Oncostatin M promotes cancer cell plasticity through cooperative STAT3-SMAD3 signaling.

Increasing evidence supports the idea that cancer cell plasticity promotes metastasis and tumor recurrence, resulting in patient mortality. While it is clear that the tumor microenvironment (TME) contributes to cancer cell plasticity, the specific TME factors most actively controlling plasticity remain largely unknown. Here, we performed a screen to identify TME cytokines and growth factors that promote epithelial-mesenchymal plasticity, and acquisition of cancer stem cell (CSC) properties. Of 28 TME cytokines and growth factors tested, we identified Oncostatin M (OSM) as the most potent inducer of mesenchymal/CSC properties. OSM-induced plasticity was Signal Transducer and Activator of Transcription 3 (STAT3)-dependent, and also required a novel intersection with transforming growth factor-ß (TGF-ß)/SMAD signaling. OSM/STAT3 activation promoted SMAD3 nuclear accumulation, DNA binding and induced SMAD3-dependent transcriptional activity. Suppression of TGF-ß receptor activity or ablation of SMAD3 or SMAD4, but not SMAD2, strongly suppressed OSM/STAT3-mediated plasticity. Moreover, removal of OSM or inhibition of STAT3 or SMAD3 resulted in a marked reversion to a non-invasive, epithelial phenotype. We propose that targeted blockade of the STAT3/SMAD3 axis in tumor cells may represent a novel therapeutic approach to prevent the plasticity required for metastatic progression and tumor recurrence.

The impact of postmastectomy and regional nodal radiation after neoadjuvant chemotherapy for clinically lymph node-positive breast cancer: a National Cancer Database (NCDB) analysis.

BACKGROUND: Following neoadjuvant chemotherapy (NAC), the optimal strategies for postmastectomy radiotherapy (PMRT) and regional nodal irradiation (RNI) after breast-conserving surgery (BCS) are controversial. In this analysis, we evaluate the impact of these radiotherapy (RT) approaches for women with clinically node-positive breast cancer treated with NAC in the National Cancer Database (NCDB). PATIENTS AND METHODS: Women with cT1-3 cN1 M0 breast cancer treated with NAC were divided into four cohorts by surgery [Mastectomy (Mast) versus BCS] and post-chemotherapy pathologic nodal status (ypN0 versus ypN+). Overall survival (OS) was estimated using the Kaplan-Meier method and RT approaches were analyzed using the log-rank test, multivariate Cox models, and propensity score-matched analyses. RESULTS: From 2003 to 2011, 15 315 cases were identified including 3040 Mast-ypN0, 7243 Mast-ypN+, 2070 BCS-ypN0, and 2962 BCS-ypN+ patients. On univariate analysis, PMRT was associated with improved OS for both Mast-ypN0 (P = 0.019) and Mast-ypN+ (P < 0.001) patients. On multivariate analyses adjusted for factors including age, comorbidity score, cT stage, in-breast pathologic complete response, axillary surgery, ypN stage, estrogen receptor status and hormone therapy, PMRT remained independently associated with improved OS among Mast-ypN0 [hazard ratio (HR) = 0.729, 95% confidence interval (CI) 0.566-0.939, P = 0.015] and Mast-ypN+ patients (HR = 0.772, 95% CI 0.689-0.866, P < 0.001). No differences in OS were observed with the addition of RNI to breast RT for BCS-ypN0 or BCS-ypN+ patients. Propensity score-matched analyses demonstrated identical patterns of significance. On subset analysis, OS was improved with PMRT in each pathologic nodal subgroup (ypN0, ypN1, and ypN2-3) (all P < 0.05). CONCLUSIONS: In the largest reported analysis of RT for cN1 patients treated with NAC, PMRT was associated with improved OS for all pathologic nodal subgroups. No OS differences were observed with the addition of RNI to breast RT.

Hyperactivation of EGFR and downstream effector phospholipase D1 by oncogenic FAM83B.

Despite the progress made in targeted anticancer therapies in recent years, challenges remain. The identification of new potential targets will ensure that the arsenal of cancer therapies continues to expand. FAM83B was recently discovered in a forward genetic screen for novel oncogenes that drive human mammary epithelial cell (HMEC) transformation. We report here that elevated FAM83B expression increases Phospholipase D (PLD) activity, and that suppression of PLD1 activity prevents FAM83B-mediated transformation. The increased PLD activity is engaged by hyperactivation of epidermal growth factor receptor (EGFR), which is regulated by an interaction involving FAM83B and EGFR. Preventing the FAM83B/EGFR interaction by site-directed mutation of lysine 230 of FAM83B suppressed PLD activity and MAPK signaling. Furthermore, ablation of FAM83B expression from breast cancer cells inhibited EGFR phosphorylation and suppressed cell proliferation. We propose that understanding the mechanism of FAM83B-mediated transformation will provide a foundation for future therapies aimed at targeting its function as an intermediary in EGFR, MAPK and mTOR activation.

An immunodominant La/SSB autoantibody proteome derives from public clonotypes.

The La/SSB autoantigen is a major target of long-term humoral autoimmunity in primary Sjögren's Syndrome (SS) and systemic lupus erythematosus. A majority of patients with linked anti-Ro60/Ro52/La responses target an NH2-terminal epitope designated LaA that is expressed on Ro/La ribonucleoprotein complexes and the surface membrane of apoptotic cells. In this study, we used high-resolution Orbitrap mass spectrometry to determine the clonality, isotype and V-region sequences of LaA-specific autoantibodies in seven patients with primary SS. Anti-LaA immunoglobulin (Ig)Gs purified from polyclonal sera by epitope-specific affinity chromatography were analysed by combined database and de-novo mass spectrometric sequencing. Autoantibody responses comprised two heavily mutated IgG1 kappa-restricted monoclonal species that were shared (public) across unrelated patients; one clonotype was specified by an IGHV3-30 heavy chain paired with IGKV3-15 light chain and the second by an IGHV3-43/IGKV3-20 pairing. Shared amino acid replacement mutations were also seen within heavy and light chain complementarity-determining regions, consistent with a common breach of B cell tolerance followed by antigen-driven clonal selection. The discovery of public clonotypic autoantibodies directed against an immunodominant epitope on La, taken together with recent findings for the linked Ro52 and Ro60 autoantigens, supports a model of systemic autoimmunity in which humoral responses against protein-RNA complexes are mediated by public sets of autoreactive B cell clonotypes.

Rescue administration of a helper-dependent adenovirus vector with long-term efficacy in dogs with glycogen storage disease type Ia.

Glycogen storage disease type Ia (GSD-Ia) stems from glucose-6-phosphatase (G6Pase) deficiency and causes hypoglycemia, hepatomegaly, hypercholesterolemia and lactic acidemia. Three dogs with GSD-Ia were initially treated with a helper-dependent adenovirus encoding a human G6Pase transgene (HDAd-cG6Pase serotype 5) on postnatal day 3. Unlike untreated dogs with GSD-Ia, all three dogs initially maintained normal blood glucose levels. After 6-22 months, vector-treated dogs developed hypoglycemia, anorexia and lethargy, suggesting that the HDAd-cG6Pase serotype 5 vector had lost efficacy. Liver biopsies collected at this time revealed significantly elevated hepatic G6Pase activity and reduced glycogen content, when compared with affected dogs treated only by frequent feeding. Subsequently, the HDAd-cG6Pase serotype 2 vector was administered to two dogs, and hypoglycemia was reversed; however, renal dysfunction and recurrent hypoglycemia complicated their management. Administration of a serotype 2 HDAd vector prolonged survival in one GSD-Ia dog to 12 months of age and 36 months of age in the other, but the persistence of long-term complications limited HDAd vectors in the canine model for GSD-Ia.

Hdm2 is a ubiquitin ligase of Ku70-Akt promotes cell survival by inhibiting Hdm2-dependent Ku70 destabilization.

Earlier, we have reported that 70 kDa subunit of Ku protein heterodimer (Ku70) binds and inhibits Bax activity in the cytosol and that ubiquitin (Ub)-dependent proteolysis of cytosolic Ku70 facilitates Bax-mediated apoptosis. We found that Hdm2 (human homolog of murine double minute) has an ability to ubiquitinate Ku70 and that Hdm2 overexpression in cultured cells causes a decrease in Ku70 expression levels. An interaction between Ku70 and Hdm2 was shown by means of immunoprecipitation, whereas none could be shown between 80 kDa subunit of Ku protein heterodimer and Hdm2. Vascular endothelial growth factor (VEGF) is known to inhibit endothelial cell (EC) apoptosis through an Akt-mediated survival kinase signal; however, the mechanism underlying this inhibition of apoptosis has not been fully elucidated. We found that VEGF inhibited cytosolic Ku70 degradation induced by apoptotic stress. It is known that Akt-dependent phosphorylation of Hdm2 causes nuclear translocation of Hdm2 followed by Hdm2-mediated inactivation of p53. We found that VEGF stimulated nuclear translocation of Hdm2 in EC and efficiently inhibited Ku70 degradation. We also found that constitutively active Akt, but not kinase-dead Akt, inhibited Ku70 degradation in the cytosol. Furthermore, Ku70 knockdown diminished antiapoptotic activity of Akt. Taken together, we propose that Hdm2 is a Ku70 Ub ligase and that Akt inhibits Bax-mediated apoptosis, at least in part, by maintaining Ku70 levels through the promotion of Hdm2 nuclear translocation.

Rb/E2F4 and Smad2/3 link survivin to TGF-beta-induced apoptosis and tumor progression.

Survivin is a prosurvival protein overexpressed in many cancers through mechanisms that remain poorly explored, and is implicated in control of tumor progression and resistance to cancer chemotherapeutics. Here, we report a critical role for survivin in the induction of apoptosis by transforming growth factor-beta (TGF-beta). We show that TGF-beta rapidly downregulates survivin expression in prostate epithelial cells, through a unique mechanism of transcriptional suppression involving Smads 2 and 3, Rb/E2F4, and the cell-cycle repressor elements CDE and CHR. This TGF-beta response is triggered through a Smad2/3-dependent hypophosphorylation of Rb and the subsequent association of the Rb/E2F4 repressive complex to CDE/CHR elements in the proximal region of the survivin promoter. Viral-mediated gene delivery experiments, involving overexpressing or silencing survivin, reveal critical roles of survivin in apoptosis induced by TGF-beta alone or in cooperation with cancer therapeutic agents. We propose a novel TGF-beta/Rb/survivin axis with a putative role in the functional switch of TGF-beta from tumor suppressor to tumor promoter.

Different temporal expression of immunodominant Ro60/60 kDa-SSA and La/SSB apotopes.

Opsonization of apoptotic cardiocytes by maternal anti-Ro/SSA and anti-La/SSB antibodies contributes to tissue injury in the neonatal lupus syndrome. The objective of the current study was to quantify the surface membrane expression of Ro/La components during different phases of apoptosis and map the Ro/La apotopes (epitopes expressed on apoptotic cells) bound by cognate antibodies. Multi-parameter flow cytometry was used to define early and late apoptotic populations and their respective binding by monospecific anti-Ro and anti-La IgGs. Anti-Ro60 bound specifically to early apoptotic Jurkat cells and remained accessible on the cell surface throughout early and late apoptosis. In contrast, anti-La bound exclusively to late apoptotic cells in experiments controlled for non-specific membrane leakage of IgG. Ro52 was not accessible for antibody binding on either apoptotic population. The immunodominant NH2-terminal and RNA recognition motif (RRM) epitopes of La were expressed as apotopes on late apoptotic cells, confirming recent in vivo findings. An immunodominant internal epitope of Ro60 that contains the RRM, and is recognized by a majority of sera from mothers of children with congenital heart block (CHB) and patients with primary Sjögren's syndrome, was also accessible as an apotope on early apoptotic cells. The distinct temporal expression of the immunodominant Ro60 and La apotopes indicates that these intracellular autoantigens translocate independently to the cell surface, and supports a model in which maternal antibody populations against both Ro60 and La apotopes act in an additive fashion to increase the risk of tissue damage in CHB.

Autoantibody-mediated bladder dysfunction in type 1 diabetes.

Bladder dysfunction is a common complication of diabetic autonomic neuropathy; however, its cause remains uncertain. We have recently identified a novel IgG autoantibody (Ab) in patients with type 1 diabetes that acts as an agonist at the dihydropyridine (DHP) site of L-type voltage-gated calcium channels (VGCC), disrupting neuronal regulation of visceral smooth muscle. In the present study, passive transfer to mice of IgG from patients with type 1 diabetes was used to investigate the role of anti-VGCC Abs in mediating diabetic bladder dysfunction. Injection of mice with diabetic immunoglobulin (IgG) with anti-VGCC activity induced features of an overactive bladder, including phasic detrusor contractions and a loss of bladder wall compliance. The bladder overactivity is mimicked by the DHP agonist Bay K8644, reversed by the DHP antagonist nicardipine, but is insensitive to the motor nerve blocker tetrodotoxin, indicating that the anti-VGCC Ab acts at the level of the bladder detrusor itself. This study reports the first evidence of Ab-mediated bladder dysfunction in type 1 diabetes, which may be part of a wider spectrum of smooth muscle and cardiac abnormalities.

Costs and benefits of cold tolerance in transgenic Arabidopsis thaliana.

Cold tolerance in plants is an ecologically important trait that has been under intensive study for basic and applied reasons. Determining the fitness benefits and costs of cold tolerance has previously been difficult because cold tolerance is normally an induced trait that is not expressed in warm environments. The recent creation of transgenic plants constitutively expressing cold tolerance genes enables the investigation of the fitness consequences of cold tolerance in multiple temperature environments. We studied three genes from the CBF (C-repeat/dehydration responsive element binding factor) cold tolerance pathway, CBF1, 2 and 3, in Arabidopsis thaliana to test for benefits and costs of constitutive cold tolerance. We used multiple insertion lines for each transgene and grew the lines in cold and control conditions. Costs of cold tolerance, as determined by fruit number, varied by individual transgene. CBF2 and 3 overexpressers showed costs of cold tolerance, and no fitness benefits, in both environments. CBF1 overexpressing plants showed no fitness cost of cold tolerance in the control environment and showed a marginal fitness benefit in the cold environment. These results suggest that constitutive expression of traits that are normally induced in response to environmental stress will not always lead to costs in the absence of that stress, and that the ecological risks of CBF transgene escape should be assessed prior to their use in commercial agriculture.

Evaluation of the clinical and allergen specific serum immunoglobulin E responses to oral challenge with cornstarch, corn, soy and a soy hydrolysate diet in dogs with spontaneous food allergy.

Fourteen dogs with known clinical hypersensitivity to soy and corn were maintained on a limited antigen duck and rice diet until cutaneous manifestations of pruritus were minimal (78 days). Sequential oral challenges with cornstarch, corn and soy were then performed. Subsequently, the dogs were fed a diet containing hydrolysed soy protein and cornstarch. Throughout the study period the dogs were examined for cutaneous manifestations of pruritus and, additionally, serum was collected for measurement of allergen-specific and total immunoglobulin (Ig)E concentrations. Intradermal testing with food antigens was performed prior to entry into the study and after 83 days. A statistically significant clinical improvement was measured between days 0 and 83. Significant pruritus was induced after oral challenge with cornstarch, corn and soy (P = 0.04, 0.002, 0.01, respectively) but not with the hydrolysed diet (P = 0.5). The positive predictive value of the skin test for soy and corn allergy was reduced after feeding a soy and corn free diet. Although increases in soy and corn-specific serum IgE concentrations were measured in individual dogs post challenge they were not statistically significant and could not be used to predict clinical hypersensitivity.

MdmX binding to ARF affects Mdm2 protein stability and p53 transactivation.

Regulation of p53 involves a complex network of protein interactions. The primary regulator of p53 protein stability is the Mdm2 protein. ARF and MdmX are two proteins that have recently been shown to inhibit Mdm2-mediated degradation of p53 via distinct associations with Mdm2. We demonstrate here that ARF is capable of interacting with MdmX and in a manner similar to its association with Mdm2, sequestering MdmX within the nucleolus. The sequestration of MdmX by ARF results in an increase in p53 transactivation. In addition, the redistribution of MdmX by ARF requires that a nucleolar localization signal be present on MdmX. Although expression of either MdmX or ARF leads to Mdm2 stabilization, coexpression of both MdmX and ARF results in a decrease in Mdm2 protein levels. Similarly, increasing ARF protein levels in the presence of constant MdmX and Mdm2 leads to a dose-dependent decrease in Mdm2 levels. Under these conditions, ARF can synergistically reverse the ability of Mdm2 and MdmX to inhibit p53-dependent transactivation. Finally, the association and redistribution of MdmX by ARF has no effect on the protein stability of either ARF or MdmX. Taken together, these results demonstrate that the interaction between MdmX and ARF represents a novel pathway for regulating Mdm2 protein levels. Additionally, both MdmX and Mdm2, either individually or together, are capable of antagonizing the effects of the ARF tumor suppressor on p53 activity.

Secondary hypoparathyroidism attributed to hypomagnesemia in a dog with protein-losing enteropathy.

Severe hypomagnesemia (0.8 mg/dl; reference range, 1.6 to 2.3 mg/dl), hypocalcemia, and protein-losing enteropathy were identified in a 5-year-old castrated male 3-kg (6.6 lb) Shih Tzu examined because of anorexia, lethargy, paresis, and abdominal distention. Histologic examination of intestinal biopsy specimens revealed lymphangiectasia and lymphocytic, plasmacytic, neutrophilic infiltrates. Initial treatment included administration of magnesium (0.80 mEq/kg [0.36 mEq/lb]) of body weight in a balanced electrolyte solution. This treatment resulted in normalization of the serum magnesium concentration (1.7 mg/dl); resolution of the lethargy, paresis, and tachycardia; and an increase in the serum parathyroid hormone and ionized calcium concentrations. Findings were consistent with secondary hypoparathyroidism attributable to hypomagnesemia. Magnesium concentration should be monitored in all dogs with gastrointestinal tract disease, especially those with protein-losing enteropathy, anorexia, and weakness.

Interactions between type III secretion apparatus components from Yersinia pestis detected using the yeast two-hybrid system.

Interactions among the Yersinia secretion (Ysc) proteins of Yersinia pestis were explored using the yeast two-hybrid system. Various pairwise combinations of the yscEFGHIKLN and Q genes fused to the DNA-binding or activation domain of the yeast GAL4 gene were introduced into yeast, and expression of a reporter gene encoding beta-galactosidase was detected. Combinations of yscN and yscL, yscL and yscQ, and yscQ and yscK resulted in high levels of reporter gene activation. These results suggest that YscL interacts with both YscN and YscQ, and that YscQ interacts with both YscL and YscK. Three-hybrid analyses using plasmid pDELA to target a third hybrid protein to the yeast nucleus was used to detect the formation of ternary protein complexes. Using the three-hybrid system, YscQ expressed from plasmid pDELA was able to bring together the YscK and YscL fusion proteins. In a similar manner, YscL expressed from plasmid pDELA was able to bring together the YscN and YscQ fusion proteins. Together, these results suggest that a complex composed of YscN, YscQ, YscK and YscL is involved in the assembly and/or function of the Y. pestis type III secretion apparatus.

MdmX protects p53 from Mdm2-mediated degradation.

The p53 tumor suppressor protein is stabilized in response to cellular stress, resulting in activation of genes responsible for either cell cycle arrest or apoptosis. The cellular pathway for releasing normal cells from p53-dependent cell cycle arrest involves the Mdm2 protein. Recently, a p53-binding protein with homology to Mdm2 was identified and called MdmX. Like Mdm2, MdmX is able to bind p53 and inhibit p53 transactivation; however, the ability of MdmX to degrade p53 has yet to be examined. We report here that MdmX is capable of associating with p53 yet is unable to facilitate nuclear export or induce p53 degradation. In addition, expression of MdmX can reverse Mdm2-targeted degradation of p53 while maintaining suppression of p53 transactivation. Using a series of MdmX deletions, we have determined that there are two distinct domains of the MdmX protein that can stabilize p53 in the presence of Mdm2. One domain requires MdmX interaction with p53 and results in the retention of both proteins within the nucleus and repression of p53 transactivation. The second domain involves the MdmX ring finger and results in stabilization of p53 and an increase in p53 transactivation. The potential basis for stabilization and increased p53 transactivation by the MdmX ring finger domain is discussed. Based on these observations, we propose that the MdmX protein may function to maintain a nuclear pool of p53 protein in undamaged cells.

Constitutive mdmx expression during cell growth, differentiation, and DNA damage.

The mdmx gene was shown to possess high homology to the mdm-2 gene and to encode a protein that can bind p53 and block p53 transactivation. Because Mdm-2 protein blocks the growth-suppressive activity of the p53 tumor-suppressor protein through similar activities, we examined the expression patterns of mdmx to determine how MdmX expression correlates with p53 protein levels. In this study, the expression pattern and protein levels of mdmx were examined in a number of cell culture systems. Like mdm-2, mdmx gene expression was constitutive during serum deprivation/restimulation of murine fibroblasts and differentiation of either murine teratocarcinoma or preadipocyte cells. In contrast, whereas mdm-2 gene expression was induced after cisplatin damage to ovarian carcinoma cells, mdmx expression remained constitutive. Because p53 transactivation is critical following a genotoxic stress, we examined p53:MdmX complexes after in vitro DNA-PK phosphorylation, a posttranslational modification that blocks p53 association with Mdm-2. The DNA-PK phosphorylation of p53 was capable of inhibiting p53:MdmX association. Thus, whereas DNA damage does not regulate mdmx mRNA levels, posttranslational modifications induced during DNA damage may block p53:MdmX association in vivo. These results demonstrate that, in the cell lines examined, mdmx gene expression remains constitutive during cell proliferation and differentiation or following DNA damage. Taken together, the data suggest that cells retain a constant level of MdmX. Thus, in undamaged cells, there exists the potential for an MdmX:p53 reservoir.

DsbA is required for stable expression of outer membrane protein YscC and for efficient Yop secretion in Yersinia pestis.

The role of the periplasmic disulfide oxidoreductase DsbA in Yop secretion was investigated in Yersinia pestis. A Y. pestis dsbA mutant secreted reduced amounts of the V antigen and Yops and expressed reduced amounts of the full-sized YscC protein. Site-directed mutagenesis of the four cysteine residues present in the YscC protein resulted in defects similar to those found in the dsbA mutant. These results suggest that YscC contains at least one disulfide bond that is essential for the function of this protein in Yop secretion.

Assessing insulin sensitivity in the cat: evaluation of the hyperinsulinemic euglycemic clamp and the minimal model analysis.

Current methods of assessing glucose tolerance in the cat are inadequate for quantifying insulin sensitivity. The hyperinsulinemic euglycemic clamp (EGC) and the minimal model analysis (MMA) of the frequently sampled intravenous glucose tolerance test (FSIGT) have been used in other species to assess the effects of insulin on glucose homeostasis. Each of these procedures was performed on healthy cats, two weeks apart, to generate indices of insulin sensitivity - M/I (amount of glucose metabolised per unit of plasma insulin concentration) from the EGC and S(I) (insulin sensitivity) from the MMA. Close and significant correlation between M/I and S(I) in individual cats was found (r=0.91, P=0.032). Use of these research methods may further our understanding of feline diabetes mellitus and other endocrinopathies.

YscB of Yersinia pestis functions as a specific chaperone for YopN.

Following contact with a eucaryotic cell, Yersinia species pathogenic for humans (Y. pestis, Y. pseudotuberculosis, and Y. enterocolitica) export and translocate a distinct set of virulence proteins (YopE, YopH, YopJ, YopM, and YpkA) from the bacterium into the eucaryotic cell. During in vitro growth at 37 degrees C in the presence of calcium, Yop secretion is blocked; however, in the absence of calcium, Yop secretion is triggered. Yop secretion occurs via a plasmid-encoded type III, or "contact-dependent," secretion system. The secreted YopN (also known as LcrE), TyeA, and LcrG proteins are necessary to prevent Yop secretion in the presence of calcium and prior to contact with a eucaryotic cell. In this paper we characterize the role of the yscB gene product in the regulation of Yop secretion in Y. pestis. A yscB deletion mutant secreted YopM and V antigen both in the presence and in the absence of calcium; however, the export of YopN was specifically reduced in this strain. Complementation with a functional copy of yscB in trans completely restored the wild-type secretion phenotype for YopM, YopN, and V antigen. The YscB amino acid sequence showed significant similarities to those of SycE and SycH, the specific Yop chaperones for YopE and YopH, respectively. Protein cross-linking and immunoprecipitation studies demonstrated a specific interaction between YscB and YopN. In-frame deletions in yopN eliminating the coding region for amino acids 51 to 85 or 6 to 100 prevented the interaction of YopN with YscB. Taken together, these results indicate that YscB functions as a specific chaperone for YopN in Y. pestis.