MMTV-induced mammary tumorigenesis: gene discovery, progression to malignancy and cellular pathways. untransfected parental MCF7 and T47D cells, as WNT1 and FGF3 are secreted factors. Proteomic analysis of this model system revealed the induction of i) EMT markers, ii) mitochondrial proteins, iii) glycolytic enzymes and iv) protein synthesis machinery, consistent with an anabolic CSC phenotype. MitoTracker staining validated the expected WNT1/FGF3-induced increase in mitochondrial mass and activity, which presumably displays increased mitochondrial biogenesis. Importantly, many of the SJB2-043 proteins that were up-regulated by WNT/FGF-signaling in MCF7 cells, were also transcriptionally over-expressed in human breast cancer cells Greater than 40 nuclear-encoded mitochondrial-related proteins were over-expressed in MCF7-WNT1/FGF3 cells. Many of these proteins were related to the TCA cycle (ACO2), oxidative phosphorylation (MT-CO2), regenerating ATP (CKMT1/2) or mitochondrial biogenesis (TOMM34). In addition, MT-CO2 (a mitochondrial DNA encoded protein) was upregulated by >2.5-fold. More than 10 enzymes related to glycolysis, the pentose phosphate pathway, glycogen metabolism and amino acid synthesis were all upregulated in MCF7-WNT1/FGF3 cells. SJB2-043 Over 35 proteins related to protein synthesis, including ribosome-related proteins, enzymes for tRNA synthesis, chaperones for protein folding and amino acid transporters, were all up upregulated in MCF7-WNT1/FGF3 SJB2-043 cells. Greater than 45 proteins known to be associated with the EMT phenotype were upregulated in MCF7-WNT1/FGF3 cells. Examples include FRS2 (FGF receptor substrate-2; >10-fold) and -catenin (>2-fold). Expression of WNT1/FGF3-related targets in patient-derived human breast cancer samples To determine the possible translational significance of our results, we intersected our WNT-FGF SLC5A5 proteomics data with human genome-wide transcriptional profiling data. These human clinical data were derived from publically available human breast malignancy samples, in which breast cancer cells were separated by laser-capture microdissection from tumor stromal cells. Transcriptional profiles were analyzed from from N=28 human breast cancer patients (See the Materials & Methods section). In this data set, gene expression was previously decided using Affymetrix U133A 2.0 GeneChips. A concise summary of these findings is offered in Tables ?Furniture5,5, ?,66 and ?and7.7. Overall, greater than sixty WNT1/FGF3 targets (related to mitochondria, glycolysis, the EMT, and protein synthesis) that we recognized in MCF7-WNT1/FGF3 cells were also transcriptionally elevated in human breast malignancy cells in vivo. These new protein targets that we recognized in MCF7-WNT1/FGF3 cells may be important for developing new strategies for the diagnosis and treatment of breast cancer. Table 5 WNT1/FGF3 Targets Increased in Human Breast Malignancy Cells in Vivo: Mitochondria and Glycolysis
Mitochondrial-related Proteins/TCA Cycle (26)ATP5OATP synthase subunit O, mitochondrial5.122.13E-06ATP5BATP synthase subunit beta, mitochondrial5.042.75E-06ATP5A1ATP synthase subunit alpha, mitochondrial5.013.09E-06COX6A1Cytochrome c oxidase subunit 6A, mitochondrial4.462.07E-05ECHS1Enoyl-CoA hydratase, mitochondrial4.058.22E-05MDH1Malate dehydrogenase, cytoplasmic3.999.88E-05PCK2Phosphoenolpyruvate carboxykinase [GTP], mitochondrial3.881.43E-04SCDAcyl-CoA desaturase3.702.55E-04HSPA9Stress-70 SJB2-043 protein, mitochondrial3.692.64E-04NQO1NAD(P)H dehydrogenase [quinone] 13.494.81E-04HSPD160 kDa warmth shock protein, mitochondrial3.425.93E-04COX4I1Cytochrome c oxidase subunit 4 isoform 1, mitochondrial3.396.61E-04TUFMElongation factor Tu, mitochondrial3.386.74E-04C21orf33ES1 protein homolog, mitochondrial3.318.40E-04NDUFS1Mitochondrial NADH-ubiquinone oxidoreductase 75 kDa subunit3.201.15E-03IDH1Isocitrate dehydrogenase [NADP] 13.181.22E-03OATOrnithine aminotransferase, mitochondrial3.171.25E-03CSCitrate synthase, mitochondrial2.665.13E-03AK2Adenylate kinase 2, mitochondrial2.201.59E-02IDH3AIsocitrate dehydrogenase [NAD] subunit alpha, mitochondrial2.161.78E-02PRKDCDNA-dependent protein kinase catalytic subunit (maintains mt-DNA copy number)2.141.85E-02CLPXATP-dependent Clp protease ATP-binding subunit clpX-like, mitochondrial2.111.96E-02ABAT4-aminobutyrate aminotransferase, mitochondrial2.082.14E-02ACO2Aconitase 2, mitochondrial1.833.64E-02DUTDeoxyuridine 5-triphosphate nucleotidohydrolase, mitochondrial1.873.37E-02ETFAElectron transfer flavoprotein subunit alpha, mitochondrial1.764.25E-02Enzymes Related to Glycolysis, the Pentose Phosphate Pathway, Glycogen, and Amino Acid Synthesis (Serine/Arginine) (4)PKM2Pyruvate kinase3.269.79E-04PGK1Phosphoglycerate kinase2.468.66E-03TKTTransketolase2.201.60E-02ENO1Enolase, alpha1.962.75E-02 Open in a separate windows -Transcriptional profiling data derived from the analysis of N=28 breast cancer patients are shown, high-lighting the levels of fold-upregulation observed in the epithelial cancer cell compartment (relative to the tumor stroma), and corresponding p-values derived from the analysis of these clinical samples. Table 6 WNT1/FGF3 Targets Increased in Human Breast Malignancy Cells in Vivo: The EMT and Cell Migration
EMT Markers, Extracellular Matrix, Cell Migration and Cytoskeletal proteins (15)FLNBFilamin-B4.816.21E-06TPT1Translationally-controlled tumor protein3.435.81E-04CDC42Cell division control protein 42 homolog3.111.48E-03S100A11Protein S100-A112.882.81E-03ANXA2Annexin A22.833.28E-03MYOFMyoferlin2.675.00E-03TUBB2ATubulin beta-2A chain2.635.56E-03SEPT2Septin-22.566.60E-03TAGLN2Transgelin-22.429.47E-03IQGAP1IQ motif containing GTPase activating protein 1 (scaffold protein for CDC42)2.321.19E-02HMGB1High mobility group protein B12.211.57E-02CAPZBF-actin-capping protein subunit beta2.191.66E-02CDV3Carnitine deficiency-associated gene expressed in cardiac ventricle 32.042.30E-02FAM82BRegulator of microtubule dynamics protein 11.972.72E-02MYH10Myosin, heavy polypeptide 10, non-muscle1.823.69E-02Miscellaneous (11)PON2Paraoxonase 2, isoform4.029.25E-05MATR3Matrin-33.455.56E-04SH3BGRLSH3 domain-binding glutamic acid-rich-like protein3.121.43E-03AHNAKNeuroblast differentiation-associated protein, AHNAK2.576.41E-03CASTCalpastatin A2.547.08E-03SEC24AProtein transport protein Sec24A2.191.65E-02PABPC4Polyadenylate-binding protein 42.151.78E-02COMTSoluble catechol-O-methyltransferase2.102.04E-02STUB1E3 SJB2-043 ubiquitin-protein ligase CHIP1.952.79E-02TFF1Trefoil factor.