Anton Wellstein

Downstream of mutant KRAS, the transcription regulator YAP is essential for neoplastic progression to pancreatic ductal adenocarcinoma.

Without the transcription regulator YAP, KRAS-mutant pancreatic neoplasia cannot progress into invasive cancer. Targeting YAP to Promote the Antitumor Response Pancreatic ductal adenocarcinoma is frequently associated with mutations in KRAS, but targeting RAS proteins is clinically challenging. From studies with genetically engineered mouse models and correlations in human samples, Zhang et al. determined that the protein YAP mediated the transition from pancreatic metaplasia to invasive adenocarcinoma by transcriptionally activating genes induced by KRAS signaling. Deleting Yap in Kras or Kras:Trp53 mutant mouse pancreatic epithelia prevented their proliferation, decreased the expression and secretion of KRAS-regulated proteins that mediate inflammation and migration, and promoted immune cell infiltration in the tumor microenvironment in mice. Because Yap deletion did not affect normal pancreatic development or function, the findings indicate that Yap may be a viable target for KRAS-mutant PDAC. Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor survival rates and frequently carries oncogenic KRAS mutation. However, KRAS has thus far not been a viable therapeutic target. We found that the abundance of YAP mRNA, which encodes Yes-associated protein (YAP), a protein regulated by the Hippo pathway during tissue development and homeostasis, was increased in human PDAC tissue compared with that in normal pancreatic epithelia. In genetically engineered KrasG12D and KrasG12D:Trp53R172H mouse models, pancreas-specific deletion of Yap halted the progression of early neoplastic lesions to PDAC without affecting normal pancreatic development and endocrine function. Although Yap was dispensable for acinar to ductal metaplasia (ADM), an initial step in the progression to PDAC, Yap was critically required for the proliferation of mutant Kras or Kras:Trp53 neoplastic pancreatic ductal cells in culture and for their growth and progression to invasive PDAC in mice. Yap functioned as a critical transcriptional switch downstream of the oncogenic KRAS–mitogen-activated protein kinase (MAPK) pathway, promoting the expression of genes encoding secretory factors that cumulatively sustained neoplastic proliferation, a tumorigenic stromal response in the tumor microenvironment, and PDAC progression in Kras and Kras:Trp53 mutant pancreas tissue. Together, our findings identified Yap as a critical oncogenic KRAS effector and a promising therapeutic target for PDAC and possibly other types of KRAS-mutant cancers.

Altered Rho GTPase Signaling Pathways in Breast Cancer Cells

The Rho family of GTPases have emerged as key players in regulating a diverse set of biological activities including actin organization, focal complex/adhesion assembly, cell motility, cell polarity, gene transcription and cell-cycle progression. Some Rho GTPases and their signaling components are overexpressed and/or are hyperactive in breast cancer and recent studies have shown a requirement for Rho GTPases in breast cancer cell metastasis in vivo. Herein we describe the contribution of Rho GTPase to the malignant phenotype of breast cancer cells and the role of these pathways as potential targets for breast cancer therapy. Rho GTPases promote cell-cycle progression through cyclin D1, and cyclin D1 in turn reduces cellular adhesion and promotes migration, an example of ‘inside-out’ signaling by cyclin D1. As cyclin D1 overexpression correlates with metastatic cancer, the ‘inside-out’ signaling function of cyclin D1 to promote cell migration may represent a useful new therapeutic target.

Chronic antidepressant treatments increase basic fibroblast growth factor and fibroblast growth factor-binding protein in neurons

One of the mechanisms proposed for antidepressant drugs is the enhancement of synaptic connections and plasticity in the hippocampus and cerebral cortex. Fibroblast growth factor 2 (FGF2) is a growth factor essential for the proper formation of synaptic connections in the cerebral cortex, maturation and survival of catecholamine neurons, and neurogenesis. In this report, we attempted to establish a correlation between antidepressant treatments and FGF2 expression in the cerebral cortex and hippocampus, two brain areas relevant for depression. Desipramine (DMI, 10mg/kg) or fluoxetine (FLU, 5mg/kg) was injected acutely (single injection) or chronically (daily injection for two weeks) in adult rats. Chronic, but not acute, antidepressant treatments increase FGF2 immunoreactivity in neurons of the cerebral cortex and in both astrocytes and neurons of the hippocampus. FGF2 immunoreactivity in the cortex was increased mainly in the cytoplasm of neurons of layer V. Western blot analyses of nuclear and cytosolic extracts from the cortex revealed that both antidepressants increase FGF2 isoforms in the cytosolic extracts and decrease accumulation of FGF2 immunoreactivity in the nucleus. To characterize the anatomical and cellular specificity of antidepressants, we examined FGF-binding protein (FBP), a secreted protein that acts as an extracellular chaperone for FGF2 and enhances its activity. DMI and FLU increased FBP immunoreactivity in both cortical and hippocampal neurons. Our data suggest that FGF2 and FBP may participate in the plastic responses underlying the clinical efficacy of antidepressants.

An FGF-binding protein (FGF-BP) exerts its biological function by parallel paracrine stimulation of tumor cell and endothelial cell proliferation through FGF-2 release

Fibroblast growth factors FGF‐1 (aFGF) and FGF‐2 (bFGF) are found in most embryonic and adult normal and tumor tissues, where they are immobilized in the extracellular matrix (ECM). Mobilization of these FGFs is part of a tightly controlled process resulting in the activation of high‐affinity FGF receptors. Recently, we have shown that a secreted FGF‐binding protein (FGF‐BP) binds non‐covalently to FGF‐2 and is able to release it from the ECM. This process of growth factor bioactivation seems to play a pivotal role in the growth of squamous cell carcinomas, especially through induction of tumor angiogenesis. Since previous studies provided only indirect evidence for the proposed mechanism of FGF‐BP‐mediated FGF‐2 release, we decided to use recombinant purified FGF‐BP to study further the underlying mechanism of FGF‐BP action. Here we show that FGF‐BP is able to bind directly to FGF‐2 without additional cofactors and to exhibit bioactivity. The purified recombinant FGF‐BP stimulates tumor cell growth as well as endothelial cell growth and chemotaxis, indicating a dual growth‐supporting role of FGF‐BP in tumors. We show that this paracrine FGF‐BP effect is dependent on endogenously expressed FGF‐2, since it can be completely blocked by anti‐FGF‐2 antibodies. In tumor xenografts and in tumor cells, we detected a pattern of specific FGF‐BP‐immunoreactive high molecular weight forms, which presumably represent stable covalent complexes of FGF‐BP and show marked differences in their occurrence in different tumors and in their heparin binding affinity. By providing further insight into the mechanism of FGF‐BP action, our results emphasize the relevance of FGF‐BP and of FGF‐2 in tumor growth.

Immunolocalization of an FGF-binding protein reveals a widespread expression pattern during different stages of mouse embryo development

Abstract. Fibroblast growth factors (FGFs) play important roles during fetal and embryonic development. FGF-2 (basic FGF, bFGF) is widely expressed in the embryo and has been linked to tissue growth and remodeling. However, it is tightly bound to heparin sulfate proteoglycans of the extracellular matrix which quenches its biological activity. We showed previously that a secreted FGF-binding protein (FGF-BP) can mobilize and activate FGF-2 from the extracellular matrix. While considerable data exist on the expression and pivotal role of FGF-BP in tumor growth, less is known about FGF-BP during embryonic development. In this immunohistochemical study in mice, we show FGF-BP protein expression in a broad spectrum of tissues at various stages between dayxa08 and dayxa016 of embryonal development, and compare FGF-BP and FGF-2 immunolocalization. FGF-BP is detected in the digestive system, thymus, skin, hair follicles, dental germ, respiratory tract, various glandular tissues, kidney, liver, and certain areas of the CNS, with immunoreactivity being mainly confined to cells of primitive epithelia. The putative significance of these findings with regard to mobilization of FGF-2 or other molecules is discussed. From the locally confined, time-dependent, and apparently tightly regulated FGF-BP expression we propose that FGF-BP may play an important role in embryonic development.

Chlorpheniramine plasma concentration and histamine H1‐receptor occupancy

The plasma concentration‐response relationship of the antihistamine chlorpheniramine is poorly characterized. This study examined concurrently the concentrations of chlorpheniramine and presence of H1‐receptor antagonist in plasma after administration of 8 mg chlorpheniramine in normal volunteers. Six extensive metabolizers and five poor metabolizers, as judged by CYP2D6 phenotype (dextromethorphan metabolic ratio), were enrolled in the study. More than 80% occupancy of H1‐receptors by antagonist in plasma was observed for 12 hours after the dose in extensive metabolizers and greater than 60% from 12 to 30 hours in poor metabolizers, when plasma concentrations had fallen below those that should result in 50% occupancy of receptors. The results suggest that (±)‐chlorpheniramine plasma concentrations do not predict H1‐receptor antagonist in plasma. In addition, a role is suggested for CYP2D6 in formation of a potent active metabolite of chlorpheniramine.

Circulating biomarkers to monitor cancer progression and treatment.

Tumor heterogeneity is a major challenge and the root cause of resistance to treatment. Still, the standard diagnostic approach relies on the analysis of a single tumor sample from a local or metastatic site that is obtained at a given time point. Due to intratumoral heterogeneity and selection of subpopulations in diverse lesions this will provide only a limited characterization of the makeup of the disease. On the other hand, recent developments of nucleic acid sequence analysis allows to use minimally invasive serial blood samples to assess the mutational status and altered gene expression patterns for real time monitoring in individual patients. Here, we focus on cell-free circulating tumor-specific mutant DNA and RNA (including mRNA and non-coding RNA), as well as current limitations and challenges associated with circulating nucleic acids biomarkers.

d‐Sotalol reduces heart rate in vivo through a β‐adrenergic receptor–independent mechanism

d‐Sotalol was developed as an antiarrhythmic agent with a relative lack of antagonist activity at β‐adrenergic receptors. Exercise heart rate reduction has been observed after administration to humans. The purpose of this study was to determine directly whether this effect of d‐sotalol was attributable to β ‐blockade. Plasma samples from normal volunteers who randomly received either atenolol, d‐sotalol, or placebo were used in an in vitro radioreceptor assay to determine occupancy of β‐adrenergic receptors by antagonist present in the plasma. Occupancy was compared with the observed pharmacologie effects. A reduction in exercise heart rate of 7.7% ± 3.8% for d‐sotalol and 15.9% ± 3.0% for atenolol occurred with β1‐adrenergic receptor occupancy of 0% and 33.9% ± 21.4%, respectively. Absence of antagonist effect in the radioreceptor assay eliminates the potential role of β1‐blockade in d‐sotalol— induced heart rate reduction. This effect is most likely a result of prolongation of the sinus node action potential duration.

An additional 5'-upstream exon exists in the human pleiotrophin-encoding gene ☆

A 2-kb 5 fragment of hPTN (human pleiotrophin-encoding) genomic DNA was sequenced. Within this region we identified a new, upstream exon (U2). Cloning and sequencing of the PCR products of cDNAs from a human melanoma cell line revealed that a 401-bp intron was spliced out between exon U2 and a previously described untranslated exon U1. Our analysis also revealed that previously reported transcription start points (tsp) of PTN are located within exon U1.

Transcriptional Repression of AIB1 by FoxG1 Leads to Apoptosis in Breast Cancer Cells

The oncogene nuclear receptor coactivator amplified in breast cancer 1 (AIB1) is a transcriptional coactivator that is overexpressed in various types of human cancers. However, the molecular mechanisms controlling AIB1 expression in the majority of cancers remain unclear. In this study, we identified a novel interacting protein of AIB1, forkhead-box protein G1 (FoxG1), which is an evolutionarily conserved forkhead-box transcriptional corepressor. We show that FoxG1 expression is low in breast cancer cell lines and that low levels of FoxG1 are correlated with a worse prognosis in breast cancer. We also demonstrate that transient overexpression of FoxG1 can suppress endogenous levels of AIB1 mRNA and protein in MCF-7 breast cancer cells. Exogenously expressed FoxG1 in MCF-7 cells also leads to apoptosis that can be rescued in part by AIB1 overexpression. Using chromatin immunoprecipitation, we determined that FoxG1 is recruited to a region of the AIB1 gene promoter previously characterized to be responsible for AIB1-induced, positive autoregulation of transcription through the recruitment of an activating, multiprotein complex, involving AIB1, E2F transcription factor 1, and specificity protein 1. Increased FoxG1 expression significantly reduces the recruitment of AIB1, E2F transcription factor 1 and E1A-binding protein p300 to this region of the endogenous AIB1 gene promoter. Our data imply that FoxG1 can function as a pro-apoptotic factor in part through suppression of AIB1 coactivator transcription complex formation, thereby reducing the expression of the AIB1 oncogene.