G to the American Joint Committee on Cancer (AJCC) TNM system. doi:10.1371/journal.pone.0048178.tHeterogeneous Twist2 Expression in Breast CancersTable 2. The expression of Twist2 in cytoplasm and nucleus of breast carcinomas.CasesCytoplasm only 2 +X10.P,0.Tumor histological type Ductal Lobular Squamous cell TNM clinical stage 0 I/II III/IV Metastasis Non-metastatic tumor Regional lymph nodes metastasis Distant lymph nodes metastasis 90 20 31 Cases 62 16 13 Nucleus only 2 Tumor histological type Ductal Lobular Squamous cell TNM clinical stage 0 I/II III/IV Metastasis Non-metastatic tumor Regional lymph nodes metastasis Distant lymph nodes metastasis 90 20 31 Cases 82 17 25 8 3 6 22 73 46 22 65 37 0 8 9 115 22 4 107 14 3 8 8 1 + 28 4 18 22 73 46 17 50 24 5 23 22 115 22 4 70 20 1 45 25..0.9.,0.X13.P,0.5..0.2..0.Both cytoplasmic and nuclear expression 2 +X1.P.0.Tumor histological type Ductal Lobular Squamous cell TNM clinical stage 0 I/II III/IV Metastasis Non-metastatic tumor Regional lymph nodes metastasis Distant lymph nodes metastasis 90 20 31 65 14 24 25 6 7 22 73 46 16 54 33 6 19 13 115 22 4 82 17 4 33 50..0.0..0.TNM clinical stage of breast cancer is according to the American Joint Committee on Cancer (AJCC) TNM system. doi:10.1371/journal.pone.0048178.twas located in cytoplasm of the cancer cells (Figure 4B, C). Most cells with cytoplasmic Twist2 mostly showed E-cadherin on cell membrane (Figure 4C), which is similar to the cancer cells at tumor center or metastases in vivo. In contrast, transiently expressed Twist2 was strongly detected in nuclei of cancer cellswith loss of E-cadherin (Figure 4D). Little is known so far on how and when Twist2 translocates into nuclei. Here, we show that EMT program may be activated transiently through nuclear Twist2, but not cytoplasmic Twist2. Taken together, our results suggest that nuclear Twist2 may activate EMT transiently in theHeterogeneous Twist2 Expression in Breast CancersTable 3. Spearman’s correlation 
between the immunostaining of Twist2, E-cadherin and Slug.Marker TwistCorrelation Correlation coefficient P value NTwist2 1.000 0.000 71 0.217 0.267 28 0.434 0.056E-cadherinSlugE-cadherinCorrelation coefficient P value N1.000 0.000 28 20.034 0.888 20 1.000 1081537 0.000SlugCorrelation coefficient P value NSpearman’s rank correlation was used to determine whether there was a positive or negative correlation. doi:10.1371/journal.pone.0048178.ttumor invasion front, while cytoplasmic Twist2 contributes to the maintenance of epithelial cancer characteristics in tumor center or LM metastases in breast cancer.DiscussionIt has been well recognized that EMT plays a critical role in cancer metastasis [1]. However, the difficulty to directly demonstrate the role of EMT in metastasis in vivo is to validate cancer cells that have undergone an EMT in primary human tumor specimens [8]. The molecular mechanism associated with the involvement of EMT in tumor metastasis is still highly debated. As clinical observations showed that the majority of human breast carcinoma cells in metastases express E-cadherin and 16574785 P7C3 maintain their epithelial morphology, cancer cells may have disseminated without switching to a mesenchymal phenotype [11,12]. The order 1454585-06-8 master regulators of tumor invasion and metastasis were largely unknown [10]. Twist1 is one of essential factors to promote tumor metastasis [25]. The hypothesis that cancer cells routinely undergo a complete EMT program is likely to be simplistic. In breast cancer, Twist1 only.G to the American Joint Committee on Cancer (AJCC) TNM system. doi:10.1371/journal.pone.0048178.tHeterogeneous Twist2 Expression in Breast CancersTable 2. The expression of Twist2 in cytoplasm and nucleus of breast carcinomas.CasesCytoplasm only 2 +X10.P,0.Tumor histological type Ductal Lobular Squamous cell TNM clinical stage 0 I/II III/IV Metastasis Non-metastatic tumor Regional lymph nodes metastasis Distant lymph nodes metastasis 90 20 31 Cases 62 16 13 Nucleus only 2 Tumor histological type Ductal Lobular Squamous cell TNM clinical stage 0 I/II III/IV Metastasis Non-metastatic tumor Regional lymph nodes metastasis Distant lymph nodes metastasis 90 20 31 Cases 82 17 25 8 3 6 22 73 46 22 65 37 0 8 9 115 22 4 107 14 3 8 8 1 + 28 4 18 22 73 46 17 50 24 5 23 22 115 22 4 70 20 1 45 25..0.9.,0.X13.P,0.5..0.2..0.Both cytoplasmic and nuclear expression 2 +X1.P.0.Tumor histological type Ductal Lobular Squamous cell TNM clinical stage 0 I/II III/IV Metastasis Non-metastatic tumor Regional lymph nodes metastasis Distant lymph nodes metastasis 90 20 31 65 14 24 25 6 7 22 73 46 16 54 33 6 19 13 115 22 4 82 17 4 33 50..0.0..0.TNM clinical stage of breast cancer is according to the American Joint Committee on Cancer (AJCC) TNM system. doi:10.1371/journal.pone.0048178.twas located in 
cytoplasm of the cancer cells (Figure 4B, C). Most cells with cytoplasmic Twist2 mostly showed E-cadherin on cell membrane (Figure 4C), which is similar to the cancer cells at tumor center or metastases in vivo. In contrast, transiently expressed Twist2 was strongly detected in nuclei of cancer cellswith loss of E-cadherin (Figure 4D). Little is known so far on how and when Twist2 translocates into nuclei. Here, we show that EMT program may be activated transiently through nuclear Twist2, but not cytoplasmic Twist2. Taken together, our results suggest that nuclear Twist2 may activate EMT transiently in theHeterogeneous Twist2 Expression in Breast CancersTable 3. Spearman’s correlation between the immunostaining of Twist2, E-cadherin and Slug.Marker TwistCorrelation Correlation coefficient P value NTwist2 1.000 0.000 71 0.217 0.267 28 0.434 0.056E-cadherinSlugE-cadherinCorrelation coefficient P value N1.000 0.000 28 20.034 0.888 20 1.000 1081537 0.000SlugCorrelation coefficient P value NSpearman’s rank correlation was used to determine whether there was a positive or negative correlation. doi:10.1371/journal.pone.0048178.ttumor invasion front, while cytoplasmic Twist2 contributes to the maintenance of epithelial cancer characteristics in tumor center or LM metastases in breast cancer.DiscussionIt has been well recognized that EMT plays a critical role in cancer metastasis [1]. However, the difficulty to directly demonstrate the role of EMT in metastasis in vivo is to validate cancer cells that have undergone an EMT in primary human tumor specimens [8]. The molecular mechanism associated with the involvement of EMT in tumor metastasis is still highly debated. As clinical observations showed that the majority of human breast carcinoma cells in metastases express E-cadherin and 16574785 maintain their epithelial morphology, cancer cells may have disseminated without switching to a mesenchymal phenotype [11,12]. The master regulators of tumor invasion and metastasis were largely unknown [10]. Twist1 is one of essential factors to promote tumor metastasis [25]. The hypothesis that cancer cells routinely undergo a complete EMT program is likely to be simplistic. In breast cancer, Twist1 only.