in various cancers (10-12). Its upregulation has been associated with drug resistance in colon cancer (13). It has prognostic significance for breast carcinomas (14).
i, Associated with poor breast cancer prognosis (15).
j, Overexpressed in inflammatory breast cancer (16).
k, Overexpressed in various cancers (17,18) and in breast cancer cells (19).
l, Overexpressed in breast cancer (20).
m, Promising drug target because of overexpression in various malignancies (21).
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Table 4. Repressed Myc-target genes found down-regulated in Myc- and N1IC-induced mouse mammary tumors*
Symbol | Gene name | Function | WT/Myc | WT/N1IC |
Ppp6c | protein phosphatase 6, catalytic subunit | Cell cycle | 2.7 | 3.3 |
Ptp4a1 | Protein tyrosine phosphatase 4a1 | Cell cycle | 2.2 | 2.4 |
Cebpa† | CCAAT/enhancer binding protein (C/EBP), alpha | Regulation of transcription | 3.3 | 3.8 |
Nr1d1 | Nuclear receptor subfamily 1, group D, member 1 | Transcription factor activity | 2.4 | 4.8 |
Spib | Spi-B transcription factor (Spi-1/PU.1 related) | Transcription factor | 12.5 | 5.7 |
Casp9 | Caspase 9 | Apoptosis | 2.3 | 2.2 |
Cradd | CASP2 and RIPK1 and death domain containing adaptor | Apoptosis | 2.0 | 3.1 |
Birc3 | Baculoviral IAP repeat-containing 3 | Anti-apoptosis | 4.2 | 3.7 |
Lsp1‡ | Lymphocyte specific protein 1 | Signal transduction | 3.2 | 2.6 |
Rab11a | RAB11a, member RAS oncogene family | Signal transduction | 2.4 | 3.7 |
Rac2 | RAS-related C3 botulinum substrate 2 | Signal transduction | 6.0 | 2.1 |
Blk | B lymphoid kinase | Intracellular signaling | 17.6 | 10.7 |
Mfng§ | Manic fringe homolog (Drosophila) | Regulation of Notch signaling | 3.2 | 3.3 |
Ier5 | Immediate early response 5 | Mitogenic signal transduction (?) | 5.1 | 3.7 |
Cd79b | CD79B antigen | Transmembrane receptor activity | 22.4 | 5.0 |
Hadhb | Hydroxyacyl dehydrogenase, subunit B | Metabolism | 4.2 | 3.0 |
Acadm | Acetyl-coenzyme A dehydrogenase, medium chain | Metabolism | 3.6 | 3.1 |
Acox1 | Acyl-coenzyme A oxidase 1, palmitoyl | Metabolism | 2.6 | 4.1 |
Acyp2 | Acylphosphatase 2, muscle type | Metabolism | 4.3 | 8.8 |
Crat | Carnitine acetyltransferase | Metabolism | 4.9 | 3.7 |
Aldh2 | Aldehyde dehydrogenase 2, mitochondrial | Metabolism | 3.1 | 2.0 |
Ampd3 | AMP deaminase 3 | Metabolism | 3.5 | 2.1 |
Fah | Fumarylacetoacetate hydrolase | Metabolism | 5.2 | 2.2 |
Mocs2 | Molybdenum cofactor synthesis 2 | Metabolism | 3.0 | 3.0 |
Bckdhb | Branched chain ketoacid dehydrogenase E1 β polypeptide | Amino acid catabolism | 2.3 | 4.4 |
Dbi | Diazepam binding inhibitor | Transport | 3.2 | 2.4 |
Slc2a4 | Solute carrier family 2, member 4 (Glut4) | Transporter | 23.5 | 16.7 |
Atp1a2 | ATPase, Na+/K+ transporting, alpha 2 polypeptide | Transporting atpase activity | 19.8 | 2.0 |
Psen2 | Presenilin 2 | Protein processing | 3.2 | 3.8 |
Rnf141 | Ring finger protein 141 | Protein ubiquitination | 2.5 | 2.3 |
Ltb | Lymphotoxin B | Immune response regulation | 22.6 | 3.9 |
Pscdbp | Pleckstrin homology, Sec7 and coiled-coil domains, bp | Cell adhesion (?) | 2.1 | 2.2 |
Dpm1 | Dolichol-phosphate (beta-D) mannosyltransferase 1 | Glycosylation | 2.0 | 2.3 |
Ndufs1 | NADH dehydrogenase (ubiquinone) Fe-S protein 1 | Mitochondrial function | 2.0 | 2.4 |
Ucp3 | Uncoupling protein 3, mitochondrial | Mitochondrial function | 2.9 | 2.7 |
Ndufb3 | NADH dehydrogenase (ubiquinone) 1 beta subcomplex 3 | Mitochondrial function | 2.6 | 2.4 |
Ubl3 | Ubiquitin-like 3 | Function unknown | 2.1 | 2.3 |
*For details, see Legend to Supplemental Table 3.
†
Reduced expression of C/EBPalpha may play a role in the development and/or progression of breast cancer (1).‡
Expressed in hematopoietic malignancies (2).§
Expressed in ovarian carcinomas (3).1. Gery, S., Tanosaki, S., Bose, S., Bose, N., Vadgamma, J. & Koeffler, H. P. (2005) Clin. Cancer Res. 11, 3184-3190.
2. Marafioti, T., Jabri, L., Pulford, K., Brousset, P., Mason, D. Y. & Delsol, G. (2003) Br. J. Haematol. 120, 671-678.
3. Hopfer, O., Zwahlen, D., Fey, M. F. & Aebi, S. (2005) Br. J. Cancer 93, 709-718.