WT1 expression in breast cancer disrupts the epithelial/mesenchymal balance of tumour cells and correlates with the metabolic response to docetaxel

WT1 is a transcription factor which regulates the epithelial-mesenchymal balance during embryonic development and, if mutated, can lead to the formation of Wilms' tumour, the most common paediatric kidney cancer. Its expression has also been reported in several adult tumour types, including breast cancer, and usually correlates with poor outcome. However, published data is inconsistent and the role of WT1 in this malignancy remains unclear. Here we provide a complete study of WT1 expression across different breast cancer subtypes as well as isoform specific expression analysis. Using in vitro cell lines, clinical samples and publicly available gene expression datasets, we demonstrate that WT1 plays a role in regulating the epithelial-mesenchymal balance of breast cancer cells and that WT1-expressing tumours are mainly associated with a mesenchymal phenotype. WT1 gene expression also correlates with CYP3A4 levels and is associated with poorer response to taxane treatment. Our work is the first to demonstrate that the known association between WT1 expression in breast cancer and poor prognosis is potentially due to cancer-related epithelial-to-mesenchymal transition (EMT) and poor chemotherapy response

Acute Multiple Organ Failure in Adult Mice Deleted for the Developmental Regulator Wt1

There is much interest in the mechanisms that regulate adult tissue homeostasis and their relationship to processes governing foetal development. Mice deleted for the Wilms' tumour gene, Wt1, lack kidneys, gonads, and spleen and die at mid-gestation due to defective coronary vasculature. Wt1 is vital for maintaining the mesenchymal–epithelial balance in these tissues and is required for the epithelial-to-mesenchyme transition (EMT) that generates coronary vascular progenitors. Although Wt1 is only expressed in rare cell populations in adults including glomerular podocytes, 1% of bone marrow cells, and mesothelium, we hypothesised that this might be important for homeostasis of adult tissues; hence, we deleted the gene ubiquitously in young and adult mice. Within just a few days, the mice suffered glomerulosclerosis, atrophy of the exocrine pancreas and spleen, severe reduction in bone and fat, and failure of erythropoiesis. FACS and culture experiments showed that Wt1 has an intrinsic role in both haematopoietic and mesenchymal stem cell lineages and suggest that defects within these contribute to the phenotypes we observe. We propose that glomerulosclerosis arises in part through down regulation of nephrin, a known Wt1 target gene. Protein profiling in mutant serum showed that there was no systemic inflammatory or nutritional response in the mutant mice. However, there was a dramatic reduction in circulating IGF-1 levels, which is likely to contribute to the bone and fat phenotypes. The reduction of IGF-1 did not result from a decrease in circulating GH, and there is no apparent pathology of the pituitary and adrenal glands. These findings 1) suggest that Wt1 is a major regulator of the homeostasis of some adult tissues, through both local and systemic actions; 2) highlight the differences between foetal and adult tissue regulation; 3) point to the importance of adult mesenchyme in tissue turnover

Linear domain interactome and biological function of anterior gradient 2

The Anterior Gradient 2 (AGR2) protein has been implicated in a variety of biological systems linked to cancer and metastasis, tamoxifen-induced drug resistance, pro-inflammatory diseases like IBD and asthma, and limb regeneration. The molecular mechanisms by which AGR2 mediates these various phenotypes in disease progression in both cancer and IBD are poorly understood, as is the biological function(s) of AGR2 under non-disease conditions. Here, we use a combination of biochemical techniques, organ culture, cell biology and mouse genetics to investigate the biological significance of AGR2 both in cell lines and in vivo. We present data based on phage-peptide inter-actomics screens suggesting a role for AGR2 in mediating the maturation and trafficking of a class of membrane and secretory proteins, and investigate a putative interaction between AGR2 and one member of this class of proteins. We also describe the construction of a universal vector for use in making a variety of transgenic animals, and then present data showing its use as a promoter reporter, and attempt to investigate the temporal and spatial expression of AGR2 in the developing and adult mouse. Further, we present data describing the localisation pattern of AGR2 in the developing murine kidney using a combination of organ culture and antibody staining, and suggest a role for AGR2 in the developing kidney based on this data that is in agreement with a chaperone function for membrane and secretory proteins. Together, these data suggest that AGR2 has an intrinsic consensus docking site for a subset of its client proteins, that AGR2 plays a role in protein maturation in ciliated cell types, and provides a novel biological model to dissect the role of AGR2 in ER-trafficking

Visceral and subcutaneous fat have different origins and evidence supports a mesothelial source

International audience: Fuelled by the obesity epidemic, there is considerable interest in the developmental origins of white adipose tissue (WAT) and the stem and progenitor cells from which it arises. Whereas increased visceral fat mass is associated with metabolic dysfunction, increased subcutaneous WAT is protective. There are six visceral fat depots: perirenal, gonadal, epicardial, retroperitoneal, omental and mesenteric, and it is a subject of much debate whether these have a common developmental origin and whether this differs from that for subcutaneous WAT. Here we show that all six visceral WAT depots receive a significant contribution from cells expressing Wt1 late in gestation. Conversely, no subcutaneous WAT or brown adipose tissue arises from Wt1-expressing cells. Postnatally, a subset of visceral WAT continues to arise from Wt1-expressing cells, consistent with the finding that Wt1 marks a proportion of cell populations enriched in WAT progenitors. We show that all visceral fat depots have a mesothelial layer like the visceral organs with which they are associated, and provide several lines of evidence that Wt1-expressing mesothelium can produce adipocytes. These results reveal a major ontogenetic difference between visceral and subcutaneous WAT, and pinpoint the lateral plate mesoderm as a major source of visceral WAT. They also support the notion that visceral WAT progenitors are heterogeneous, and suggest that mesothelium is a source of adipocytes

Primers and pDONR vectors used in the generation of pENTR vectors.

<p>Primers and pDONR vectors used in the generation of pENTR vectors.</p

The <i>Six2</i>^+/GCiP allele based on the pMULTIrec system.

<p><b>A.</b> Retrival of the <i>Six2</i>-GCIP targeting vector. <b>B.</b> Targeting of the <i>Six2</i> locus. <b>C.</b> Confirmation of correct targeting of the <i>Six2</i> locus (restriction enzyme and probe indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062054#pone-0062054-g004" target="_blank">figure 4B</a>). <b>D.. </b><i>Six2</i>^+/GCiP<i>Rosa26</i>^tdRFP kidney in culture showing GFP and RFP signals. <b>E.. </b><i>Six2</i>^+/GCiP<i>Rosa26</i>^tdRFP kidney in culture showing GFP and RFP signals and Cdh1 antibody staining. CM: cap mesenchyme; UB: ureteric bud; N: nephron.</p

The <i>Six2</i>-GCiP BAC construct made using pMULTIrec.

<p><b>A.</b> The four-fragment and dual selection cassette from pMULTIrec was cloned in a <i>Six2</i> containing BAC replacing the start codon. <b>B.</b> GFP imaging of a E13.5 embryo carrying the <i>Six2</i>-GCiP BAC. <b>C.</b> GFP imaging of whole mount E13.5 kidneys. <b>D.</b> E13.5 <i>Six2</i>-GCiP BAC kidneys cultured for four days showing GFP expression in the cap mesenchyme and Cdh1 expression in the ureteric bud.</p

Efficiency of the Gateway reactions used.

<p>Efficiency of the Gateway reactions used.</p

The <i>Nanog</i>-KiP BAC made from a modified pMULTIrec vector.

<p><b>A.</b> Generation of the <i>Nanog</i>-KiP BAC. <b>B, C.</b> mKate2 (B) and brightfield signals of iPS clones derived from <i>Nanog</i>-KiP BAC MEFs. <b>D, E.</b> mKate2 (B) and brightfield signals of iPS clones derived from wild type MEFs.</p

Efficiency of the recombineering reactions used.

<p>Efficiency of the recombineering reactions used.</p