Epithelial-mesenchymal transition (EMT) is a tightly regulated process that is critical for embryogenesis but is abnormally activated during cancer metastasis and recurrence. Here we show that a switch in CD44 alternative splicing is required for EMT. Using both in vitro and in vivo systems, we have demonstrated a shift in CD44 expression from variant isoforms (CD44v) to the standard isoform (CD44s) during EMT. This isoform switch to CD44s was essential for cells to undergo EMT and was required for the formation of breast tumors that display EMT characteristics in mice. Mechanistically, the splicing factor epithelial splicing regulatory protein 1 (ESRP1) controlled the CD44 isoform switch and was critical for regulating the EMT phenotype. Additionally, the CD44s isoform activated Akt signaling, providing a mechanistic link to a key pathway that drives EMT. Finally, CD44s expression was upregulated in high-grade human breast tumors and was correlated with the level of the mesenchymal marker N-cadherin in these tumors. Together, our data suggest that regulation of CD44 alternative splicing causally contributes to EMT and breast cancer progression.

Breast cancer recurrence is a fundamental clinical manifestation of tumor progression and represents the principal cause of death from this disease. Using a conditional transgenic mouse model for the recurrence of HER2/neu-induced mammary tumors, we demonstrate that the transcriptional repressor Snail is spontaneously upregulated in recurrent tumors in vivo and that recurrence is accompanied by epithelial-to-mesenchymal transition (EMT). Consistent with a causal role for Snail in these processes, we show that Snail is sufficient to induce EMT in primary tumor cells, that Snail is sufficient to promote mammary tumor recurrence in vivo, and that high levels of Snail predict decreased relapse-free survival in women with breast cancer. In aggregate, our observations strongly implicate Snail in the process of breast cancer recurrence.

Abstract. Expression of the adenovirus E1A oncogene stimulates both cell proliferation and p53-dependent apoptosis in rodent cells, p53 implements apoptosis in all or in part through transcriptional activation of bax, the product of which promotes cell death. The adenovirus E1B 19K product is homologous in sequence and in function to Bcl-2, both of which bind to and inhibit the activity of Bax and thereby suppress apoptosis. The E1B 19K protein also interacts with the nuclear lamins, but the role of this interaction in the regulation of apoptosis is not known. Lamins are, however, substrates for members of the interleukin-ll3--converting enzyme (ICE) family of cysteine proteases that are activated during apoptosis and function downstream of Bcl-2 in the cell death pathway. Lamins are degraded during E1A-induced p53-dependent apoptosis. Lamin A and C are cleaved into 47-and 37-kD fragments, respectively, and the site of proteolysis is mapped to a conserved aspartic acid residue at position 230. The cleavage of lamins during apoptosis is consistent with the activation of an ICE-related cysteine protease downstream of p53. No lamin protease activity was detected in cells expressing the E1B 19K protein, indicating that 19K functions upstream of protease activation in inhibiting apoptosis. Substitution of the aspartic acid at the cleavage site produced a mutant lamin protein that was resistant to proteolysis both in vitro and in vivo. Expression of uncleavable mutant lamin A or B attenuated apoptosis, delaying cell death and the associated DNA fragmentation by 12 h. Mutant lamin expressing cells failed to show the signs of chromatin condensation and nuclear shrinkage typical of cell death by apoptosis. Instead, the nuclear envelope collapsed and the nuclear lamina remained intact. However, the late stage of apoptosis was morphologically unaltered and formation of apoptotic bodies was evident. Thus, lamin breakdown by proteolytic degradation facilitates the nuclear events of apoptosis perhaps by facilitating nuclear breakdown.

The E1B 19K protein is a potent apoptosis inhibitor and the putative adenovirus Bcl-2 homolog. To investigate the mechanism of apoptosis regulation, 19K-interacting cellular proteins were identified using the yeast two-hybrid system, and