Differential Proteome Analysis Identifies TGF-β-Related Pro-Metastatic Proteins in a 4T1 Murine Breast Cancer Model.

Transforming growth factor-β (TGF-β) has a dual role in tumorigenesis, acting as either a tumor suppressor or as a pro-oncogenic factor in a context-dependent manner. Although TGF-β antagonists have been proposed as anti-metastatic therapies for patients with advanced stage cancer, how TGF-β mediates metastasis-promoting effects is poorly understood. Establishment of TGF-β-related protein expression signatures at the metastatic site could provide new mechanistic information and potentially allow identification of novel biomarkers for clinical intervention to discriminate TGF-β oncogenic effects from tumor suppressive effects. In the present study, we found that systemic administration of the TGF-β receptor kinase inhibitor, SB-431542, significantly inhibited lung metastasis from transplanted 4T1 mammary tumors in Balb/c mice. The differentially expressed proteins in the comparison of lung metastases from SB-431542 treated and control vehicle-treated groups were analyzed by a quantitative LTQ Orbitrap Velos system coupled with stable isotope dimethyl labeling. A total of 36,239 peptides from 6,694 proteins were identified, out of which 4,531 proteins were characterized as differentially expressed. A subset of upregulated proteins in the control group was validated by western blotting and immunohistochemistry. The eukaryotic initiation factor (eIF) family members constituted the most enriched protein pathway in vehicle-treated compared with SB-43512-treated lung metastases, suggesting that increased protein expression of specific eIF family members, especially eIF4A1 and eEF2, is related to the metastatic phenotype of advanced breast cancer and can be down-regulated by TGF-β pathway inhibitors. Thus our proteomic approach identified eIF pathway proteins as novel potential mediators of TGF-β tumor-promoting activity

Validation of protein expression using western blot and immunohistochemistry.

<p>(A) Metastases lysates were prepared from three mice per group and evaluated by western blot with anti-vimentin, anti-Hsp90a/b, and anti-Eno1 antibodies. β-actin was used as internal normalization. (B) Representative image of positive vimentin staining is shown exclusively in the metastases lesion and not in the surrounding lung tissue. White dotted lines represent a boundary of tumor and surrounding normal lung tissue. M, metastases; NT, normal lung tissue; PV, pulmonary vein. (C) Randomly selected high power fields were immunostained with vimentin and were quantitated using Zeiss software with the % area occupied by metastasized nodules in 4T1 metastasized tumors. A significant reduction in the number of vimentin positive cells was seen in the SB-431542-treated tumors. Scale bars represent 100 μm; hpf, high power fields.</p

Protein expression profiling of 4T1 metastases tumor using dimethyl labeling with triplex stable isotopes.

<p>(A) Schematic representation of the proteomics approach. Lung-metastasized 4T1 tumor samples were isolated from each group (control and SB-431542-treated tumors). Subsequently, the tumor protein was lysed, and differential protein expression was detected by relative quantification using dimethyl-labeling (light, medium and high) followed by an SCX-LC-MS/MS (LTQ Orbitrap Velos) analysis for each set of sample quadruplicates. Isotope-light was used as the reference sample and contained mixed aliquots of all control and SB-431542-treated tumor samples. Four sets of dimethyl-labeled experiments were performed to compare the protein profiles of 8 metastasized tumor samples. (B) Representative dimethyl labeled-based LC-MS/MS spectrum for one of the peptides from vimentin (P20152) showing RQVQSLTCEVDALK, a double charged peptide.</p

Orthotopic metastasis model demonstrates that a specific inhibitor of TGF-β receptor kinase, SB-431542, decreases lung metastasis but does not significantly alter growth of the primary tumor.

<p>(A) Scheme for the experimental approach using 4T1 metastasis model. 4T1 tumor cells (1×10⁴ cells) were transplanted into the mammary fat pad of Balb/c mice. Mice bearing 4T1 mammary tumors were treated three times weekly with SB-431542 (10 mg/kg body weight) or vehicle (20% DMSO/80% corn oil). At day 10 post-injection of 4T1 cells, primary tumors were surgically excised, and the mice were kept alive to allow the tumor to metastasize to the lung. (B) Graph showing relative primary tumor growth of 4T1 cells over time. Data are presented as mean ± SEM. (ns = not significant, control, n = 14; SB-431542, n = 15, unpaired <i>t</i>-test). (C) The number of gross metastasis (control, n = 14; SB-431542-treated, n = 15) was counted. The administration of SB-431542 markedly reduced both the number and the size of the metastasized tumor compared to the control group. Lungs were collected after 40 days and the lung surface was examined for the metastasis. The number of visible lung metastasis was counted. ** p<0.001.; Mann Whitney <i>U</i>-test. Data are presented as the median ± SEM. (D) Representative gross lung images from control and SB-431542-treated groups are shown from control (top) and SB-431542-treated (bottom) animals, with metastases visible at the lung surface marked by bold black arrows. (E) Representative histological view of lung metastases treated with vehicle, DMSO (left) and SB-431542 (right). H&E staining; black dotted lines demarcate tumor parenchyma (*) from the normal lung tissue.</p

EIF signaling was the most enriched canonical IPA pathway of the down-regulated proteins in SB-431542-treated groups.

<p>(A) Pathway analysis conducted using IPA (<a href="http://www.ingenuitypathway.com" target="_blank">www.ingenuitypathway.com</a>) showed a ranking of the most enriched pathways from the down-regulated proteins in SB-431542-treated groups. (B) Whole cell lysates from each group were evaluated by western blot with the EIF family of proteins: anti-eIF4G1, anti-eIF4E, and anti-Eef2 antibodies. (n = 3/group).β -actin was used as a loading control. (C) Sections were assessed by H&E staining (top panel), and immunohistochemistry was performed using anti-eIF4G1 (middle panel) and anti-eIF4E (bottom panel) antibodies. Both eIF4G1 and eIF4E immunostaining in 4T1 lung metastasis showed the heterogeneity of staining patterns seen among individual metastases. 100× magnification. Tumor sections are boxed with a black dotted line, M, metastases; NT, normal lung tissue. (D) eIF4G1- and eIF4E-positive areas were semiquantified using Image Pro Premier Software in three randomly selected high power fields from each samples. Data are represented as box plots (n = 3/group), ** p<0.001 using an unpaired <i>t</i>-test.</p