Figure 3

Doxycycline-induced FAK increases tumorigenesis in xenograft mice model in vivo in contrast to FAK-CD. A, MCF-7-Tet-ON-TRE-2 control cell line, MCF-7-Tet-ON-FAK and MCF-7-Tet-ON-FAK-CD cells were injected into the nude mice. Mice were divided on two groups (n = 5–7) that were given either drinking water without or with doxycycline. Doxycycline significantly increased volume and weight, in mice with injected MCF-7-Tet-ON-FAK cells in contrast to FAK-CD. Bars show means ± standard errors. *, p < 0.05, Student's t-test. B, Doxycycline induced FAK expression in xenograft tumors with injected MCF-7-Tet-ON-FAK cells and induced FAK-CD expression in case of MCF-7-Tet-ON-FAK-CD. Western blotting was performed on tumor lysates with FAK-C20, C-terminal antibody for detecting FAK and FAK-CD. C, TaqMan RT-PCR analysis of gene expression in MCF-7-Tet-ON cells and in MCF-7-Tet-ON xenograft tumors. Real-time PCR analysis was performed on breast cancer cell lines MCF-7-Tet-ON-TRE-2, -FAK or -FAK-CD grown for 6 days either without or with doxycyclin (2 μg/ml) (left panel) and on tumor samples from these injected cell lines without or with doxycycline (right panel) (Materials and Methods). Expression of genes was calculated relative to GAPDH control (RQ) and normalized to MCF-7 cells in case of cell lines and relatively to MCF-7-Tet-ON-TRE-2 without doxycycline in case of tumor samples. Bars show means ± standard errors of two independent experiments. *p < 0.05, FAK mRNA level in MCF-7-Tet-ON-FAK (+Dox) versus MCF-7-Tet-ON-FAK(-Dox) samples Student's t-test. Left upper panel: FAK expression in MCF-7-Tet-ON breast cell lines, Right upper panel: FAK expression in tumor samples (two tumors for each group was used without doxycycline and with doxycycline). Left lower panel: Pyk-2 expression in MCF-7-Tet-ON breast cell lines. Right lower panel: Pyk-2 expression in tumor samples (two tumors for each group was used without doxycycline and with doxycycline). D, Immunohistochemical analysis of MCF-7-Tet-ON tumors. MCF-7-Tet-ON-FAK tumors demonstrate increased FAK expression in the presence of doxycycline. E, Decreased expression of DMP1 gene in MCF-7-Tet-ON-FAK tumors in the presence of doxycycline. Upper panel: The Real-Time PCR analysis by TaqMan array assay on tumor samples. Bars show average ± standard errors of two independent experiments. * p < 0.05, FAK +Dox tumors versus FAK-Dox tumors, Student's t-test. Lower panel: Western with DMP1 antibody was performed on tumor cell lysates. DMP1 protein level was significantly lower in MCF-7-Tet-ON-FAK tumors in the presence of doxycycline compared with MCF-7-Tet-ON-FAK tumors without doxycycline. F, Decreased expression of AKT1 in MCF-7-Tet-ON-FAK-CD tumors in the presence of doxyxycline. Western blotting with AKT1 antibody was performed on tumor cell lysates, as described above. AKT 1 expression is significantly decreased in MCF-7-Tet-ON-FAK-CD tumors in the presence of doxycycline. * p < 0.05, FAK-CD Dox⁺ tumors versus FAK-CD Dox^- tumors, Student's t-test.