The basal-like subtype of breast cancer has an aggressive clinical behavior compared to that of the luminal subtype. We identified the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) as basal-like subtype-specific miRNAs and showed that expression of miR-221/222 decreased expression of epithelial-specific genes and increased expression of mesenchymal-specific genes, and increased cell migration and invasion in a manner characteristic of the epithelial-to-mesenchymal transition (EMT). The transcription factor FOSL1 (also known as Fra-1), which is found in basal-like breast cancers but not in the luminal subtype, stimulated the transcription of miR-221/222, and the abundance of these miRNAs decreased with inhibition of the epidermal growth factor receptor (EGFR) or MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), placing miR-221/222 downstream of the RAS pathway. Furthermore, miR-221/222-mediated reduction in E-cadherin abundance depended on their targeting the 3' untranslated region of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1), which inhibited EMT by decreasing ZEB2 (zinc finger E-box-binding homeobox2) expression. We conclude that by promoting EMT, miR-221/222 may contribute to the more aggressive clinical behavior of basal-like breast cancers.

Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn's disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.

OTULIN (OTU deubiquitinase with linear linkage specificity) removes linear polyubiquitin from proteins that have been modified by LUBAC (linear ubiquitin chain assembly complex) and is critical for preventing auto-inflammatory disease and embryonic lethality during mouse development. Here we show that OTULIN promotes rather than counteracts LUBAC activity by preventing its auto-ubiquitination with linear polyubiquitin. Thus, knock-in mice that express catalytically inactive OTULIN, either constitutively or selectively in endothelial cells, resembled LUBAC-deficient mice and died midgestation as a result of cell death mediated by TNFR1 (tumour necrosis factor receptor 1) and the kinase activity of RIPK1 (receptor-interacting protein kinase 1). Inactivation of OTULIN in adult mice also caused pro-inflammatory cell death. Accordingly, embryonic lethality and adult auto-inflammation were prevented by the combined loss of cell death mediators: caspase 8 for apoptosis and RIPK3 for necroptosis. Unexpectedly, OTULIN mutant mice that lacked caspase 8 and RIPK3 died in the perinatal period, exhibiting enhanced production of type I interferon that was dependent on RIPK1. Collectively, our results indicate that OTULIN and LUBAC function in a linear pathway, and highlight a previously unrecognized interaction between linear ubiquitination, regulators of cell death, and induction of type I interferon.

No abstract

The epithelial-to-mesenchymal transition (EMT) is a highly conserved physiological program involved in development and tissue repair; however, its aberrant activation has been implicated in accelerating the progression of a variety of cancers. In breast cancer, the microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) are differentially expressed in the clinically more aggressive basal-like subtype compared to luminal subtype of breast cancer and upregulation of miR-221/222 induces the EMT by targeting the 3′ untranslated region (3′UTR) of the GATA family transcriptional repressor TRPS1 (tricho-rhino-phalangeal syndrome type 1). The complete mechanism through which miR-221/222 promotes the EMT, however, is not fully understood. We identified adiponectin receptor 1 (ADIPOR1), a receptor for the adipocytokine adiponectin, as a direct target of miR-221/222. ADIPOR1 is expressed at higher levels in the luminal compared to the basal-like subtype of breast cancer cell lines, which can be reduced by miR-221/222 targeting of its 3’UTR. In addition, miR-221/222 were negatively correlated with ADIPOR1 expression across breast cancer cell lines and tumors. ADIPOR1 depletion by siRNA in MCF10A cells induced the EMT and increased cell invasion. Depletion of ADIPOR1 by siRNA induced activation of the canonical nuclear factor-kappaB (NF-κB) and subsequent phosphorylation of signal transducer and activator of transcription 3 (STAT3) in an interleukin 6 (IL6)-dependent manner. Finally, overexpression of ADIPOR1 in the basal-like cell line, MDA-MB-231, attenuated cell invasion and promoted the mesenchymal-to-epithelial transition (MET). We conclude that ADIPOR1 negatively regulates EMT in breast cancer and provides an additional node by which miR-221/222 induces the EMT. These results suggest that ADIPOR1 may play an important role in breast cancer progression and metastasis, and could potentially offer an alternative therapeutic strategy for basal-like breast cancer patients.

No abstract

Compared with the luminal subtype, the basal-like subtype of breast cancer has an aggressive clinical behavior, but the reasons for this difference between the two subtypes are poorly understood. We identified microRNAs (miRNAs) miR-221 and miR-222 (miR-221/222) as basal-like subtype-specific miRNAs that decrease expression of epithelial-specific genes and increase expression of mesenchymal-specific genes. In addition, expression of these miRNAs increased cell migration and invasion, which collectively are characteristics of the epithelial-to-mesenchymal transition (EMT). The basal-like transcription factor FOSL1 (also known as Fra-1) directly stimulated the transcription of miR-221/222, and the abundance of these miRNAs decreased with inhibition of MEK (mitogen-activated or extracellular signal-regulated protein kinase kinase), placing miR-221/222 downstream of the RAS pathway. The miR-221/222-mediated reduction in E-cadherin abundance depended on their targeting of the 3' untranslated region (3'UTR) of TRPS1 (trichorhinophalangeal syndrome type 1), which is a member of the GATA family of transcriptional repressors. TRPS1 inhibited EMT by directly repressing expression of ZEB2 (Zinc finger E-box-binding homeobox 2). Therefore, miR-221/222 may contribute to the aggressive clinical behavior of basal-like breast cancers.

Highlights d HPK1 kinase activity limits TCR signaling and effector cytokine secretion d Loss of HPK1 kinase activity enhances anti-viral responses in preclinical models d Loss of HPK1 kinase function suppresses tumor growth in preclinical tumor models d Therapeutic co-blockade of HPK1 kinase and PD-L1 enhances anti-tumor responses