Soluble expression, purification, and secondary structure determination of human PDX1 transcription factor.

The transcription factor PDX1 is a master regulator essential for proper development of the pancreas, duodenum and antrum. Furthermore, it is an indispensable reprogramming factor for the derivation of human ß-cells, and recently, it has been identified as a tumor suppressor protein in gastric cancer. Here, we report the soluble expression and purification of the full-length human PDX1 protein from a heterologous system. To achieve this, the 849 bp coding sequence of the PDX1 gene was first codon-optimized for expression in Escherichia coli (E. coli). This codon-optimized gene sequence was fused to a protein transduction domain, a nuclear localization sequence, and a His-tag, and this insert was cloned into the protein expression vector for expression in E. coli strain BL21(DE3). Next, screening and identification of the suitable gene construct and optimal expression conditions to obtain this recombinant fusion protein in a soluble form was performed. Further, we have purified this recombinant fusion protein to homogeneity under native conditions. Importantly, the secondary structure of the protein was retained after purification. Further, this recombinant PDX1 fusion protein was applied to human cells and showed the ability to enter the cells as well as translocate to the nucleus. This recombinant tool can be used as a safe tool and can potentially replace its genetic and viral forms in the reprogramming process to induce a ß-cell-specific transcriptional profile in an integration-free manner. Additionally, it can also be used to elucidate its role in cellular processes and for structural and biochemical studies.

Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy.

AIM: To detect hyper-conserved regions in the hepatitis B virus (HBV) X gene (HBX) 5' region that could be candidates for gene therapy. METHODS: The study included 27 chronic hepatitis B treatment-naive patients in various clinical stages (from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeAg-negative and HBeAg-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/mL, the HBX 5' end region [nucleotide (nt) 1255-1611] was PCR-amplified and submitted to next-generation sequencing (NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences (haplotypes) obtained by NGS. Conservation at the HBx protein amino acid (aa) level was also analyzed. RESULTS: NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample (2487-9279, IQR 2817). In 14/27 patients (51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region (nt 1255-1286) and the other in the 5' end coding region (nt 1519-1603). This last region coded for a conserved amino acid region (aa 63-76) that partially overlaps a Kunitz-like domain. CONCLUSION: Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.

Hepatitis B Virus X Protein Modulates Apoptosis in NRK-52E Cells and Activates Fas/FasL Through the MLK3-MKK7-JNK3 Signaling Pathway.

BACKGROUND/AIMS: The hepatitis B virus X protein (HBx) contributes to HBV-induced injury of renal tubular cells and induces apoptosis via Fas/FasL up-regulation. However, the mechanism of Fas/FasL activation is unknown. Recent studies indicated that HBx induction of apoptosis in hepatic cells depends on activating the MLK3-MKK7-JNKs signaling module, which then up-regulates FasL expression. In this study, we used NRK-52E cells transfected an HBx expression vector to examine the role of the MLK3-MKK7-JNKs signaling pathway on HBx-induced renal tubular cell injury. METHODS: NRK-52E cells were transfected with pc-DNA3.1(+)-HBx to establish an HBx over-expression model, and with pc-DNA3.1(+)-HBx and pSilencer3.1-shHBx to establish an HBx low expression model. One control group was not transfected and another control group was transfected with an empty plasmid. Cell proliferation was determined by the formazan dye method (Cell Counting Kit-8) and apoptosis was measured by flow cytometry and fluorescence microscopy. Western blotting was used to measure the expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins. The activity of caspase-8 was measured by spectrophotometry. RESULTS: Transfection of NRK-52E cells with pc-DNA3.1(+)-HBx inhibited cell proliferation and increased apoptosis and caspase-8 activity. The expression of Fas, FasL, and MLK3-MKK7-JNKs signaling pathway-related proteins were also greater in the pc-DNA3.1(+)-HBx group, but lower in RNAi group. Furthermore, the activity of MLK3-MKK7-JNKs signaling pathway, expression of Fas/FasL, and apoptosis were significantly lower in the pc-DNA3.1(+)-HBx group when treated with K252a, a known inhibitor of MLK3. CONCLUSIONS: Our results show that HBx induces apoptosis in NRK-52E cells and activates Fas/FasL via the MLK3-MKK7-JNK3-c-Jun signaling pathway.

In vitro and in vivo methodologies for studying the Sigma 54-dependent transcription.

Here we describe approaches and methods to assaying in vitro the major variant bacterial sigma factor, Sigma 54 (σ(54)), in a purified system. We include the complete transcription system, binding interactions between σ54 and its activators, as well as the self-assembly and the critical ATPase activity of the cognate activators which serve to remodel the closed promoter complexes. We also present in vivo methodologies that are used to study the impact of physiological processes, metabolic states, global signalling networks, and cellular architecture on the control of σ(54)-dependent gene expression.

Growth phase-dependent modulation of Rgg binding specificity in Streptococcus pyogenes.

Streptococcus pyogenes Rgg is a transcriptional regulator that interacts with the cofactor LacD.1 to control growth phase-dependent expression of genes, including speB, which encodes a secreted cysteine protease. LacD.1 is thought to interact with Rgg when glycolytic intermediates are abundant in a manner that prevents Rgg-mediated activation of speB expression via binding to the promoter region. When the intermediates diminish, LacD.1 dissociates from Rgg and binds to the speB promoter to activate expression. The purpose of this study was to determine if Rgg bound to chromatin during the exponential phase of growth and, if so, to identify the binding sites. Rgg bound to 62 chromosomal sites, as determined by chromatin immunoprecipitation coupled with DNA microarrays. Thirty-eight were within noncoding DNA, including sites upstream of the genes encoding the M protein (M49), serum opacity factor (SOF), fibronectin-binding protein (SfbX49), and a prophage-encoded superantigen, SpeH. Each of these sites contained a promoter that was regulated by Rgg, as determined with transcriptional fusion assays. Purified Rgg also bound to the promoter regions of emm49, sof, and sfbX49 in vitro. Results obtained with a lacD.1 mutant showed that both LacD.1 and Rgg were necessary for the repression of emm49, sof, sfbX49, and speH expression. Overall, the results indicated that the DNA binding specificity of Rgg is responsive to environmental changes in a LacD.1-dependent manner and that Rgg and LacD.1 directly control virulence gene expression in the exponential phase of growth.

Isolation and functional characterization of a transcription factor VpNAC1 from Chinese wild Vitis pseudoreticulata.

NAC (for NAM, ATAF1, 2, and CUC2) family genes encode plant-specific transcription factors that play important roles in plant development regulation and in abiotic and biotic stresses. However, the function of NAC genes in grapevines is not clear. A novel NAC transcription factor, designated as VpNAC1, was isolated from Chinese wild Vitis pseudoreticulata. It belongs to the TERN subgroup and is a nuclear-targeting protein and functions as a transcriptional activator. Moreover, VpNAC1 was induced by the fungus Erysiphe necator and the exogenous hormones, particularly salicylic acid, methyl jasmonate and ethylene. Over-expression of VpNAC1 in tobacco plants enhanced their resistance to Erysiphe cichoracearum and Phytophthora parasitica var. nicotianae Tucker. These results suggest that VpNAC1 acts as a positive regulator in biotic stresses.

High productivity chromatography refolding process for Hepatitis B Virus X (HBx) protein guided by statistical design of experiment studies.

The Hepatitis B Virus X (HBx) protein is a potential therapeutic target for the treatment of hepatocellular carcinoma. However, consistent expression of the protein as insoluble inclusion bodies in bacteria host systems has largely hindered HBx manufacturing via economical biosynthesis routes, thereby impeding the development of anti-HBx therapeutic strategies. To eliminate this roadblock, this work reports the development of the first 'chromatography refolding'-based bioprocess for HBx using immobilised metal affinity chromatography (IMAC). This process enabled production of HBx at quantities and purity that facilitate their direct use in structural and molecular characterization studies. In line with the principles of quality by design (QbD), we used a statistical design of experiments (DoE) methodology to design the optimum process which delivered bioactive HBx at a productivity of 0.21 mg/ml/h at a refolding yield of 54% (at 10 mg/ml refolding concentration), which was 4.4-fold higher than that achieved in dilution refolding. The systematic DoE methodology adopted for this study enabled us to obtain important insights into the effect of different bioprocess parameters like the effect of buffer exchange gradients on HBx productivity and quality. Such a bioprocess design approach can play a pivotal role in developing intensified processes for other novel proteins, and hence helping to resolve validation and speed-to-market challenges faced by the biopharmaceutical industry today.

Purification, crystallization and preliminary X-ray diffraction studies of the arsenic repressor ArsR from Corynebacterium glutamicum.

ArsR is a member of the SmtB/ArsR family of metalloregulatory proteins that regulate prokaryotic arsenic-resistance operons. Here, the crystallization and preliminary X-ray diffraction studies of a cysteine-free derivative of ArsR from Corynebacterium glutamicum (CgArsR-C15/16/55S) are reported. CgArsR-C15/16/55S was expressed, purified, crystallized and X-ray diffraction data were collected to 1.86 Å resolution. The protein crystallized in a tetragonal space group (P4), with unit-cell parameters a = b = 41.84, c = 99.47 Å.

RepC protein of the octopine-type Ti plasmid binds to the probable origin of replication within repC and functions only in cis.

Vegetative replication and partitioning of many plasmids and some chromosomes of alphaproteobacteria are directed by their repABC operons. RepA and RepB proteins direct the partitioning of replicons to daughter cells, while RepC proteins are replication initiators, although they do not resemble any characterized replication initiation protein. Here we show that the replication origin of an Agrobacterium tumefaciens Ti plasmid resides fully within its repC gene. Purified RepC bound to a site within repC with moderate affinity, high specificity and with twofold cooperativity. The binding site was localized to an AT-rich region that contains a large number of GANTC sites, which have been implicated in replication regulation in related organisms. A fragment of RepC containing residues 26-158 was sufficient to bind DNA, although with limited sequence specificity. This portion of RepC is predicted to have structural homology to members of the MarR family of transcription factors. Overexpression of RepC in A. tumefaciens caused large increases in copy number in cis but did not change the copy number of plasmids containing the same oriV sequence in trans, confirming other observations that RepC functions only in cis.

Development of a high-throughput screen for inhibitors of Epstein-Barr virus EBNA1.

Latent infection with Epstein-Barr virus (EBV) is a carcinogenic cofactor in several lymphoid and epithelial cell malignancies. At present, there are no small-molecule inhibitors that specifically target EBV latent infection or latency-associated oncoproteins. EBNA1 is an EBV-encoded sequence-specific DNA binding protein that is consistently expressed in EBV-associated tumors and required for stable maintenance of the viral genome in proliferating cells. EBNA1 is also thought to provide cell survival function in latently infected cells. In this work, the authors describe the development of a biochemical high-throughput screening (HTS) method using a homogeneous fluorescence polarization (FP) assay monitoring EBNA1 binding to its cognate DNA binding site. An FP-based counterscreen was developed using another EBV-encoded DNA binding protein, Zta, and its cognate DNA binding site. The authors demonstrate that EBNA1 binding to a fluorescent-labeled DNA probe provides a robust assay with a Z factor consistently greater than 0.6. A pilot screen of a small-molecule library of ~14,000 compounds identified 3 structurally related molecules that selectively inhibit EBNA1 but not Zta. All 3 compounds had activity in a cell-based assay specific for the disruption of EBNA1 transcription repression function. One of the compounds was effective in reducing EBV genome copy number in Raji Burkitt lymphoma cells. These experiments provide a proof of concept that small-molecule inhibitors of EBNA1 can be identified by biochemical HTS of compound libraries. Further screening in conjunction with medicinal chemistry optimization may provide a selective inhibitor of EBNA1 and EBV latent infection.

Expression, purification, and characterization of recombinant human pancreatic duodenal homeobox-1 protein in Pichia pastoris.

Pancreatic duodenal hemeobox-1 (PDX1) is essential for the development of the embryonic pancreas and plays a key role in pancreatic beta-cell differentiation, maturation, regeneration, and maintenance of normal pancreatic beta-cell insulin-producing function. Purified recombinant PDX1 (rPDX1) may be a useful tool for many research and clinical applications, however, using the Escherichia coli expression system has several drawbacks for producing quality PDX1 protein. To explore the yeast expression system for generating rPDX1 protein, the cDNA coding for the full-length human PDX1 gene was cloned into the secreting expression organism Pichia pastoris. SDS-PAGE and western blotting analysis of culture medium from methanol-induced expression yeast clones demonstrated that the rPDX1 was secreted into the culture medium, had a molecular weight by SDS-PAGE of 50kDa, and was glycosylated. The predicted size of the mature unmodified PDX1 polypeptide is 31kDa, suggesting that eukaryotic post-translational modifications are the result of the increased molecular weight. The recombinant protein was purified to greater than 95% purity using a combined ammonium sulfate precipitation with heparin-agarose chromatography. Finally, 120mug of the protein was obtained in high purity from 1L of the culture supernatant. Bioactivity of the rPDX1 was confirmed by the ability to penetrate cell membranes and activation of an insulin-luciferase reporter gene. Our results suggest that the P. pastoris expression system can be used to produce a fully functional human rPDX1 for both research and clinical application.

Purification and identification of estrogen receptor alpha co-regulators in osteoclasts.

Mature osteoclasts are multinuclear, macrophage-like cells derived from hematopoietic stem cells in the bone marrow. Several transcription factors regulating osteoclast differentiation have been identified. However, the molecular basis of transcriptional regulation in osteoclasts at epigenetic levels is largely unknown. In fact, no osteoclast-specific transcriptional co-regulators have been characterized. Recently, selective ablation of estrogen receptor alpha (ERalpha) in mature osteoclasts derived from female mice (ERalpha(Deltaoc/Deltaoc)) exhibited trabecular bone loss due to induced apoptosis via upregulated expression of Fas ligand mRNA. In general, the component composition of the ERalpha-associated co-activator complex and its expression levels are distinct among tissues. However, ERalpha transcriptional co-regulators in mature osteoclasts remain unclear. In the present study, we achieved large-scale cultivation of mature, multinucleated osteoclasts and established a purification system for ERalpha-associated proteins. In addition to co-regulators previously found in other ERalpha target cells, several unexpected factors were found such as CAP-H. The mRNA expression level of CAP-H was high during osteoclast differentiation. These results demonstrate the existence of osteoclast-specific transcriptional co-regulators supporting ERalpha function.

GST-tagged mouse estrogen receptor alpha-transactivation domain fusion protein is specifically degraded during its over-expression in E. coli and purification.

Escherichia coli BL21 (DE3) is commonly used for the overproduction of fusion proteins. Using this system, we recently reported the overproduction of histidine-tagged mouse estrogen receptor (ER) alpha-ligand binding domain as an intact 30 kD protein and its inhibitory effect on the growth of bacteria. However, when GST-tagged mouse ERalpha transactivation domain (TAD) was overproduced using this system, it showed no effect on the growth of bacteria but was specifically degraded during its expression and purification. Here we report the expression of 47 kD GST-tagged mouse ERalpha-TAD protein, which was degraded partially and specifically into 46 and 43 kD fragments. This fusion protein was further degraded into 37, 31, 29 and 26 kD fragments during its purification by affinity chromatography. Such specific degradation of GST-tagged mouse ERalpha-TAD during its overproduction in E. coli and purification indicates the induction of specific protease and suggests the modification of expression system.

Construction and expression of a spliced variant of Epstein-Barr virus bzlf1 and preparation of its polyclonal antibody.

The BZLF1 gene-encoded protein, Zta (EB1, ZEBRA), is a key transcriptional activator of induction of the lytic cycle of EBV. Zta; it contains a basic region with homology to the DNA binding domains of the AP-1 family. In this study, an alternatively spliced BZLF1 (Delta BZLF1) cDNA lacking exon 2, which encodes the DNA-binding domain of Zta, was isolated from B95-8 marmoset cell line releasing EBV. The cDNA was inserted into a prokaryotic expression vector pET-28a+. The His-tagged recombinant protein was overproduced in E. coli BL21(DE3) and purified by nickel affinity chromatography. The purified fraction was characterized by Western blot and MALDI-TOF-MS analysis and used as an antigen to immunize mice. The antibody against Delta Zta can recognize both denatured and natural Zta protein. The Delta Zta protein and its antibody can be used to further investigate its unknown functions.

Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition.

The cytolytic activity of natural killer (NK) cells is regulated by inhibitory receptors that detect the absence of self molecules on target cells. Structural studies of missing self recognition have focused on NK receptors that bind MHC. However, NK cells also possess inhibitory receptors specific for non-MHC ligands, notably cadherins, which are downregulated in metastatic tumors. We determined the structure of killer cell lectin-like receptor G1 (KLRG1) in complex with E-cadherin. KLRG1 mediates missing self recognition by binding to a highly conserved site on classical cadherins, enabling it to monitor expression of several cadherins (E-, N-, and R-) on target cells. This site overlaps the site responsible for cell-cell adhesion but is distinct from the integrin alpha(E)beta(7) binding site. We propose that E-cadherin may coengage KLRG1 and alpha(E)beta(7) and that KLRG1 overcomes its exceptionally weak affinity for cadherins through multipoint attachment to target cells, resulting in inhibitory signaling.

The roles of the PDZ-containing proteins bridge-1 and PDZD2 in the regulation of insulin production and pancreatic beta-cell mass.

PDZ domains are versatile protein interaction modules with the ability to dimerize and to recognize internal and carboxy-terminal peptide motifs. Their function in mediating the formation of multi-molecular signaling complexes is best understood at neuronal and epithelial membranes. In a screen for interactors that regulate transcription factor function in pancreatic beta cells, we isolated two PDZ-containing proteins Bridge-1 (PSMD9) and PDZD2, which contain one and six PDZ domains, respectively. Here, we review their functions in the regulation of pancreatic beta cells as a nuclear coactivator or extracellular signaling molecule. Bridge-1 interacts with both E12 and PDX-1 to stimulate insulin promoter activity. Recent gain-of-function analysis in both cell and transgenic models has revealed its functions to regulate both insulin gene expression and pancreatic beta-cell survival. Little is known about the intracellular function of PDZD2 that is predominantly localized to the endoplasmic reticulum of INS-1E cells. Interestingly, PDZD2 is proteolytically processed by caspase-3 to generate a carboxy-terminal secreted protein (sPDZD2) containing two PDZ domains. Expressed in fetal pancreatic progenitor and INS-1E cells, sPDZD2 when added as recombinant protein exerts concentration-dependent mitogenic effects on beta-like cells. We propose that the PDZ domain proteins Bridge-1 and PDZD2 likely transduce signals that regulate insulin production, proliferation, and survival of pancreatic beta cells.

Comparison of Escherichia coli uropathogenic genes (kps, usp and ireA) and enteroaggregative genes (aggR and aap) via multiplex polymerase chain reaction from suprapubic urine specimens of young children with fever.

OBJECTIVE: Escherichia coli is the most frequently identified microbiological agent in childhood urinary tract infections (UTIs). However, the pathogenic role of this organism in young children remains to be clearly elucidated. So far, no studies have been conducted in which multiplex polymerase chain reaction (PCR) has been applied to determine the association between childhood UTIs and E. coli and urovirulent genes. MATERIAL AND METHODS: Altogether, 330 suprapubic urine specimens from febrile young children were cultured. In 33 of the cases, E. coli was identified; among these cases, 18 had a UTI (>10(4)-10(5) cfu/ml), four had a suspected UTI (>10(2)-10(3) cfu/ml) and 11 did not have UTIs (10(2) cfu/ml). Using multiplex PCR, three uropathogenic E. coli (UPEC) genes and two enteroaggregative E. coli (EAEC) genes were detected. RESULTS: In the UTI-UPEC cases, the kps gene was detected in 18 of 22 cases (82%) and the usp gene in 16 of 22 cases (73%). Among the 18 cases of children with UTIs characterized by 10(4)-10(5) E. coli cfu/ml, urinary tract abnormalities were identified via dimercaptosuccinic acid scans in seven of 18 cases (39%) and via voiding cystourethrograms in four of the 18 cases (22%). CONCLUSIONS: The UPEC kps and usp genes were clearly associated with childhood UTIs, and may also be associated with kidney or urinary tract dysfunctions in young children. Escherichia coli colony count numbers in excess of 10(4)-10(5) cfu/ml in the suprapubic urine were considered to be strong evidence of UTI in infants.

A cytoplasmic variant of the KH-type splicing regulatory protein serves as a decay-promoting factor for phosphoglycerate kinase 2 mRNA in murine male germ cells.

Phosphoglycerate kinase 2 (PGK2) is a germ cell-specific protein whose mRNA is translationally regulated in the mammalian testis. Using RNA affinity chromatography with the 3'-untranslated region (UTR) of Pgk2 mRNA and adult testis extracts, several associated proteins including a novel isoform of the AU-rich element RNA-binding protein and KH-type splicing regulatory protein (KSRP) were identified. KSRP, a protein of approximately 75 kDa, is widely expressed in somatic and germ cells where it is primarily nuclear. In addition to the approximately 75-kDa KSRP, a approximately 52-kD KSRP, t-KSRP, is present in the cytoplasm of a subpopulation of germ cells. t-KSRP binds directly to a 93-nt sequence (designated the F1 region) of the 3'-UTR of the Pgk2 mRNA and destabilizes Pgk2 mRNA constructs in testis extracts and in transfected cells. We conclude that this testicular variant of the multifunctional nucleic acid-binding protein, KSRP, serves as a decay-promoting factor for Pgk2 mRNA in male germ cells.

Mechanism of homotropic control to coordinate hydrolysis in a hexameric AAA+ ring ATPase.

AAA(+) proteins are ubiquitous mechanochemical ATPases that use energy from ATP hydrolysis to remodel their versatile substrates. The AAA(+) characteristic hexameric ring assemblies raise important questions about if and how six often identical subunits coordinate hydrolysis and associated motions. The PspF AAA(+) domain, PspF(1-275), remodels the bacterial sigma(54)-RNA polymerase to activate transcription. Analysis of ATP substrate inhibition kinetics on ATP hydrolysis in hexameric PspF(1-275) indicates negative homotropic effects between subunits. Functional determinants required for allosteric control identify: (i) an important link between the ATP bound ribose moiety and the SensorII motif that would allow nucleotide-dependent *-helical */beta subdomain dynamics; and (ii) establishes a novel regulatory role for the SensorII helix in PspF, which may apply to other AAA(+) proteins. Consistent with functional data, homotropic control appears to depend on nucleotide state-dependent subdomain angles imposing dynamic symmetry constraints in the AAA(+) ring. Homotropic coordination is functionally important to remodel the sigma(54) promoter. We propose a structural symmetry-based model for homotropic control in the AAA(+) characteristic ring architecture.

Genome analysis identifies the p15ink4b tumor suppressor as a direct target of the ZNF217/CoREST complex.

The ZNF217 oncoprotein is a constituent of a core transcriptional complex that includes CoREST, histone deacetylase 1/2, lysine demethylase 1, and the C-terminal binding protein 1/2. We have combined genome-wide expression profiling and chromatin immunoprecipitation with directed selection and ligation (ChIP-DSL) to identify a subset of genes directly regulated by ZNF217. Our results establish p15(ink4b) as a direct target of the ZNF217 complex. Downregulation of ZNF217 in MCF-7 breast cancer cells resulted in a dramatic increase in p15(ink4b) expression and coincided with increases in dimethylation of H3-K4 and, surprisingly, a decrease in K9/K14-H3 acetylation. Stimulation of HaCaT cells with transforming growth factor beta (TGF-beta) resulted in a release of ZNF217 and a concomitant binding of SMAD2 to the proximal promoter, which preceded increases in ink4b protein expression. Furthermore, the changes in chromatin marks at the p15(ink4b) promoter following TGF-beta stimulation were similar to those observed following ZNF217 downregulation. Collectively, these results establish the ZNF217 complex as a novel negative regulator of the p15(ink4b) gene and may constitute an important link between amplification of ZNF217 and the loss of TGF-beta responsiveness in breast cancer.