Figure 1.
Bit1 expression is downregulated in invasive breast tumors.
A. Breast tumor tissue array slides were stained with affinity purified anti-Bit1 (Sigma). Images are representative of each respective case type: normal breast 10X (i,ii), Ductal carcinoma in situ (DCIS) 10X (iii, iv), node negative invasive breast carcinoma 10X (v, vi), and node positive invasive breast carcinoma 10x (vii, viii). B. The average staining intensity of each subgroup was determined. While no significant difference was found between normal and DCIS subgroups, the normal/DCIS was statistically significant (P<0.01) from the nodal negative- and nodal positive-invasive breast carcinomas using the ANOVA and subsequent Tukey post-hoc analysis (see Materials and Methods). Further Tukey post-hoc analysis indicated a significant difference (*, P<0.05) between the invasive node negative and invasive node positive breast carcinoma tissues. C. The distribution of staining intensity scores shows the change in staining pattern between normal/DCIS and invasive case types. Scores were grouped as low (0–1), medium (2), and high (3). Data represent the ratio of the number of samples in each group (low, medium, high) to the total number of samples.
Figure 2.
Suppression of Bit1 expression enhances anoikis resistance.
A. Total cell lysate derived from each respective breast cancer cell line was subjected to SDS-PAGE and immunoblotting using a specific antibody to Bit1. The membrane was then reprobed with anti-β-actin antibody to confirm equal loading of protein. B. Stable MCF7controlshRNA and Bit1shRNA knockdown pools were generated as described in Materials and Methods, and the total cell lysates derived from controlshRNA and Bit1shRNA knockdown pools were subjected to immunobloting using a specific antibody to Bit1. C and D. MCF7controlshRNA and Bit1shRNA knockdown pools were plated onto a polyhema coated or uncoated tissue culture plates. Following 48 h in culture, cells were the stained with Annexin V, a marker of apoptosis, and the relative fluorescence intensities are shown (C). In D, level of apoptosis was also quantified by measuring the amount of DNA histone fragments (Cell Death Elisa). E and F. The stable control shRNA and Bit1shRNA knockdown pools were also generated from the B16F1 cell line. The resulting B16F1 controlshRNA and Bit1shRNA knockdown pools were subjected to total cell lysate isolation, SDS-PAGE, and immunoblotting against a specific Bit1 antibody to confirm Bit1 downregulation (E). In F, the B16F1 controlshRNA and Bit1shRNA knockdown pools were plated onto a polyhema coated or uncoated tissue culture plates for 48 h, and the level of apoptosis was the quantified by measuring the amount of DNA histone fragments. In C, D, and F, three independent experiments were performed in triplicates. *p<0.05 as compared with control cells (Student's t test).
Figure 3.
Downregulation of Bit1 results in morphological changes and enhances cellular adhesion and migration.
A and B. The morphology of controlshRNA and Bit1shRNA knockdown pools derived from MCF7 (A) and B16F1 (B) was examined by phase contrast microscopy (100× magnification) under normal culture conditions. C and D. Stable control shRNAcontrol and Bit1shRNA knockdown pools derived from MCF7 (C) and B16F1(D) were seeded in 96-well plates precoated with fibronectin, collagen I, or BSA. After 15 min of incubation at 37°C, the number of adherent cells was determined by staining with green fluorescent dye (calcein-AM) followed by fluorescence measurement as described under Materials and Methods (Innocyte Cell Adhesion Assay Kit, EMD Biosciences). E. Stable control and Bit1 Hela knockdown clones were subjected to a wound repair assay. The wound was generated at time 0 h and cell migration into the wound was analyzed by phase contrast microscopy at 16 h. F, G, and H. Control and Bit1 knockdown cells derived from Hela (F), MCF7 (G), and B16F1 (H) were subjected to a QCM 96-well migration boyden chamber assay wherein the number of cells that migrated to the bottom of the insert membrane was quantified by CyQuant Gr dye (Molecular Probes) as described in Materials and Methods. In C, D, F, G, and H, results are representative of three independent experiments, *p<0.05 as compared with the control cells (Student's t test).
Figure 4.
Bit1 negatively regulates Erk activation through decreased Erk phosphatase activity, and Erk down-regulation attenuates the enhanced cell adhesion and motility of Bit1 knockdown cells.
A and B. Exponentially growing stable controlshRNA and Bit1shRNA knockdown pools derived from MCF7(A) and B16F1(B) were lysed, and the total lysate was subjected to immunoblotting to detect the phosphorylated Erk (pErk), total Erk(tErk), active Mek (pMek), total Mek (tMek), Bit1, and β-actin. C and D. Total cell lysates from stable MCF7controlshRNA and Bit1shRNA knockdown pools were subjected to an Erk phosphatase assay as described in Materials and Methods. A representative immunoblot of isolated His-6-tagged Erk2 is shown to reveal pErk2 or total Erk2 levels (C). The relative intensity of pErk2/tErk was determined using NIH Image J software, and the values represent the average of at least three independent experiments (D). E, F and G. Stable Hela control clone#1 and Bit1RNAi#21 clone were transfected with control- or Erk2-specific siRNAs; 48 h post-transfection, cells were harvested and subjected to immunoblotting (E) with antibodies against total Erk2 and phosphorylated Erk1/2 (pErks). In parallel, cells were subjected to a fibronectin cell adhesion (F) and QCM boyden chamber migration assays (G) as described in Materials and Methods. In D, F and G, results are representative of three independent experiments, *p<0.05 (Student's t test).
Figure 5.
Suppression of Bit1 enhances tumor metastasis with no significant effect on tumor growth in vivo.
A, B, C, and D. Stable B16F1 controlshRNA and Bit1shRNA knockdown pools were injected subcutaneously in BALB/c nude mice. A total of 20 mice were analyzed with 10 mice injected with control shRNA pool cells while another 10 mice injected with Bit1shRNA pool cells. At the indicated times after injection, tumor volumes were measured (A). At the end of study, the lungs from mice were harvested and photographed (the representative lungs are shown in (B) and random serial sections of paraffin-embedded lung tissue were examined by H&E staining to detect the presence of tumor foci (C) and the numbers of pulmonary metastatic foci were counted (D), *P<0.05 as compared to control shRNA pool. E, F, G and H. Stable control and Bit1 knockdown pools from B16F1 (E and F) and MCF7 (G and H) were injected into the tail vein in BALB/c nude mice. For B16F1, a total of 20 mice were analyzed with 10 tail vein injected with controlshRNA pool and another 10 mice injected with Bit1shRNA. A similar number of mice was used for control shRNA and Bit1shRNA pools derived from MCF7. The mice were sacrificed 30 days post-injection for MCF7cells (20 days for B16F1), and the lungs were harvested and metastatic colonies quantified in serial sections of H&E-stained, paraffin-embedded lung tissue (F and H), *P<0.05 as compared with controlshRNA pool. In E, the representative lungs from mice injected with B16F1 Bit1 knockdown pool showed an obvious increase in metastatic foci on the lung surface relative to that of controlshRNA pool. In G, representative serial sections of H&E-stained, paraffin-embedded lung tissue from mice injected with stable MCF7 controlshRNA or Bit1shRNA knockdown pool are shown. I and J. B16F10 cells were transfected with vector control or C-terminally myc tagged, mitochondrial localized myc-tagged Bit1 construct (Bit1 mito), and 24 h post-transfection, adherent cells were harvested and subjected to immunoblotting against the antibody to myc, pErk, tErk, and β-actin (I). In parallel, vector and mitochondrial Bit1 transfected cells were injected into the tail vein (J) with 10 mice injected with vector control cells and another 10 mice injected with mitochondrial Bit1 transfected cells. 20 days following injection, lungs were harvested and metastatic foci in lung tissue was quantified as described above, *P<0.05 as compared with vector control cells.
Figure 6.
Erk phosphorylation is up-regulated in pulmonary metastatic foci of Bit1 knockdown cells.
A. Random serial sections of paraffin-embedded lung tissue derived from mice injected tail vein with stable B16F1controlshRNA or Bit1shRNA knockdown pools were subjected to immunofluorescence analysis of active Erk using anti-phosphorylated Erk antibody (1∶100) as the primary antibody followed by incubation with FITC-conjugated secondary antibody as described in Materials and Methods. Representative staining are shown in (A). B. The number of metastatic tumor foci staining positive for pErk was quantified by examining serial sections of paraffin-embedded lung tissue of mice tail vein injected with stable B16F1controlshRNA or Bit1shRNA knockdown pools. A total of sixty metastatic lesions (30 controlshRNA and 30 Bit1shRNA) from 10 animals (5 tail vein injected with control shRNA pool and 5 tail injected with Bit1shRNA pool) were analyzed, *p<0.01 (Student's t test).