Impact of mutational profiles on response of primary oestrogen receptor-positive breast cancers to oestrogen deprivation.

Pre-surgical studies allow study of the relationship between mutations and response of oestrogen receptor-positive (ER+) breast cancer to aromatase inhibitors (AIs) but have been limited to small biopsies. Here in phase I of this study, we perform exome sequencing on baseline, surgical core-cuts and blood from 60 patients (40 AI treated, 20 controls). In poor responders (based on Ki67 change), we find significantly more somatic mutations than good responders. Subclones exclusive to baseline or surgical cores occur in ∼30% of tumours. In phase II, we combine targeted sequencing on another 28 treated patients with phase I. We find six genes frequently mutated: PIK3CA, TP53, CDH1, MLL3, ABCA13 and FLG with 71% concordance between paired cores. TP53 mutations are associated with poor response. We conclude that multiple biopsies are essential for confident mutational profiling of ER+ breast cancer and TP53 mutations are associated with resistance to oestrogen deprivation therapy.

A gene responsible for prolyl-hydroxylation of moss-produced recombinant human erythropoietin.

Recombinant production of pharmaceutical proteins is crucial, not only for personalized medicine. While most biopharmaceuticals are currently produced in mammalian cell culture, plant-made pharmaceuticals gain momentum. Post-translational modifications in plants are similar to those in humans, however, existing differences may affect quality, safety and efficacy of the products. A frequent modification in higher eukaryotes is prolyl-4-hydroxylase (P4H)-catalysed prolyl-hydroxylation. P4H sequence recognition sites on target proteins differ between humans and plants leading to non-human posttranslational modifications of recombinant human proteins produced in plants. The resulting hydroxyprolines display the anchor for plant-specific O-glycosylation, which bears immunogenic potential for patients. Here we describe the identification of a plant gene responsible for non-human prolyl-hydroxylation of human erythropoietin (hEPO) recombinantly produced in plant (moss) bioreactors. Targeted ablation of this gene abolished undesired prolyl-hydroxylation of hEPO and thus paves the way for plant-made pharmaceuticals humanized via glyco-engineering in moss bioreactors.

p63 is an alternative p53 repressor in melanoma that confers chemoresistance and a poor prognosis.

The role of apoptosis in melanoma pathogenesis and chemoresistance is poorly characterized. Mutations in TP53 occur infrequently, yet the TP53 apoptotic pathway is often abrogated. This may result from alterations in TP53 family members, including the TP53 homologue TP63. Here we demonstrate that TP63 has an antiapoptotic role in melanoma and is responsible for mediating chemoresistance. Although p63 was not expressed in primary melanocytes, up-regulation of p63 mRNA and protein was observed in melanoma cell lines and clinical samples, providing the first evidence of significant p63 expression in this lineage. Upon genotoxic stress, endogenous p63 isoforms were stabilized in both nuclear and mitochondrial subcellular compartments. Our data provide evidence of a physiological interaction between p63 with p53 whereby translocation of p63 to the mitochondria occurred through a codependent process with p53, whereas accumulation of p53 in the nucleus was prevented by p63. Using RNA interference technology, both isoforms of p63 (TA and ΔNp63) were demonstrated to confer chemoresistance, revealing a novel oncogenic role for p63 in melanoma cells. Furthermore, expression of p63 in both primary and metastatic melanoma clinical samples significantly correlated with melanoma-specific deaths in these patients. Ultimately, these observations provide a possible explanation for abrogation of the p53-mediated apoptotic pathway in melanoma, implicating novel approaches aimed at sensitizing melanoma to therapeutic agents.

A HIF-regulated VHL-PTP1B-Src signaling axis identifies a therapeutic target in renal cell carcinoma.

Metastatic renal cell carcinoma (RCC) is a molecularly heterogeneous disease that is intrinsically resistant to chemotherapy and radiotherapy. Although therapies targeted to the molecules vascular endothelial growth factor and mammalian target of rapamycin have shown clinical effectiveness, their effects are variable and short-lived, underscoring the need for improved treatment strategies for RCC. Here, we used quantitative phosphoproteomics and immunohistochemical profiling of 346 RCC specimens and determined that Src kinase signaling is elevated in RCC cells that retain wild-type von Hippel-Lindau (VHL) protein expression. RCC cell lines and xenografts with wild-type VHL exhibited sensitivity to the Src inhibitor dasatinib, in contrast to cell lines that lacked the VHL protein, which were resistant. Forced expression of hypoxia-inducible factor (HIF) in RCC cells with wild-type VHL diminished Src signaling output by repressing transcription of the Src activator protein tyrosine phosphatase 1B (PTP1B), conferring resistance to dasatinib. Our results suggest that a HIF-regulated VHL-PTP1B-Src signaling pathway determines the sensitivity of RCC to Src inhibitors and that stratification of RCC patients with antibody-based profiling may identify patients likely to respond to Src inhibitors in RCC clinical trials.

Complex and dynamic patterns of Wnt pathway gene expression in the developing chick forebrain.

BACKGROUND: Wnt signalling regulates multiple aspects of brain development in vertebrate embryos. A large number of Wnts are expressed in the embryonic forebrain; however, it is poorly understood which specific Wnt performs which function and how they interact. Wnts are able to activate different intracellular pathways, but which of these pathways become activated in different brain subdivisions also remains enigmatic. RESULTS: We have compiled the first comprehensive spatiotemporal atlas of Wnt pathway gene expression at critical stages of forebrain regionalisation in the chick embryo and found that most of these genes are expressed in strikingly dynamic and complex patterns. Several expression domains do not respect proposed compartment boundaries in the developing forebrain, suggesting that areal identities are more dynamic than previously thought. Using an in ovo electroporation approach, we show that Wnt4 expression in the thalamus is negatively regulated by Sonic hedgehog (Shh) signalling from the zona limitans intrathalamica (ZLI), a known organising centre of forebrain development. CONCLUSION: The forebrain is exposed to a multitude of Wnts and Wnt inhibitors that are expressed in a highly dynamic and complex fashion, precluding simple correlative conclusions about their respective functions or signalling mechanisms. In various biological systems, Wnts are antagonised by Shh signalling. By demonstrating that Wnt4 expression in the thalamus is repressed by Shh from the ZLI we reveal an additional level of interaction between these two pathways and provide an example for the cross-regulation between patterning centres during forebrain regionalisation.

Physcomitrella HMGA-type proteins display structural differences compared to their higher plant counterparts.

High mobility group (HMG) proteins of the HMGA family are chromatin-associated proteins that act as architectural factors in nucleoprotein structures involved in gene transcription. To date, HMGA-type proteins have been studied in various higher plant species, but not in lower plants. We have identified two HMGA-type proteins, HMGA1 and HMGA2, encoded in the genome of the moss model Physcomitrella patens. Compared to higher plant HMGA proteins, the two Physcomitrella proteins display some structural differences. Thus, the moss HMGA proteins have six (rather than four) AT-hook DNA-binding motifs and their N-terminal domain lacks similarity to linker histone H1. HMGA2 is expressed in moss protonema and it localises to the cell nucleus. Typical of HMGA proteins, HMGA2 interacts preferentially with A/T-rich DNA, when compared with G/C-rich DNA. In cotransformation assays in Physcomitrella protoplasts, HMGA2 stimulated reporter gene expression. In summary, our data show that functional HMGA-type proteins occur in Physcomitrella.