All authors read and approved the final manuscript. Notes Ethics approval and consent to participate Investigation has been conducted in accordance with the ethical standards and according to the Declaration of Helsinki and national and international guidelines. using GraphPad Prism5 statistical software. Results were considered as statistically significant when value was 0.05. Results Expression of Gab1 is positively associated with BCa metastasis clinically To investigate expressional level of Gab1 PHA 408 in BCa, we first analyzed two independent datasets from Oncomine database (Ma Breast 4 et al.  and Richardson et al. ). Data from both of datasets indicated that expression of Gab1 was significantly elevated in PHA 408 BCa samples when compared to normal control samples (Fig.?1a). Next, we wondered whether expressional level of Gab1 is correlated with metastasis in BCa. By analyzing another Oncomine dataset (Nikolsky et al. ), we found that patients with lymph node metastasis (LNM) showed increased expression of Gab1, compared to patients without metastasis (Fig. ?(Fig.1b).1b). To validate these results, we then examined Gab1 expression in patient samples collected from our hospital by western blot assay. We found that expression of Gab1 was indeed upregulated in BCa tissues (n?=?8) when compared to the paired adjacent normal control tissues (Additional file 2: Figure S1a and Additional file 3: Table S1). Furthermore, we carried out IHC staining for Gab1 and IF co-staining for Gab1 and EpCAM to further determine Gab1 expression in these clinical tumor samples from three major subtypes of BCa, i.e. luminal BCa (n?=?6 for IHC and n?=?6 for IF), HER2 BCa (n?=?6 for IHC and n?=?6 for IF) and triple negative breast cancer (TNBC, n?=?6 for IHC and n?=?6 for IF), respectively (Additional file 2: Figure S1b, S1c and Additional file 3: Table S2, Table S3). Comparison to benign mammary hyperplastic control samples (n?=?6 for IHC and n?=?6 for Rabbit Polyclonal to SCTR IF), significantly elevated Gab1 expression was observed in all of the BCa subtypes (Fig. ?(Fig.1c,1c, Additional file 2: Figure S1d). Importantly, in either HER2 BCa (n?=?4) or TNBC subtype (n?=?2) our IHC staining assessment confirmed a further upregulated Gab1 expression in metastatic samples (Fig. ?(Fig.1d1d and Additional file 3: Table S4). Support for our findings also came from the result of Oncomine data analysis (Ma Breast 3 et al. ), which showed a positive association of Gab1 expressional level with malignant grade progression in BCa (Additional file 2: Figure S1e). In addition, patients with high expressional PHA 408 level of Gab1 displayed a lower rate of overall survival via data assay using The Cancer Genome Atlas (TCGA) database (Additional file 2: Figure S1f). Taken together, these results indicate that expression of Gab1 is not only upregulated in BCa patients with malignant tumor growth and a poor prognosis but also positively associated with tumor metastasis. Open PHA 408 in a separate window Fig. 1 Expression of Gab1 is upregulated in metastatic BCa tissues. a Analysis of datasets from Oncomine database shows that Gab1 expression is upregulated in BCa tissues when compared to the normal mammary tissues. b Expression of Gab1 is significantly upregulated in BCa tissues with lymph node metastasis when compared to that with primary tumor only. c Expression of Gab1 is measured by IHC staining in tumor tissues from a luminal, HER2 or TNBC subtype BCa patient and in mammary tissue from a benign mammary hyperplastic control respectively. d Expression of PHA 408 Gab1 is measured by IHC staining in tumor tissues with or without metastasis from HER2 or TNBC subtype BCa patients. Scale Bar: 100?m, P: patient, Data are presented as means SEM. *: p?0.05, **: p?0.01 Elevated expression of Gab1 enhances BCa cell migration by dissociating the PAR complex in vitro To investigate what role of Gab1 plays in regulation of BCa metastasis, we construct Gab1 stable overexpression and related control subclones in MDA-MB-231 (a TNBC cell line) and SK-BR3 (a HER2 BCa cell line) cells respectively (named 231-Gab1OE vs. 231-vec and SK-Gab1OE vs. SK-vec cell, see Additional.