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The results of clinical trials evaluating the efficacy of HER2 inhibitors in patients with breast cancer indicate that the correlation between HER2 receptor levels and patient outcomes is as low as 50%. The relatively weak correlation between HER2 status and response to HER2-targeting drugs suggests that measurement of HER2 signaling activity, rather than absolute HER2 levels, may more accurately diagnose HER2-driven breast cancer. A new diagnostic test, the CELx HER2 Signaling Profile (CELx HSP) test, is demonstrated to measure real-time HER2 signaling function in live primary cells. In the present study, epithelial cells extracted fresh from breast cancer patient tumors classified as HER2 negative (HER2-, n = 34 of which 33 were estrogen receptor positive) and healthy subjects (n = 16) were evaluated along with reference breast cancer cell lines (n = 19). Live cell response to specific HER2 agonists (NRG1b and EGF) and antagonist (pertuzumab) was measured. Of the HER2- breast tumor cell samples tested, 7 of 34 patients (20.5%; 95% CI = 10%-37%) had HER2 signaling activity that was characterized as abnormally high. Amongst the tumor samples there was no correlation between HER2 protein status (by cell cytometry) and HER2 signaling activity (hyperactive or normal) (Regression analysis P = 0.144, R2 = 0.068). One conclusion is that measurement of HER2 signaling activity can identify a subset of breast cancers with normal HER2 receptor levels with abnormally high levels of HER2 signaling. This result constitutes a new subtype of breast cancer that should be considered for treatment with HER2 pathway inhibitors.

Transforming growth factor (TGF)-[[beta].sub.1] is one of the most pleiotropic and multifunctional peptides known. While the cardioprotective effect of TGF-[[beta].sub.1] during ischemia is well known, the specific role of TGF-[[beta].sub.1] in altering the cardiac remodeling process remains unclear. This study was designed to examine the regulation of hypoxia-reoxygenation-mediated collagen type I expression and activity of matrix metalloproteinases (MMPs) by overexpression of TGF-[[beta].sub.1] in cultured HL-1 mouse cardiomyocytes. TGF-[[beta].sub.1] was overexpressed in cardiomyocytes by transfection with adeno-associated virus (AAV)/TGF-[[beta].sup.Latent.sub.1] or with AAV/TGF-[[beta].sup.ACT.sub.1] (active TGF-[[beta].sub.1]). Twenty-four hours of hypoxia followed by 3 h of reoxygenation (H-R) markedly enhanced (pro)collagen type I expression and activity of MMPs concomitant with an increase in reactive oxygen species (ROS) release and LOX-I expression. Over-expression of TGF-[[beta].sub.1] reduced these alterations induced by H-R. TGF-[[beta].sub.1] overexpression also blocked H-R-mediated p38 and p44/42 MAPK activation. Transfection with AAV/TGF-[[beta].sup.ACT.sub.1] was superior to that with AAV/TGF-[[beta].sup.Latent.sub.1]. These data for the first time demonstrate that H-R induces signals for cardiac remodeling in cardiomyocytes and TGF-[[beta].sub.1] can modulate, possibly via antioxidant mechanism, these signals. These findings contribute to further understanding of the role of TGF-[[beta].sub.1] in the cardiac remodeling process. transforming growth factor-[[beta].sub.1]; HL-1 adult murine cardiomyocytes; metalloproteinases

Background: Approximately 18-20% of all human breast cancers have overexpressed human epidermal growth factor receptor 2 (HER2). Standard clinical practice is to treat only overexpressed HER2 (HER2+) cancers with targeted anti-HER2 therapies. However, recent analyses of clinical trial data have found evidence that HER2-targeted therapies may benefit a sub-group of breast cancer patients with non-overexpressed HER2. This suggests that measurement of other biological factors associated with HER2 cancer, such as HER2 signaling pathway activity, should be considered as an alternative means of identifying patients eligible for HER2 therapies.Methods: A new biosensor-based test (CELxTM HSF) that measures HER2 signaling activity in live cells is demonstrated using a set of 19 human HER2+ and HER2- breast cancer reference cell lines and primary cell samples derived from two fresh patient tumor specimens. Pathway signaling is elucidated by use of highly specific agonists and antagonists. The test method relies upon well-established phenotypic, adhesion-related, impedance changes detected by the biosensor.Results: The analytical sensitivity and analyte specificity of this method was demonstrated using ligands with high affinity and specificity for HER1 and HER3. The HER2-driven signaling quantified ranged 50-fold between the lowest and highest cell lines. The HER2+ cell lines were almost equally divided into high and low signaling test result groups, suggesting that little correlation exists between HER2 protein expression and HER2 signaling level. Unexpectedly, the highest HER2-driven signaling level recorded was with a HER2- cell line.Conclusions: Measurement of HER2 signaling activity in the tumor cells of breast cancer patients is a feasible approach to explore as a biomarker to identify HER2-driven cancers not currently diagnosable with genomic techniques. The wide range of HER2-driven signaling levels measured suggests it may be possible to make a distinction between normal and abnormal levels of activity. Analytical validation studies and clinical trials treating HER2- patients with abnormal HER2-driven signaling would be required to evaluate the analytical and clinical validity of using this functional biomarker as a diagnostic test to select patients for treatment with HER2 targeted therapy. In clinical practice, this method would require patient specimens be delivered to and tested in a central lab. [ABSTRACT FROM AUTHOR]

The article discusses a study done on the link between angiotensin II and lipoprotein receptor-1 (LOX-1) activation. Angiotensin II upregulates lectin-like oxidized LOX-1 receptors, which in turn activate angiotensin II. The study tested whether an interruption to this cycle could cause hypertension. These tests showed that LOX-1 is an important modulator of angiotensin II hypertension.

Transforming growth factor β1 (TGFβ1) activation leads to tissue fibrosis. Here, we report on the role of LOX-1, a lectin-like 52-kDa receptor for oxidized low density lipoprotein, in TGFβ1- mediated collagen expression and underlying signaling in mouse cardiac fibroblasts. TGFβ1 was overexpressed in wild- type (WT) and LOX-1 knock-out mouse cardiac fibroblasts by transfection with adeno-associated virus type 2 vector carrying the active TGFβ1 moiety (AAV/TGFβ1ACT). Transfection of WT mouse cardiac fibroblasts with AAV/TGFβ1ACT markedly enhanced the expression of NADPH oxidases (p22phox, p47phox, and gp91phox subunits) and LOX-1, formation of reactive oxygen species, and collagen synthesis, concomitant with an increase in the activation of p38 and p44/42 mitogen-activated protein kinases (MAPK). The TGFβ1-mediated increase in collagen synthesis was markedly attenuated in the LOX-1 knock-out mouse cardiac fibroblasts as well as in WT mouse cardiac fibroblasts treated with a specific anti-LOX-1 antibody. Treatment with anti-LOX-1 antibody also reduced NADPH oxidase expression and MAPK activation. The NADPH oxidase inhibitors and gp91phox small interfering RNA reduced LOX-1 expression, MAPK activation, and collagen formation. The p38 MAPK inhibitors as well as the p44/42 MAPK inhibitors reduced collagen formation without affecting LOX-1 expression in cardiac fibroblasts. These observations suggest that collagen synthesis in cardiac fibroblasts involves a facilitative interaction between TGFβ1-NADPH oxidase and LOX-1. Further, the activation of MAPK pathway appears to be downstream of TGFβ1-reactive oxygen species-LOX-1 cascade. [ABSTRACT FROM AUTHOR]

Abstract: Objective: Chronic ischemia is associated with alterations in genes that result in myocardial remodeling. An important biochemical basis of cardiac remodeling is generation of reactive oxygen species (ROS). A few studies have suggested that acute ischemia triggers signals for remodeling. We examined the hypothesis that targeted deletion of lectin-like oxidized-LDL receptor (LOX-1) may inhibit signals related to cardiac remodeling. Methods and results: We generated LOX-1 knockout (KO) mice on C57BL/6 (wild-type mice) background, and subjected wild-type and KO mice to ischemia–reperfusion (I–R). The wild-type mice developed a marked reduction in left ventricular systolic pressure and ±dp/dtmax and an increase in left ventricular end-diastolic pressure following I–R, and this change was much less in the LOX-1 KO mice, indicating preservation of left ventricular function with LOX-1 deletion. There was evidence for marked oxidative stress (NADPH oxidase expression, malondialdehyde and 8-isoprostane) following I–R in the wild-type mice, much less so in the LOX-1 KO mice (P <0.01). In concert, collagen deposition (Masson''s trichrome and Picro-sirius red staining) increased dramatically in the wild-type mice, but only half as much in the LOX-1 KO mice (P <0.01). Collagen staining data was corroborated with procollagen-I expression. Further, fibronectin and osteopontin expression increased in the wild-type mice, but to a much smaller extent in the LOX-1 KO mice (P <0.01). Conclusions: These findings provide compelling evidence that LOX-1 is a key modulator of cardiac remodeling which starts immediately following I–R. [Copyright &y& Elsevier]

The article discusses a study which identified the role of angiotensin II in capillary formation from endothelial cells through the LOX-1 dependent redox-sensitive pathway. LOX-1 is a lectin-like receptor for oxidized low-density lipoprotein. It was found that angiotensin II inhibited mitogen-activated protein kinase inhibitor. The article concludes that small concentrations of angiotensin II promote capillary formation by inducing the expresion of vascular endothelial growth factor (VEGF).

Transforming growth factor (TGF)-β1 is one of the most pleiotropic and multifunctional peptides known. While the cardioprotective effect of TGF-β1 during ischemia is well known, the specific role of TGF-β1 in altering the cardiac remodeling process remains unclear. This study was designed to examine the regulation of hypoxia-reoxygenation-mediated collagen type I expression and activity of matrix metalloproteinases (MMPs) by overexpression of TGF-β1 in cultured HL-1 mouse cardiomyocytes. TGF-β1 was overexpressed in cardiomyocytes by transfection with adeno-associated virus (AAV)/TGF-β1Latent or with AAV/TGF-β1ACT (active TGF-β1). Twenty-four hours of hypoxia followed by 3 h of reoxygenation (H-R) markedly enhanced (pro)collagen type I expression and activity of MMPs concomitant with an increase in reactive oxygen species (ROS) release and LOX-1 expression. Over-expression of TGF-β1 reduced these alterations induced by H-R. TGF-β1 overexpression also blocked H-R-mediated p38 and p44/42 MAPK activation. Transfection with AAV/TGF-β1ACT was superior to that with AAV/TGFβ1Latent. These data for the first time demonstrate that H-R induces signals for cardiac remodeling in cardiomyocytes and TGF-β1 can modulate, possibly via antioxidant mechanism, these signals. These findings contribute to further understanding of the role of TGF-β1 in the cardiac remodeling process. [ABSTRACT FROM AUTHOR]

Misexpression of the double homeodomain protein DUX4 in muscle is believed to cause facioscapulohumeral muscular dystrophy (FSHD). Although strategies are being devised to inhibit DUX4 activity in FSHD, there is little known about the normal function of this protein. Expression of DUX4 has been reported in pluripotent cells and testis. To test the idea that DUX4 may be involved in initiating a germ lineage program in pluripotent cells, we interrogated the effect of expressing the human DUX4 gene at different stages during in vitro differentiation of murine embryonic stem (ES) cells. We find that expression of even low levels of DUX4 is incompatible with pluripotency: DUX4-expressing ES cells downregulate pluripotency markers and rapidly differentiate even in the presence of leukemia inhibitory factor (LIF) and bone morphogenetic protein 4 (BMP4). Transcriptional profiling revealed unexpectedly that DUX4 induced a neurectodermal program. Embryoid bodies exposed to a pulse of DUX4 expression displayed severely inhibited mesodermal differentiation, but acquired neurogenic potential. In a serum-containing medium in which neurogenic differentiation is minimal, DUX4 expression served as a neural-inducing factor, enabling the differentiation of Tuj1+ neurites. These data suggest that besides effects in muscle and germ cells, the involvement of DUX4 in neurogenesis should be considered as anti-DUX4 therapies are developed.

The lipid peroxidation product 4-hydroxynon-2-enal (4-HNE) forms as a consequence of oxidative stress, and acts as a signaling molecule or, at superphysiological levels, as a toxicant. The steady-state concentration of the compound reflects the balance between its generation and its metabolism, primarily through glutathione conjugation. Using an RNAi-based screen, we identified in Caenorhabditis elegans five glutathione transferases (GSTs) capable of catalyzing 4-HNE conjugation. RNAi knock-down of these GSTs (products of the gst-5, gst-6, gst-8, gst-10, and gst-24 genes) sensitized the nematode to electrophilic stress elicited by exposure to 4-HNE. However, interference with the expression of only two of these genes (gst-5 and gst-10) significantly shortened the life span of the organism. RNAi knock-down of the other GSTs resulted in at least as much 4-HNE adducts, suggesting tissue specificity of effects on longevity. Our results are consistent with the oxidative stress theory of organismal aging, broadened by considering electrophilic stress as a contributing factor. According to this extended hypothesis, peroxidation of lipids leads to the formation of 4-HNE in a chain reaction which amplifies the original damage. 4-HNE then acts as an "aging effector" via the formation of 4-HNE-protein adducts, and a resulting change in protein function.