Escape from HER-family tyrosine kinase inhibitor therapy by the kinase-inactive HER3

Oncogenic tyrosine kinases have proved to be promising targets for the development of highly effective anticancer drugs. However, tyrosine kinase inhibitors (TKIs) against the human epidermal growth factor receptor (HER) family show only limited activity against HER2-driven breast cancers, despite effective inhibition of epidermal growth factor receptor (EGFR) and HER2 in vivo. The reasons for this are unclear. Signalling in trans is a key feature of this multimember family and the critically important phosphatidylinositol-3-OH kinase (PI(3)K)/Akt pathway is driven predominantly through transphosphorylation of the kinase-inactive HER3 (refs 9, 10). Here we show that HER3 and consequently PI(3)K/Akt signalling evade inhibition by current HER-family TKIs in vitro and in tumours in vivo. This is due to a compensatory shift in the HER3 phosphorylation-dephosphorylation equilibrium, driven by increased membrane HER3 expression driving the phosphorylation reaction and by reduced HER3 phosphatase activity impeding the dephosphorylation reaction. These compensatory changes are driven by Akt-mediated negative-feedback signalling. Although HER3 is not a direct target of TKIs, HER3 substrate resistance undermines their efficacy and has thus far gone undetected. The experimental abrogation of HER3 resistance by small interfering RNA knockdown restores potent pro-apoptotic activity to otherwise cytostatic HER TKIs, re-affirming the oncogene-addicted nature of HER2-driven tumours and the therapeutic promise of this oncoprotein target. However, because HER3 signalling is buffered against an incomplete inhibition of HER2 kinase, much more potent TKIs or combination strategies are required to silence oncogenic HER2 signalling effectively. The biologic marker with which to assess the efficacy of HER TKIs should be the transphosphorylation of HER3 rather than autophosphorylation.     

Measurement of urinary steroid production rates using stable-isotopes and GC-MS

Summary The urinary production rate of pregnenolone has been determined for a male subject using 7,7-d₂-pregnenolone as an isotopic tracer.To whom correspondence should be addressed.     

The mas oncogene encodes an angiotensin receptor

The class of receptors coupled to GTP-binding proteins share a conserved structural motif which is described as a 'seven-transmembrane segment' following the prediction that these hydrophobic segments form membrane-spanning alpha-helices. Identified examples include the mammalian opsins, alpha 1-, alpha 2-, beta 1- and beta 2-adrenergic receptors, the muscarinic receptor family, the 5-HT1C-receptor, and the substance-K receptor. In addition, two mammalian genes have been identified that code for predicted gene products with sequence similarity to these receptors, but whose ligand specificity is unknown namely, G21 and the mas oncogene. The mas oncogene shows the greatest sequence similarity to the substance-K receptor, and on this basis it was predicted that it would encode a peptide receptor with mitogenic activity which would act through the inositol lipid signalling pathways. The mas oncogene product was transiently expressed in Xenopus oocytes, and stably expressed in a transfected mammalian cell line. The results demonstrate that the mas gene product is a functional angiotensin receptor.     

Pathogenic mutation in the ALS/FTD gene, <Emphasis Type="Italic">CCNF,</Emphasis> causes elevated Lys48-linked ubiquitylation and defective autophagy

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are fatal neurodegenerative disorders that have common molecular and pathogenic characteristics, such as aberrant accumulation and ubiquitylation of TDP-43; however, the mechanisms that drive this process remain poorly understood. We have recently identified CCNF mutations in familial and sporadic ALS and FTD patients. CCNF encodes cyclin F, a component of an E3 ubiquitin–protein ligase (SCF^{cyclin F}) complex that is responsible for ubiquitylating proteins for degradation by the ubiquitin–proteasome system. In this study, we examined the ALS/FTD-causing p.Ser621Gly (p.S621G) mutation in cyclin F and its effect upon downstream Lys48-specific ubiquitylation in transfected Neuro-2A and SH-SY5Y cells. Expression of mutant cyclin F^S621G caused increased Lys48-specific ubiquitylation of proteins in neuronal cells compared to cyclin F^WT. Proteomic analysis of immunoprecipitated Lys48-ubiquitylated proteins from mutant cyclin F^S621G-expressing cells identified proteins that clustered within the autophagy pathway, including sequestosome-1 (p62/SQSTM1), heat shock proteins, and chaperonin complex components. Examination of autophagy markers p62, LC3, and lysosome-associated membrane protein 2 (Lamp2) in cells expressing mutant cyclin F^S621G revealed defects in the autophagy pathway specifically resulting in impairment in autophagosomal–lysosome fusion. This finding highlights a potential mechanism by which cyclin F interacts with p62, the receptor responsible for transporting ubiquitylated substrates for autophagic degradation. These findings demonstrate that ALS/FTD-causing mutant cyclin F^S621G disrupts Lys48-specific ubiquitylation, leading to accumulation of substrates and defects in the autophagic machinery. This study also demonstrates that a single missense mutation in cyclin F causes hyper-ubiquitylation of proteins that can indirectly impair the autophagy degradation pathway, which is implicated in ALS pathogenesis.