Figure 1.
Twist2 is up-regulated in breast cancer.
A. Immuboblot analysis of Twist2 expression in fresh breast cancer tissues. Twist2 was up-regulated in breast carcinomas relative to matched normal breast tissues. B. Immunohistochemical (IHC) staining of Twist2 on sections of paraffin-embedded breast carcinomas and normal breast tissues. Twist2 was localized in the cytoplasm (Tumor 10) or nucleus (Tumor 9), depending on the tumor specimen (magnification:×400).
Table 1.
Clinical pathological characteristics of Twist2-associated breast carcinomas.
Table 2.
The expression of Twist2 in cytoplasm and nucleus of breast carcinomas.
Table 3.
Spearman's correlation between the immunostaining of Twist2, E-cadherin and Slug.
Figure 2.
The expression patterns of ErbB2, Twist2 and E-cadherin in human breast carcinomas.
Representative images ErbB2, Twist2 and E-cadherin IHC staining in tumor central areas (TC, first and second row) and invasive front (IF, third and fourthrow) of the primary tumor, and surrounding lymph metastasis (LM, fifth and sixth row) are shown. Boxes indicate magnified regions in stained serial sections. Specific positive staining is shown in brown color, and nuclei were counterstained in blue. Tumor cells are clearly polarized in the differentiated gland-like or tubular structures areas in both primary tumor center and metastases. A high ErbB2 indicating strong proliferation (star) was detected only in differentiated area of TC and LM. Loss of gland-like growth tumor cells did not express ErbB2 in the corresponding IF. Twist2 is only detectable in the cytoplasm (arrows) in TC and LM. In contrast, tumor cells at the invasive front lose their polar orientation and display fibroblast-like shape. This morphological change is accompanied by nuclear accumulation of Twist2 (arrows). Consistently, tumor cells in differentiated areas of the primary tumor and in metastases express E-cadherin on cell membrane (arrowheads). Disseminating tumor cells at the invasive fronts with nuclear Twist2 completely lost E-cadherin (arrowheads).
Figure 3.
The association between subcellular localization of Twist2 and expression of E-cadherin in cells of the tumor center (TC) and invasive front (IF).
Twist2 in the nucleus was related to the loss of E-cadherin expression. Aberrant E-cadherin expression was defined as weak or loss of expression, normal was defined as normal level and subcellular localization of E-cadherin. Cytoplasm indicated “Cytoplasm only”. Nucleus indicated “Nucleus only”.
Table 4.
Spearman's correlation between the regulation of Twist2 and E-cadherin.
Figure 4.
The regulation of E-cadherin expression by Twist2 in breast cancer cells.
A. Immunoblot analysis showing that strong expression of E-cadherin was found with cytoplasic Twist2 expression. Stable expression of ectopic Twist2 in breast cancer cells (Twist2/MCF-7) were verified by western blot with anti- Twist2 and anti-flag antibodies. No obvious changes of E-cadherin was detected between Twist2/MCF-7, Vec/MCF-7 (the vector control), and the parental group. B. Immunoblot analysis of Twist2 in subcellular fractions showing that Twist2 was localized in the cytoplasm. Oct-1 indicated nuclear fraction and IkB-α indicated cytoplasmic fraction. The cells were from the stably transfected samples. C. Immunofluorescent staining of Twist2 and E-cadherin in MCF-7 cells showing cells with Twist2 (in red) in cytoplasm expressed E-cadherin (in green) on cell membrane. Nuclei were counterstained with DAPI (in blue). The cells were from the stably transfected samples. D. Immunofluorescent staining showing that transient over-expression of Twist2 (in red) in nuclei caused loss of E-cadherin in the same cancer cells. Cells without nuclear Twist2 retained expression of E-cadherin on membrane. Nuclei were counterstained with DAPI (in blue).