1601

DAB2

BACKGROUND: Metastatic tumour cells are characterised by acquisition of migratory and invasive properties; properties shared by cells, which have undergone epithelial-to-mesenchymal transition (EMT). Disabled-2 (Dab2) is a putative tumour suppressor whose expression has been shown to be downregulated in various cancer types including breast cancer; however, its exact function in suppressing tumour initiation or progression is unclear. METHODS: Disabled-2 isoform expression was determined by RT-PCR analysis in human normal and breast tumour samples. Using shRNA-mediated technology, Dab2 was stably downregulated in two cell model systems representing nontumourigenic human mammary epithelial cells. These cells were characterised for expression of EMT markers by RT-PCR and western blot analysis. RESULTS: Decreased expression of the p96 and p67 isoforms of Dab2 is observed in human breast tumour samples in comparison to normal human breast tissue. Decreased Dab2 expression in normal mammary epithelial cells leads to the appearance of a constitutive EMT phenotype. Disabled-2 downregulation leads to increased Ras/MAPK signalling, which facilitates the establishment of an autocrine transforming growth factor beta (TGFbeta) signalling loop, concomitant with increased expression of the TGFbeta2 isoform. CONCLUSION: Loss of Dab2 expression, commonly observed in breast cancer, may facilitate TGFbeta-stimulated EMT, and therefore increase the propensity for metastasis.

Patients with esophageal squamous cell carcinomas (ESCCs) have poor survival and high recurrence rate, but lack a prognostic biomarker. Disabled-2 (DAB2) is a crucial tumor suppressor, but its roles in ESCCs are uncertain. We investigated whether low DAB2 expression in ESCCs could lead into tumor progression and poor prognosis. Our results found patients with low-DAB2 expression ESCCs had significantly larger tumor size, deeper tumor invasion depth, lymph node metastasis, worse survival, and higher recurrence rate (P<0.05). The Cox-regression model revealed low-DAB2 expression was an independent factor of poor survival (P<0.05), and also of tumor recurrence with the predictive performance superior to clinical TNM stage (P<0.05). Low-DAB2 cancer cells, validated by DAB2 knockdown or over-expression, had higher phosphorylated ERK and migration abilities, which could be suppressed by ERK inhibitor treatment. TGF-beta-induced epithelial-to-mesenchymal transition (EMT) only existed in the high-DAB2 cells, and related to worse prognosis of high-DAB2 ESCCs (P<0.05). In conclusion, DAB2 can suppress the ERK signaling, but correlate to have TGF-beta-induced EMT in ESCCs. DAB2 expression could be a biomarker to identify patients with worse survival and high recurrence. Our data suggest DAB2 expression can stratify patients in need of aggressive surveillance and with possible benefit from anti-ERK or anti-TGF-beta therapies.

The effects of different substrate stiffness were investigated on epithelial-mesenchymal transition (EMT) of cervical cancer cell lines and the role of miR-106b and its target protein DAB2 therein. Cervical cancer cell lines HeLa and SiHa were cultured on artificial substrates with different stiffness prepared using different ratios of acrylamide and bis-acrylamide. Changes of microRNA profiles were detected using microRNA chip analysis, and the expression levels of EMT-related markers E-cadherin and vimentin were detected using western blotting and real-time PCR. In addition, the effects of miR-106b overexpression as well as miR-106b and DAB2 knockdown on expression of E-cadherin and vimentin were also examined using western blotting and real-time PCR. The results showed that i) cervical cancer cell lines SiHa and HeLa cultured on substrate with stiffness of 20 kPa had the strongest EMT ability, showed the highest levels of vimentin and lowest levels of E-cadherin, compared with cells cultured on substrate with stiffness of 1 kPa; ii) miR-106b knockdown reversed the effects of substrate stiffness on EMT of cervical cancer cells, while miR-106 overexpression and DAB2 knockdown induced EMT of cervical cancer cells cultured on substrate with stiffness of 20 kPa. Overall, the results indicated that substrate stiffness could regulate EMT of cervical cancer cell lines HeLa and SiHa at least partially through miR-106b and its downstream target DAB2.

Transforming growth factor beta (TGFbeta) induces an epithelial to mesenchymal transition (EMT) during both physiological and pathological processes; however, the mechanism underlying this transition is not fully elucidated. Here, we have demonstrated that TGFbeta induces the expression of the adaptor molecule disabled-2 (Dab2) concomitant with the promotion of EMT. We show that TGFbeta induces a transient accumulation of Dab2 to the membrane and increases Dab2 binding to beta1 integrin. Furthermore, small interfering RNA (siRNA)-mediated silencing of Dab2 expression in mouse mammary gland epithelial cells results in inhibition of integrin activation, shown by a decrease of both TGFbeta-induced focal adhesion kinase phosphorylation and cellular adherence, leading to apoptosis and inhibition of EMT. Forced re-expression of human Dab2, not targeted by the mouse siRNA sequence, rescues cells from apoptosis and restores TGFbeta-mediated integrin activation and EMT. These results are confirmed in the F9 teratocarcinoma cell line, a model for retinoic acid-induced visceral endoderm differentiation in which we demonstrate that ablation of retinoic acid-induced Dab2 expression levels, by stable siRNA silencing of Dab2, blocks visceral endoderm differentiation. Our findings indicate that Dab2 plays an important regulatory role during cellular differentiation and that induction of differentiation in the absence of Dab2 expression commits the cell to apoptosis.