The contribution of lncRNAs to tumour progression and the regulatory mechanisms driving their expression are areas of intense investigation. Here, we characterize the binding of heterogeneous nuclear ribonucleoprotein E1 (hnRNP E1) to a nucleic acid structural element located in exon 12 of PNUTS (also known as PPP1R10) pre-RNA that regulates its alternative splicing. HnRNP E1 release from this structural element, following its silencing, nucleocytoplasmic translocation or in response to TGFβ, allows alternative splicing and generates a non-coding isoform of PNUTS. Functionally the lncRNA-PNUTS serves as a competitive sponge for miR-205 during epithelial-mesenchymal transition (EMT). In mesenchymal breast tumour cells and in breast tumour samples, the expression of lncRNA-PNUTS is elevated and correlates with levels of ZEB mRNAs. Thus, PNUTS is a bifunctional RNA encoding both PNUTS mRNA and lncRNA-PNUTS, each eliciting distinct biological functions. While PNUTS mRNA is ubiquitously expressed, lncRNA-PNUTS appears to be tightly regulated dependent on the status of hnRNP E1 and tumour context.

Signaling by the transforming growth factor–β (TGF-β) receptors I and II (TβRI/II) and the primary cilia-localized sonic hedgehog (Shh) pathway promote cell migration and, consequently, tumor metastasis. In contrast, the sphingolipid ceramide inhibits cell proliferation and tumor metastasis. We investigated whether ceramide metabolism inhibited TβRI/II trafficking to primary cilia to attenuate cross-talk between TβRI/II and the Shh pathway. We found that ceramide synthase 4 (CerS4)–generated ceramide stabilized the association between TβRI and the inhibitory factor Smad7, which limited the trafficking of TβRI/II to primary cilia. Expression of a mutant TβRI that signals but does not interact with Smad7 prevented the CerS4-mediated inhibition of migration in various cancer cells. Genetic deletion or knockdown of CerS4 prevented the formation of the Smad7-TβRI inhibitory complex and increased the association between TβRI and the transporter Arl6 through a previously unknown cilia-targeting signal (Ala³¹Thr³²Ala³³Leu³⁴Gln³⁵) in TβRI. Mutating the cilia-targeting signal abolished the trafficking of TβRI to the primary cilia. Localization of TβRI to primary cilia activated a key mediator of Shh signaling, Smoothened (Smo), which stimulated cellular migration and invasion. TβRI-Smo cross-talk at the cilia in CerS4-deficient 4T1 mammary cancer cells induced liver metastasis from orthotopic allografts in both wild-type and CerS4-deficient mice, which was prevented by overexpression of Smad7 or knockdown of intraflagellar transport protein 88 (IFT88). Overall, these data reveal a ceramide-dependent mechanism that suppresses cell migration and invasion by restricting TβRI/II-Shh signaling selectively at the plasma membrane of the primary cilium.

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In order to better understand the process of breast cancer metastasis, we have generated a mammary epithelial progression series of increasingly aggressive cell lines that metastasize to lung. Here we demonstrate that upregulation of an endoplasmic reticulum (ER) to Golgi trafficking gene signature in metastatic cells enhances transport kinetics, which promotes malignant progression. We observe increased ER–Golgi trafficking, an altered secretome and sensitivity to the retrograde transport inhibitor brefeldin A (BFA) in cells that metastasize to lung. CREB3 was identified as a transcriptional regulator of upregulated ER–Golgi trafficking genes ARF4, COPB1, and USO1, and silencing of these genes attenuated the metastatic phenotype in vitro and lung colonization in vivo. Furthermore, high trafficking gene expression significantly correlated with increased risk of distant metastasis and reduced relapse-free and overall survival in breast cancer patients, suggesting that modulation of ER–Golgi trafficking plays an important role in metastatic progression.

The Fbxo4 tumour suppressor is a component of an Skp1-Cul1-F-box E3 ligase for which two substrates are known. Here we show purification of SCF^Fbxo4 complexes results in the identification of fragile X protein family (FMRP, Fxr1 and Fxr2) as binding partners. Biochemical and functional analyses reveal that Fxr1 is a direct substrate of SCF^Fbxo4. Consistent with a substrate relationship, Fxr1 is overexpressed in Fbxo4 knockout cells, tissues and in human cancer cells, harbouring inactivating Fbxo4 mutations. Critically, in head and neck squamous cell carcinoma, Fxr1 overexpression correlates with reduced Fbxo4 levels in the absence of mutations or loss of mRNA, suggesting the potential for feedback regulation. Direct analysis reveals that Fbxo4 translation is attenuated by Fxr1, indicating the existence of a feedback loop that contributes to Fxr1 overexpression and the loss of Fbxo4. Ultimately, the consequence of Fxr1 overexpression is the bypass of senescence and neoplastic progression.