Table S1. Relevant proteins identified by SILAC and LC-MS/MS. The protein expression profile of MDA-MB-468 and BC-M1 was compared in 4 biological replicates. aFor detailed information of the protein identification results, see additional Supplementary data files. Proteins that may be combined to superordinate functional groups are labeled with equal colors. bA positive value of the average signal ratio signifies an increased protein expression in BC-M1 and a negative value signifies an increased protein expression in MDA-468. cFor LANCL2 no specific signals in BC-M1 were detected. dQuantification was performed manually.

Swiss-Prot acc no.a / recommended protein name by UniProtKB/Swiss-Prot (short name) / total number of peptides analyzed / number of different peptides analyzed / number of biological replicates in which peptides were detected / average valueb / standard deviation / Student’s t-test / Function/relevance
P21291 / Cysteine and glycine-rich protein 1 (CSRP1) / 13 / 5 / 4 / 4.75d / 1.81 / p < 0.001 / Cellular differentiation
P02751 / Fibronectin (FINC) / 52 / 15 / 4 / 10.56 / 5.20 / p < 0.001 / Cell adhesion
P09382 / Galectin-1 (Gal-1) / 56 / 10 / 4 / 6.85 / 3.26 / p < 0.001 / Ligand for FINC
P17931 / Galectin-3 (Gal-3) / 12 / 5 / 4 / -2.76 / 0.37 / p < 0.001 / Ligand for FINC
P13473 / Lysosome-associated membrane glycoprotein 2 (Lamp-2) / 5 / 1 / 3 / -2.02 / 0.31 / p < 0.001 / Tumor cell metastasis
Q05682 / Caldesmon (CDM) / 9 / 2 / 3 / 4.32d / 0.93 / p < 0.001 / Myoepithelial marker
P50454 / Serpin H1 (AsTP3) / 39 / 10 / 4 / 5.66 / 1.89 / p < 0.001 / Protection against cell stress
P02794 / Ferritin heavy chain (FTH1) / 6 / 3 / 4 / 4.54 / 0.92 / p < 0.001 / Iron reservoir: receives the iron from PCBP1 and 2
Q15365 / Poly(rC)-binding protein 1 (PCBP1) / 7 / 4 / 4 / -2.69 / 0.26 / p < 0.001 / Iron Chaperones
Q15366 / Poly(rC)-binding protein 2 (PCBP2) / 14 / 3 / 4 / -1.34 / 0.25 / p < 0.001
P21796 / Voltage-dependent anion-selective channel protein 1 (VDAC-1) / 9 / 3 / 4 / -1.44 / 0.16 / p < 0.001 / Transport of small metabolites into the mitochondrion
P45880 / Voltage-dependent anion-selective channel protein 2 (VDAC-2) / 16 / 2 / 4 / -2.52 / 0.39 / p < 0.001
Q9Y277 / Voltage-dependent anion-selective channel protein 3 (VDAC-3) / 16 / 1 / 4 / -1.49 / 0.16 / p < 0.001
P54819 / Adenylate kinase 2, mitochondrial (AK2) / 10 / 3 / 4 / -2.65 / 0.64 / p < 0.001 / Provides the ATP to the UPR chaperones
P40925 / Malate dehydrogenase, cytoplasmic (MDH1) / 20 / 2 / 4 / -4.04 / 2.36 / p < 0.001 / component of the malate-aspartate shuttle; balances the redox states between cytoplasm and mitochondrion
P40926 / Malate dehydrogenase, mitochondrial (MDH2) / 65 / 9 / 4 / -1.62 / 0.23 / p < 0.001
P00505 / Aspartate aminotransferase, mitochondrial (GOT2) / 17 / 3 / 4 / -2.10 / 0.43 / p < 0.001
P49327 / Fatty acid synthase (FAS) / 87 / 23 / 4 / -3.91 / 2.62 / p < 0.001 / One of the key metabolic proteins in breast cancer
Q9BWD1 / Acetyl-CoA acetyltransferase (THIC) / 10 / 3 / 4 / -3.39 / 0.56 / p < 0.001 / Involved in lipid metabolism
P11413 / Glucose-6-phosphate 1-dehydrogenase (G6PD) / 9 / 4 / 3 / -3.58 / 0.92 / p < 0.001 / Pentose phosphate pathway (PPP), Rate limiting step
P52209 / 6-phosphogluconate dehydrogenase, decarboxylating (6PGD) / 10 / 5 / 3 / -3.86 / 0.95 / p < 0.001 / PPP.
P29401 / Transketolase (TKT) / 32 / 8 / 4 / -3.19 / 0.92 / p < 0.001 / PPP
P04406 / Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) / 400 / 47 / 4 / 1.06 / 0.21 / p < 0.001 / Glycolysis. Reference for PPP in this work.
P04075 / Fructose-bisphosphate aldolase A (ALDOA) / 66 / 18 / 4 / -1.28 / 0.20 / p < 0.001
P06733 / Alpha-enolase (ENOA) / 106 / 33 / 4 / -1.27 / 0.42 / p < 0.001
Q12905 / Interleukin enhancer-binding factor 2 (ILF2) / 6 / 2 / 3 / -3.84 / 0.46 / p < 0.001 / Part of a purine-box regulator complex
Q12906 / Interleukin enhancer-binding factor 3 (ILF3) / 14 / 4 / 4 / -2.36 / 0.66 / p = 0.001
P13010 / X-ray repair cross-complementing protein 5 (Ku80) / 10 / 2 / 3 / -1.78 / 0.41 / p < 0.001
P12956 / X-ray repair cross-complementing protein 6 (Ku70) / 13 / 5 / 4 / -2.10 / 0.50 / p < 0.001
Q08211 / ATP-dependent RNA helicase A (DHX9) / 11 / 6 / 3 / -2.13 / 0.57 / p < 0.001 / Can interact with Ku80
P12004 / Proliferating cell nuclear antigen (PCNA) / 7 / 3 / 4 / -2.68 / 1.01 / p < 0.001 / DNA replication
P63244 / Guanine nucleotide-binding protein subunit beta-2-like 1 (RACK1) / 20 / 9 / 4 / -2.02 / 0.74 / p < 0.001 / Associated with lung cancer metastasis
P06702 / Protein S100-A9 (S10A9) / 8 / 2 / 4 / -38.84d / 10.94 / p < 0.001 / Tumor growth and metastasis; Inflammation
P05109 / Protein S100-A8 (S10A8) / 5 / 3 / 3 / -10.16d / 2.88 / p = 0.002 / Heterotetramer with S100A9
P31151 / Protein S100-A7 (S10A7) / 10 / 2 / 4 / -17.88d / 7.03 / p < 0.001 / Induced by oncostatin-M and interleukin-6
P04040 / Catalase (CAT) / 11 / 6 / 3 / -9.79d / 3.98 / p < 0.001 / Detoxification of hydrogen peroxide
Q9NS86 / LanC-like protein 2 (LANCL2) / 10 / 4 / 3 / MDA-468 onlyc / - / - / Sensitivity to Adriamycin

Remarks to potential biomedical relevance of differently expressed proteins between MDA-MB-468 and BC-M1

Differentially expressed proteina / Potential biological function / Reference
Ferritin heavy chain (FTH1): 4.54 / FTH1 serves as iron storage and keeps it in soluble and available form. High levels of FTH1 were associated with the mesenchymal breast cancer phenotype and the acquisition of metastatic and multidrug-resistant phenotype. See also PCBP. / (1)
Poly(rC)-binding protein 1 (PCBP1): -2.69;
PCBP2: -1.34 / Iron chaperones. BC-M1 appears to have well pronounced iron storages (FTH1), but the low expression value of PCBP1 that delivers the iron to FTH1 point towards in diminished iron refilling capacity in BC-M1. The PCBPs, in particular PCBP1, provide the iron to the catalytic centre of prolyl hydroxylases that hydroxylate HIF1α and thus marques Hif1α for proteosomal degradation. When PCBP1 expression is low, the activity of the prolyl hydroxylases is reduced and Hif1α can accumulate leading to activation of Hif1α-dependent cellular programs. Potentially this is a not yet well recognized regulation strategy of DTC that might grant attributes of a hypoxic phenotype even when oxygen is not limiting. / (2)
Catalase (CAT): -9.79 / Catalase catalyzes the detoxification of hydrogen peroxide to water. There is a controversy if hypoxia increases the generation of reactive oxygen species of if hypoxia does not affect this. / (3)
Fatty acid synthase (FAS): -3.91 / The value of -3.9 for FAS is very similar to the values detected for the pentose phosphate pathway (PPP), e. g. -3.6 for G6PD. Supports the assumption that in BC-M1 the glycolysis and ATP producing metabolic pathways may be favored over metabolic synthesis reactions compared with MDA-468.
X-ray repair cross-complementing protein 6 (Ku70): -2.10
X-ray repair cross-complementing protein 5 (Ku80): -1.78 / Ku70 and Ku80 are ATP-dependent DNA helicases that belong to the DNA-dependent protein kinase structure. The Ku proteins are involved in DNA double strand repair pathways, the non-homologous end joining and the homologous recombination. Elevated levels of Ku activity are associated with a gain of oncogenic function like increased proliferation and losses are associated with a loss of their caretaker function leading to elevated genomic instability. See also ILF2 and ILF3. / (4)(5)
Interleukin enhancer-binding factor 2 (ILF2):
-3.84
Interleukin enhancer-binding factor 3 (ILF3):
-2.36 / Part of a transcriptional regulator complex for the activation of Interleukin-2, which is composed of Ku70, Ku80, ILF2 and ILF3. The fifth protein of this complex, DNA-PKcs, was not detected in our analyses. Involved in activation of T-cells and has been implicated in beast cancer metastasis by this effect. / (6)

aA positive value of the average signal ratio signifies an increased protein expression in BC-M1 and a negative value signifies an increased protein expression in MDA-468

Literature

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2.Nandal A, Ruiz JC, Subramanian P, Ghimire-Rijal S, Sinnamon RA, Stemmler TL, et al. Activation of the HIF prolyl hydroxylase by the iron chaperones PCBP1 and PCBP2. Cell Metab 2011;14(5):647-57.

3.Denko NC. Hypoxia, HIF1 and glucose metabolism in the solid tumour. Nat Rev Cancer 2008;8(9):705-13.

4.Gullo C, Au M, Feng G, Teoh G. The biology of Ku and its potential oncogenic role in cancer. Biochim Biophys Acta 2006;1765(2):223-34.

5.Locasale JW, Cantley LC. Metabolic flux and the regulation of mammalian cell growth. Cell Metab 2011;14(4):443-51.

6.Shi L, Qiu D, Zhao G, Corthesy B, Lees-Miller S, Reeves WH, et al. Dynamic binding of Ku80, Ku70 and NF90 to the IL-2 promoter in vivo in activated T-cells. Nucleic Acids Res 2007;35(7):2302-10.

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