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Epithelial-mesenchymal transition (EMT) is a crucial event in wound healing, tissue repair and cancer progression in adult tissues. We have recently shown that transforming growth factor (TGF)-β-induced EMT involves isoform switching of fibroblast growth factor receptors by alternative splicing. We performed a microarray-based analysis at single exon level to elucidate changes in splicing variants generated during TGF-β-induced EMT, and found that TGF-β induces broad alteration of splicing patterns by downregulating epithelial splicing regulatory proteins (ESRPs). This was achieved by TGF-β-mediated upregulation of δEF1 family proteins, δEF1 and SIP1. δEF1 and SIP1 each remarkably repressed ESRP2 transcription through binding to the ESRP2 promoter in NMuMG cells. Silencing of both δEF1 and SIP1, but not either alone, abolished the TGF-β-induced ESRP repression. The expression profiles of ESRPs were inversely related to those of δEF1 and SIP in human breast cancer cell lines and primary tumor specimens. Further, overexpression of ESRPs in TGF-β-treated cells resulted in restoration of the epithelial splicing profiles as well as attenuation of certain phenotypes of EMT. Therefore, δEF1 family proteins repress the expression of ESRPs to regulate alternative splicing during TGF-β-induced EMT and the progression of breast cancers.

Transforming growth factor (TGF)‐β acts as a tumor suppressor during cancer initiation, but as a tumor promoter during tumor progression. It has become increasingly clear that TGF‐β plays fundamental roles in multiple steps of tumor progression, including epithelial‐mesenchymal transition (EMT). The EMT, first described by developmental biologists at the beginning of the 1980s, plays crucial roles in appropriate embryonic development, but also functions in adults during wound healing, organ fibrosis, and tumor progression. During EMT, epithelial cells lose their epithelial polarity and acquire mesenchymal phenotypes, endowing them with migratory and invasive properties. Many secreted polypeptides are implicated in this process, and act in a sequential or cooperative manner. TGF‐β induces EMT by propagating intracellular signaling pathways and activating transcriptional factors. Here, I discuss new insights into the molecular mechanisms underlying induction of EMT by TGF‐β in cooperation with Ras or growth factors, along with the signals that induce EMT through transcriptional and post‐transcriptional regulation.

Abnormal DNA methylation at the C‐5 position of cytosine (5mC) of CpG dinucleotides is a well‐known epigenetic feature of cancer. Levels of E‐cadherin, which is regularly expressed in epithelial tissues, are frequently reduced in epithelial tumors due to transcriptional repression, sometimes accompanied by hypermethylation of the promoter region. δEF1 family proteins (δEF1/ZEB1 and SIP1/ZEB2), key regulators of the epithelial‐mesenchymal transition (EMT), suppress E‐cadherin expression at the transcriptional level. We recently showed that levels of mRNAs encoding δEF1 proteins are regulated reciprocally with E‐cadherin level in breast cancer cells. Here, we examined the mechanism underlying downregulation of E‐cadherin expression in three basal‐type breast cancer cells in which the E‐cadherin promoter region is hypermethylated (Hs578T) or moderately methylated (BT549 and MDA‐MB‐231). Regardless of methylation status, treatment with 5‐aza‐2′‐deoxycytidine (5‐aza), which inhibits DNA methyltransferases, had no effect on E‐cadherin expression. Knockdown of δEF1 and SIP1 resulted in recovery of E‐cadherin expression in cells lacking hypermethylation, whereas combined treatment with 5‐aza synergistically restored E‐cadherin expression, especially when the E‐cadherin promoter was hypermethylated. Moreover, δEF1 interacted with DNA methyltransferase 1 (DNMT1) through the Smad‐binding domain. Sustained knockdown of δEF1 family proteins reduced the number of 5mC sites in the E‐cadherin promoter region, suggesting that these proteins maintain 5mC through interaction with DNMT1 in breast cancer cells. Thus, δEF1 family proteins appear to repress expression of E‐cadherin during cancer progression, both directly at the transcriptional level and indirectly at the epigenetic level.

Snail, also called Snai1, is a key regulator of EMT. Snail plays crucial roles in cancer progression, including resistance to anti-tumor drugs and invasion by various cancer cells. Slug, also known as Snai2, is also involved in the aggravation of certain tumors. In this study, we examined the roles of Slug in human oral squamous cell carcinoma (OSCC) cells. Slug is highly expressed in these cells, and Slug siRNA effectively represses anti-tumor drug resistance and invasive properties. In addition, transforming growth factor (TGF)-β upregulates the expression of Snail and Slug and promotes resistance to anti-tumor drugs in OSCC cells. Surprisingly, Slug siRNA appears to upregulate Snail expression considerably in OSCC cells. Snail siRNA also appears to upregulate Slug expression. Thus, either Slug or Snail siRNA alone partially mitigates malignant phenotypes in the presence of TGF-β, whereas both Slug and Snail siRNAs together dramatically suppress them. Therefore, Slug and Snail in tandem, but not alone, are potential therapeutic targets for nucleic acid medicines to treat oral cancer.