Transforming growth factor–β (TGF-β) is a pluripotent cytokine that regulates cell fate and plasticity in normal tissues and tumors. The multifunctional cellular responses evoked by TGF-β are mediated by the canonical SMAD pathway and by noncanonical pathways, including mitogen-activated protein kinase (MAPK) pathways and the phosphatidylinositol 3′-kinase (PI3K)–protein kinase B (AKT) pathway. We found that TGF-β activated PI3K in a manner dependent on the activity of the E3 ubiquitin ligase tumor necrosis factor receptor–associated factor 6 (TRAF6). TRAF6 polyubiquitylated the PI3K regulatory subunit p85α and promoted the formation of a complex between the TGF-β type I receptor (TβRI) and p85α, which led to the activation of PI3K and AKT. Lys⁶³-linked polyubiquitylation of p85α on Lys⁵¹³ and Lys⁵¹⁹ in the iSH2 (inter–Src homology 2) domain was required for TGF-β–induced activation of PI3K-AKT signaling and cell motility in prostate cancer cells and activated macrophages. Unlike the activation of SMAD pathways, the TRAF6-mediated activation of PI3K and AKT was not dependent on the kinase activity of TβRI. In situ proximity ligation assays revealed that polyubiquitylation of p85α was evident in aggressive prostate cancer tissues. Thus, our data reveal a molecular mechanism by which TGF-β activates the PI3K-AKT pathway to drive cell migration.

Transforming growth factor β (TGFβ) is a pluripotent cytokine promoting epithelial cell plasticity during morphogenesis and tumour progression. TGFβ binding to type II and type I serine/threonine kinase receptors (TβRII and TβRI) causes activation of different intracellular signaling pathways. TβRI is associated with the ubiquitin ligase tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6). Here we show that TGFβ, via TRAF6, causes Lys63-linked polyubiquitination of TβRI, promoting cleavage of TβRI by TNF-alpha converting enzyme (TACE), in a PKCζ-dependent manner. The liberated intracellular domain (ICD) of TβRI associates with the transcriptional regulator p300 to activate genes involved in tumour cell invasiveness, such as Snail and MMP2. Moreover, TGFβ-induced invasion of cancer cells is TACE- and PKCζ- dependent and the TβRI ICD is localized in the nuclei of different kinds of tumour cells in tissue sections. Thus, our data reveal a specific role for TβRI in TGFβ mediated tumour invasion.

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.

The underlying mechanisms linking antisense RNA, chromatin architecture and gene expression have not been fully elucidated. Here we show that long transcripts encoded from the Kcnq1ot1 antisense promoter silence the flanking genes more efficiently than short antisense transcripts. Interestingly, the antisense RNA‐mediated deposition of inactive chromatin‐specific histone modifications was higher with the longer antisense transcripts than with the shorter antisense transcripts. The kinetic studies of expression and chromatin remodeling of overlapping and nonoverlapping genes in response to antisense transcription revealed that the overlapping gene was rapidly silenced due to decrease in the occupancy of basal transcription machinery and simultaneous enrichment of its promoter with inactive chromatin modifications. The nonoverlapping gene, initially enriched with histone modifications specific to active chromatin, was subsequently silenced. Surprisingly, the flanking sequences were initially enriched with H3K9 monomethylation, as compared to di‐ and trimethylation, with a subsequent shift to trimethylated H3K9 enrichment. Our data provide a new perspective into antisense RNA‐mediated gene silencing, and, more importantly, provide an explanation for why the antisense transcripts encoded from imprinting control regions are of significant length.

High levels of transforming growth factor-β (TGFβ) correlate with poor prognosis for patients with prostate cancer and other cancers. TGFβ is a multifunctional cytokine and crucial regulator of cel...