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Thoracic high-dose radiation therapy (RT) for cancer has been associated with early and late cardiac toxicity. To assess altered rates of cardiomyocyte cell death due to RT we monitored changes in cardiomyocyte-specific, cell-free methylated DNA (cfDNA) shed into the circulation. Eleven patients with distal esophageal cancer treated with neoadjuvant chemoradiation to 50.4 Gy (RT) and concurrent carboplatin and paclitaxel were enrolled. Subjects underwent fasting blood draws prior to the initiation and after completion of RT as well as 4–6 months following RT. An island of six unmethylated CpGs in the FAM101A locus was used to identify cardiomyocyte-specific cfDNA in serum. After bisulfite treatment this specific cfDNA was quantified by amplicon sequencing at a depth of &gt, 35,000 reads/molecule. Cardiomyocyte-specific cfDNA was detectable before RT in the majority of patient samples and showed some distinct changes during the course of treatment and recovery. We propose that patient-specific cardiac damages in response to the treatment are indicated by these changes although co-morbidities may obscure treatment-specific events.

Fenofibrate, a peroxisome proliferator-activated receptors α (PPARα) agonist, reduces vascular complications of diabetic patients but its protective mechanisms are not fully understood. Here we tested the hypothesis that fenofibrate improves vascular endothelial dysfunction by balancing endothelium-dependent relaxation and contractility of the aorta in diabetes mellitus (DM). In streptozotocin-induced diabetic mice, eight weeks of fenofibrate treatment (100 mg/Kg/d) improved endothelium dependent relaxation in the macro- and microvessels, increased nitric oxide (NO) levels, reduced renal damage markers and effects of the vasoconstrictor prostaglandin. Levels of superoxide dismutase and catalase were both reduced and hydrogen peroxide was increased in vehicle-treated DM, but these changes were reversed by fenofibrate treatment. Vasodilation of the aorta after fenofibrate treatment was reversed by PPARα or AMPKα inhibitors. Western blots showed that fenofibrate treatment elevated PPARα expression, induced liver kinase B1 (LKB1) translocation from the nucleus to the cytoplasm and activated AMP-activated protein kinase-α (AMPKα), thus activating endothelial NO synthase (eNOS). Also, fenofibrate treatment decreased NF-κB p65 and cyclooxygenase 2 proteins in aortas. Finally, incubation with indomethacin in vitro improved aortic contractility in diabetic mice. Overall, our results show that fenofibrate treatment in diabetic mice normalizes endothelial function by balancing vascular reactivity via increasing NO production and suppressing the vasoconstrictor prostaglandin, suggesting mechanism of action of fenofibrate in mediating diabetic vascular complications., Graphical abstract fx1, Highlights • Fenofibrate improves diabetic endothelial function is via PPAR/LKB1/AMPK/eNOS signal. • Fenofibrate reduces diabetic endothelial contractility is via NF-κB/COX-2 pathway. • Diabetes-associated oxidative stress is attenuated by fenofibrate treatment.

AIB1Δ4 is an N-terminally truncated isoform of the oncogene amplified in breast cancer 1 (AIB1) with increased expression in high-grade human ductal carcinoma in situ (DCIS). However, the role of AIB1Δ4 in DCIS malignant progression has not been defined. Here we CRISPR-engineered RNA splice junctions to produce normal and early-stage DCIS breast epithelial cells that expressed only AIB1Δ4. These cells showed enhanced motility and invasion in 3D cell culture. In zebrafish, AIB1Δ4-expressing cells enabled invasion of parental cells when present in a mixed population. In mouse xenografts, a subpopulation of AIB1Δ4 cells mixed with parental cells enhanced tumor growth, recurrence, and lung metastasis. AIB1Δ4 chromatin immunoprecipitation sequencing revealed enhanced binding to regions including peroxisome proliferator-activated receptor (PPAR) and glucocorticoid receptor (GR) genomic recognition sites. H3K27ac and H3K4me1 genomic engagement patterns revealed selective activation of breast cancer-specific enhancer sites by AIB1Δ4. AIB1Δ4 cells displayed upregulated inflammatory response genes and downregulated PPAR signaling gene expression patterns. In the presence of AIB1Δ4 enabler cells, parental cells increased NF-κB and WNT signaling. Cellular cross-talk was inhibited by the PPARγ agonist efatutazone but was enhanced by treatment with the GR agonist dexamethasone. In conclusion, expression of the AIB1Δ4-selective cistrome in a small subpopulation of cells triggers an “enabler” phenotype hallmarked by an invasive transcriptional program and collective malignant progression in a heterogeneous tumor population. Significance: A minor subset of early-stage breast cancer cells expressing AIB1Δ4 enables bulk tumor cells to become invasive, suggesting that selective eradication of this population could impair breast cancer metastasis.

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells. Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

High salt, Ang II (angiotensin II), and reactive oxygen species enhance progression of chronic kidney disease. We tested the hypothesis that a high salt intake generates specific reactive oxygen species to enhance Ang II contractions of afferent arterioles from mice with reduced renal mass (RRM). C57BL/6 mice were subjected to surgical RRM or sham operations and received 6% or 0.4% NaCl salt diet for 3 months. Ang II contractions were measured in perfused afferent arterioles and superoxide (O 2 − ) and hydrogen peroxide (H 2 O 2 ) by fluorescence microscopy. RRM enhanced the afferent arteriolar gene expression for p47 phox and neutrophil oxidase (NOX) 2 and high salt intake in RRM mice enhanced gene expression for angiotensin type 1 receptors, POLDIP2 and NOX4 and reduced catalase. High salt in mice with RRM enhanced arteriolar O 2 − and H 2 O 2 generation and maximal contractions to Ang II (10 –6 mol/L) that were dependent on O 2 − because they were prevented by gene deletion of p47 phox and on H 2 O 2 because they were prevented by transgenic smooth muscle cell expression of catalase (tg CAT-SMC ) and POLDIP2 gene deletion. Three months of tempol normalized arteriolar reactive oxygen species and Ang II contractions. However, arteriolar contractions to lower concentrations of Ang II (10 –8 to 10 –11 mol/L) were paradoxically inhibited by H 2 O 2 and POLDIP2. In conclusion, both O 2 − from p47 phox /NOX2 and H 2 O 2 from NOX4/POLDIP2 enhance maximal arteriolar Ang II contractions from RRM mice during high salt, but H 2 O 2 and NOX4/POLDIP2 reduce the sensitivity to lower concentrations of Ang II by >100-fold. Tempol prevents all of these changes in function.

Secreted FGF binding proteins (FGFBP) mobilize locally-acting paracrine FGFs from their extracellular storage. Here, we report that FGFBP3 (BP3) modulates fat and glucose metabolism in mouse models of metabolic syndrome. BP3 knockout mice exhibited altered lipid metabolism pathways with reduced hepatic and serum triglycerides. In obese mice the expression of exogenous BP3 reduced hyperglycemia, hepatosteatosis and weight gain, blunted de novo lipogenesis in liver and adipose tissues, increased circulating adiponectin and decreased NEFA. The BP3 protein interacts with endocrine FGFs through its C-terminus and thus enhances their signaling. We propose that BP3 may constitute a new therapeutic to reverse the pathology associated with metabolic syndrome that includes nonalcoholic fatty liver disease and type 2 diabetes mellitus.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinomas (PDAC) are characterized by fibrosis and an abundance of cancer-associated fibroblasts (CAFs). We investigated strategies to disrupt interactions among CAFs, the immune system, and cancer cells, focusing on adhesion molecule cadherin 11 (CDH11), which has been associated with other fibrotic disorders and is expressed by activated fibroblasts. METHODS: We compared levels of CDH11 mRNA in human pancreatitis and pancreatic cancer tissues and cells, compared with normal pancreas, and measured levels of CDH11 protein in human and mouse pancreatic lesions and normal tissues. We crossed p48-Cre;LSL-Kras(G12D/+);LSL-Trp53(R172H/+) (KPC) mice with CDH11-knockout mice and measured survival times of offspring. Pancreata were collected and analyzed by histology, immunohistochemistry, and (single-cell) RNA sequencing; RNA and proteins were identified by imaging mass cytometry. Some mice were given injections of PD1 antibody or gemcitabine and survival was monitored. Pancreatic cancer cells from KPC mice were subcutaneously injected into Cdh11(+/+) and Cdh11(−/−) mice and tumor growth was monitored. Pancreatic cancer cells (mT3) from KPC mice (C57BL/6), were subcutaneously injected into Cdh11(+/+) (C57BL/6J) mice and mice were given injections of antibody against CDH11, gemcitabine, or small molecule inhibitor of CDH11 (SD133) and tumor growth was monitored. RESULTS: Levels of CDH11 mRNA and protein were significantly higher in CAFs than in pancreatic cancer epithelial cells, human or mouse pancreatic cancer cell lines, or immune cells. KPC/Cdh11(+/−) and KPC/Cdh11(−/−) mice survived significantly longer than KPC/Cdh11(+/+) mice. Markers of stromal activation entirely surrounded pancreatic intraepithelial neoplasias in KPC/Cdh11(+/+) mice and incompletely in KPC/Cdh11(+/−) and KPC/Cdh11(−/−) mice, whose lesions also contained fewer FOXP3(+) cells in the tumor center. Compared with pancreatic tumors in KPC/Cdh11(+/+) mice, tumors of KPC/Cdh11(+/−) mice had increased markers of antigen processing and presentation; more lymphocytes and associated cytokines; decreased extracellular matrix components; and reductions in markers and cytokines associated with immunosuppression. Administration of the PD1 antibody did not prolong survival of KPC mice with 0, 1, or 2 alleles of Cdh11. Gemcitabine extended survival only of KPC/Cdh11(+/−) and KPC/Cdh11(−/−) mice or reduced subcutaneous tumor growth in mT3 engrafted Cdh11(+/+) mice given in combination with the CDH11 antibody. A small molecule inhibitor of CDH11 reduced growth of pre-established mT3 subcutaneous tumors only if T and B cells were present in mice. CONCLUSIONS: Knockout or inhibition of CDH11, which is expressed by CAFs in the pancreatic tumor stroma, reduces growth of pancreatic tumors, increases their response to gemcitabine, and significantly extends survival of mice. CDH11 promotes immunosuppression and extracellular matrix deposition, and might be developed as a therapeutic target for pancreatic cancer.

Cancer cell vascular invasion is a crucial step in the malignant progression towards metastasis. Here we used a genome-wide RNAi screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to a large protein family that is implicated in crosslinking keratin intermediate filaments during hair formation, yet these keratin-associated proteins have no reported role in cancer. Depletion of Krtap5-5 from cancer cells led to cell blebbing and a loss of keratins 14 and 18, in addition to the upregulation of vimentin intermediate filaments. This intermediate filament subtype switching induced dysregulation of the actin cytoskeleton and reduced the expression of hemidesmosomal α6/β4-integrins. We further demonstrate that knockdown of keratin 18 phenocopies the loss of Krtap5-5, suggesting that Krtap5-5 crosstalks with keratin 18 in E0771 cells. Disruption of the keratin cytoskeleton by perturbing Krtap5-5 function broadly altered the expression of cytoskeleton regulators and the localization of cell surface markers. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extracellular matrix invasion, as well as extravasation of cancer cells into tissues in zebrafish and mice. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates motility and vascular invasion. Thus, in addition to its physiologic function, a keratin-associated protein can serve as a switch towards malignant progression.

High salt intake, angiotensin II (Ang II), and reactive oxygen species (ROS) enhance progression of chronic kidney disease (CKD). We reported that myogenic contractions of renal afferent arterioles (RAAs) were enhanced by superoxide (O 2 ·- ) generated from p47 phox /NOX2 but inhibited by H 2 O 2 generated from POLDIP2/NOX4. We tested the hypothesis that feeding a high salt diet to mice with the reduced renal mass (RRM) model of CKD generates specific ROS in their RAAs that enhances Ang II contractions. Methods and Results: C57BL/6 mice received surgical RRM or sham operations and 6% or 0.4% NaCl salt for 3 months. Ang II contractions were measured in RAAs perfused at 45 mmHg and superoxide (O 2 ·- ) and H 2 O 2 by fluorescence microscopy. RRM enhanced the gene expression in RAAs for p47 phox and NOX2 and high salt intake in mice with RRM enhanced the gene expression for AT1Rs, POLDIP2 and NOX4 and reduced the gene expression for catalase. Mice with RRM fed a normal salt diet had contractions to 10 -6 mol·l -1 Ang II similar to sham (-56 ± 5 vs -52 ± 5 %; NS). However, RRM mice fed a high salt diet had an enhanced O 2 ·- and H 2 O 2 generation (P2 ·- from NOX2 since they were prevented in p47 phox -/- mice and on H 2 O 2 from NOX4 since they were prevented in mice with transgenic smooth muscle cell expression of catalase (tg CAT-SMC ), and in POLDIP2 +/- mice. However, RAA contractions to lower concentrations of Ang II (10 -8 to 10 -11 mol·l -1 ) were paradoxically enhanced in tg CAT-SMC vs Wt mice (-17 ± 2 vs -1 ± 1%; P2 ·- from p47 phox /NOX2 and H 2 O 2 from NOX4/POLDIP2 enhance maximal Ang II contractions of RAAs from mice with RRM fed a high salt diet but H 2 O 2 from NOX4/POLDIP2 reduces the sensitivity to lower concentrations of Ang II by >100-fold and tempol prevents all of these changes Thus, although a high salt intake reduces circulating Ang II, blockade of angiotensin receptors or ROS may prove beneficial for patients with CKD unable to restrict salt.

Cardiovascular disease is frequent in chronic kidney disease and has been related to angiotensin II, endothelin-1 (ET-1), thromboxane A 2 , and reactive oxygen species (ROS). Because activation of thromboxane prostanoid receptors (TP-Rs) can generate ROS, which can generate ET-1, we tested the hypothesis that chronic kidney disease induces cyclooxygenase-2 whose products activate TP-Rs to enhance ET-1 and ROS generation and contractions. Mesenteric resistance arterioles were isolated from C57/BL6 or TP-R+/+ and TP-R−/− mice 3 months after SHAM-operation (SHAM) or surgical reduced renal mass (RRM, n=6/group). Microvascular contractions were studied on a wire myograph. Cellular (ethidium: dihydroethidium) and mitochondrial (mitoSOX) ROS were measured by fluorescence microscopy. Mice with RRM had increased excretion of markers of oxidative stress, thromboxane, and microalbumin; increased plasma ET-1; and increased microvascular expression of p22 phox , cyclooxygenase-2, TP-Rs, preproendothelin and endothelin-A receptors, and increased arteriolar remodeling. They had increased contractions to U-46,619 (118±3 versus 87±6, P P P

Nuclear factor erythyroid factor 2 (Nrf2) transcribes genes in cultured endothelial cells that reduce reactive oxygen species (ROS) and generate nitric oxide (NO) or metabolize asymmetric dimethylarginine (ADMA), which inhibits NO synthase (NOS). Therefore, we undertook a functional study to test the hypothesis that activation of Nrf2 by tert-butylhydroquinone (tBHQ) preserves microvascular endothelial function during oxidative stress. Wild-type CB57BL/6 (wt), Nrf2 wt (+/+), or knockout (−/−) mice received vehicle (Veh) or tBHQ (0.1%; activator of Nrf2) during 14-day infusions of ANG II (to induce oxidative stress) or sham. MAP was recorded by telemetry. Mesenteric resistance arterioles were studied on isometric myographs and vascular NO and ROS by fluorescence microscopy. ANG II increased the mean arterial pressure (112 ± 5 vs. 145 ± 5 mmHg; P < 0.01) and excretion of 8-isoprostane F2α (2.8 ± 0.3 vs. 3.8 ± 0.3 ng/mg creatinine; P < 0.05) at 12–14 days. However, 12 days of ANG II reduced endothelium-derived relaxation (27 ± 5 vs. 17 ± 3%; P < 0.01) and NO (0.38 ± 0.07 vs. 0.18 ± 0.03 units; P < 0.01) but increased microvascular remodeling, endothelium-derived contractions (7.5 ± 0.5 vs. 13.0 ± 1.7%; P < 0.01), superoxide (0.09 ± 0.03 vs. 0.29 ± 0.08 units; P < 0.05), and contractions to U-46,619 (87 ± 6 vs. 118 ± 3%; P < 0.05), and endothelin-1(89 ± 4 vs. 123 ± 12%; P < 0.05). tBHQ prevented all of these effects of ANG II at 12–14 days in Nrf2+/+ mice but not in Nrf2−/− mice. In conclusion, tBHQ activates Nrf2 to prevent microvascular endothelial dysfunction, remodeling, and contractility, and moderate ADMA and hypertension at 12–14 days of ANG II infusion, thereby preserving endothelial function and preventing hypertension.

Fibroblast growth factors (FGFs) participate in organ development and tissue maintenance, as well as the control of vascular function. The paracrine-acting FGFs are stored in the extracellular matrix, and their release is controlled by a secreted FGF-binding protein (FGF-BP, FGFBP1, and BP1) that modulates FGF receptor signaling. A genetic polymorphism in the human FGFBP1 gene was associated with higher gene expression and an increased risk of familial hypertension. Here, we report on the effects of inducible BP1 expression in a transgenic mouse model. Induction of BP1 expression in adult animals leads to a sustained rise in mean arterial pressure by >30 mm Hg. The hypertensive effect of BP1 expression is prevented by candesartan, an angiotensin II (AngII) receptor antagonist, or by tempol, an inhibitor of reactive oxygen species. In vivo, BP1 expression sensitizes peripheral resistance vessels to AngII constriction by 20-fold but does not alter adrenergic vasoconstriction. FGF receptor kinase inhibition reverses the sensitization to AngII. Also, constriction of isolated renal afferent arterioles by AngII is enhanced after BP1 expression and blocked by FGF receptor kinase inhibition. Furthermore, AngII-mediated constriction of renal afferent arterioles is abolished in FGF2 −/− mice but can be restored by add-back of FGF2 plus BP1 proteins. In contrast to AngII, adrenergic constriction is not affected in the FGF2 −/− model. Proteomics and gene expression analysis of kidney tissues after BP1 induction show that MAPK (mitogen-activated protein kinase) signaling via MKK4 (MAPK kinase 4), p38, and JNK (c-Jun N-terminal kinase) integrates the crosstalk of the FGF receptor and AngII pathways and thus impact vascular tone and blood pressure.

Successful treatment of cancers with Immune Checkpoint Inhibitors (ICIs) has been associated with the mutational load of tumors. The biological rationale for this association between mutational load and ICI response is that neoantigens are generated by mutations in protein coding sequences that provide a steady flow of neoantigens to prime the immune system for the production of antigen-specific tumor-infiltrating lymphocytes (TILs). It is thought that mutant protein fragments will lead to altered MHC/peptide recognition and immune cell activation; ICI treatment enhances TIL functionality. Neoantigens are also relevant for an alternative, cell-based immunotherapeutic approach, i.e. Adoptive Cell Transfer (ACT). This concept of neoantigens derived from DNA mutations has led to an intense line of investigation to uncover relevant neoantigens. However, there has been mixed success with the current neoantigen discovery approach based on DNA mutation analysis of tumor samples by exome sequencing of genomic DNA. The current concept of neoantigens derived from mutant DNA ignores an alternative mechanism that can also generate neoantigens in cancers: Posttranscriptional editing of primary RNA. Here we propose to use full-length Single Molecule Real Time (SMRT) RNAseq to uncover pathologically edited mRNAs in cancers and complement the discovery of pathologic mRNA. We will discuss the respective algorithms and propose the combination with identification of candidate neoantigen peptides by mass spectrometry.

The combination of irradiated fibroblast feeder cells and Rho kinase inhibitor, Y-267362, converts primary epithelial cells growing in vitro into an undifferentiated adult stem cell-like state that is characterized by long-term proliferation. This cell culture method also maintains the proliferation of adult epithelial stem cells from various tissues. Both primary and adult stem cells retain their tissue-specific differentiation potential upon removal of the culture conditions. Due to the ability to modulate the proliferation and differentiation of the cells, this method is referred to as conditional reprogramming and it is increasingly being used in studies of tumor heterogeneity, personalized medicine and regenerative medicine. However, little is known about the biology of these conditionally reprogrammed (CR) cells. Previously we showed that β-catenin activation, a hallmark of stem cells in vivo, occurs in CR human ectocervical cells (HECs). Here we show that β-catenin-dependent transcription is necessary for the induction of epithelial stem cell markers, and that β-catenin is activated via a non-canonical pathway that is independent of Wnt and Akt/GSK-3. Active Akt actually decreases due to increased mTOR signaling, with a consequent increase in dephosphorylated, active GSK-3. Despite the increase in active GSK-3, β-catenin associates with protein phosphatase 2A (PP2A) and is activated. Inhibition of PP2A catalytic activity reduces both the level of active β-catenin and the acute induction of stem cell markers, suggesting an important role for PP2A in the activation of β-catenin. Moreover, we demonstrate similar results using human prostate and breast cells, indicating that these changes are not restricted to ectocervical epithelial cells and may represent a more fundamental property of conditional reprogramming.

Background Midkine (MDK) and pleiotrophin (PTN) are heparin-binding growth factors that, in rodents, are highly expressed in early life and decrease to undetectable levels by adulthood. The potential roles of MDK and PTN in human growth and development are not completely elucidated. Method and Findings To delineate the role of MDK and PTN in human development, we developed high sensitivity assays to measure their concentrations in amniotic fluid (AF) at various gestational ages in both healthy and complicated pregnancies. We found that both of these growth factors could be readily measured in AF and that the concentrations were higher than most cytokines previously reported in AF. Conclusion The concentration of MDK but not that of PTN declined with gestational age. Both MDK and PTN concentrations were found to be lower in pregnancies that were complicated by chorioamnionitis at term, raising the possibility that these growth factors might be useful as markers for infection.

We tested the hypothesis that reactive oxygen species (ROS) contributed to renal hypoxia in C57BL/6 mice with ⅚ surgical reduction of renal mass (RRM). ROS can activate the mitochondrial uncoupling protein 2 (UCP-2) and increase O2usage. However, UCP-2 can be inactivated by glutathionylation. Mice were fed normal (NS)- or high-salt (HS) diets, and HS mice received the antioxidant drug tempol or vehicle for 3 mo. Since salt intake did not affect the tubular Na+transport per O2consumed (TNa/QO2), further studies were confined to HS mice. RRM mice had increased excretion of 8-isoprostane F2αand H2O2, renal expression of UCP-2 and renal O2extraction, and reduced TNa/QO2(sham: 20 ± 2 vs. RRM: 10 ± 1 μmol/μmol; P < 0.05) and cortical Po2(sham: 43 ± 2, RRM: 29 ± 2 mmHg; P < 0.02). Tempol normalized all these parameters while further increasing compensatory renal growth and glomerular volume. RRM mice had preserved blood pressure, glomeruli, and patchy tubulointerstitial fibrosis. The patterns of protein expression in the renal cortex suggested that RRM kidneys had increased ROS from upregulated p22phox, NOX-2, and -4 and that ROS-dependent increases in UCP-2 led to hypoxia that activated transforming growth factor-β whereas erythroid-related factor 2 (Nrf-2), glutathione peroxidase-1, and glutathione- S-transferase mu-1 were upregulated independently of ROS. We conclude that RRM activated distinct processes: a ROS-dependent activation of UCP-2 leading to inefficient renal O2usage and cortical hypoxia that was offset by Nrf-2-dependent glutathionylation. Thus hypoxia in RRM may be the outcome of NADPH oxidase-initiated ROS generation, leading to mitochondrial uncoupling counteracted by defense pathways coordinated by Nrf-2.

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, 10 mg/kg) or fluoxetine (FLU, 5 mg/kg) were 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.

Myogenic contraction is the principal component of renal autoregulation that protects the kidney from hypertensive barotrauma. Contractions are initiated by a rise in perfusion pressure that signals a reduction in membrane potential ( Em) of vascular smooth muscle cells to activate voltage-operated Ca2+ channels. Since ROS have variable effects on myogenic tone, we investigated the hypothesis that superoxide (O2·−) and H2O2 differentially impact myogenic contractions. The myogenic contractions of mouse isolated and perfused single afferent arterioles were assessed from changes in luminal diameter with increasing perfusion pressure (40–80 mmHg). O2·−, H2O2, and Em were assessed by fluorescence microscopy during incubation with paraquat to increase O2·− or with H2O2. Paraquat enhanced O2·− generation and myogenic contractions (−42 ± 4% vs. −19 ± 4%, P < 0.005) that were blocked by SOD but not by catalase and signaled via PKC. In contrast, H2O2 inhibited the effects of paraquat and reduced myogenic contractions (−10 ± 1% vs. −19 ± 2%, P < 0.005) and signaled via PKG. O2·− activated Ca2+-activated Cl− channels that reduced Em, whereas H2O2 activated Ca2+-activated and voltage-gated K+ channels that increased Em. Blockade of voltage-operated Ca2+ channels prevented the enhanced myogenic contractions with paraquat without preventing the reduction in Em. Myogenic contractions were independent of the endothelium and largely independent of nitric oxide. We conclude that O2·− and H2O2 activate different signaling pathways in vascular smooth muscle cells linked to discreet membrane channels with opposite effects on Em and voltage-operated Ca2+ channels and therefore have opposite effects on myogenic contractions.

Collecting repeat samples of blood ("liquid biopsies") is a broadly used clinical approach for serial monitoring of disease or response to treatments. In patients with cancer the most distinct molecular feature are somatic mutations acquired by cancer cells present in the diseased tissue. Indeed, mutant DNA derived from dying or lysed cancer cells can be isolated from patient serum samples, subjected to DNA sequencing and to analysis of abundance as a measure of tumor burden. Also, changes in the DNA mutation patterns in serum samples collected over time can indicate altered pathways or clonal evolution of the disease and altered abundance of mutant DNA suggests an altered disease burden. In addition, during the course of treatment, changes in circulating DNA mutation patterns can indicate the emergence of resistant clones and prompt changes in treatment. In contrast to mutant DNA, microRNAs (miR) are transcribed, processed, packaged and released from cells in normal and in diseased tissues as part of the extracellular crosstalk between cells. Interestingly, released miR can function in cell-to-cell communication and as hormone-like signals that operate at a distance through their release into the circulation and subsequent uptake into cells in distant tissues. Circulating miR expression patterns can be established from serial serum samples and monitored for alterations over time. Circulating miR provide a readout of the organism's steady state and serial analyses will indicate changes in the response to therapy or an altered physiologic or disease state. Furthermore, changes in circulating miR patterns can indicate treatment efficacy or resistance as well as adverse effects associated with the respective intervention. Thus, the combined serial analysis of mutant DNA and miR in the circulation has the potential to provide a molecular footprint of pancreatic cancer and can be used to monitor treatment responses or resistance to treatment in real time with a minimally invasive procedure.

Previously, we have demonstrated that the two mitogenic growth factors epidermal growth factor and IGF-I can activate Akt and estrogen receptor-alpha (ERalpha) in the hormone-dependent breast cancer cell line, MCF-7. In this report we now show that estradiol can also rapidly activate phosphatidylinositol 3-kinase (PI 3-K)/Akt and that this effect is mediated by the ErbB2 signaling pathway. Treatment of cells with estradiol resulted in phosphorylation of Akt and a 9-fold increase in Akt activity in 10 min. Akt activation was blocked by wortmannin and LY 294,002, two inhibitors of PI 3-K; by genistein, a protein tyrosine kinase inhibitor and an ER agonist; by AG825, a selective ErbB2 inhibitor; and by the antiestrogens ICI 182,780 and 4-hydroxy-tamoxifen; but not by rapamycin, an inhibitor of the ribosomal protein kinase p70S6K; nor by AG30, a selective epidermal growth factor receptor inhibitor. Akt activation by estradiol was abrogated by an arginine-to-cysteine mutation in the pleckstrin homology domain of Akt (R25C). Growth factors also activated Akt in the ER-negative variant of MCF-7, MCF-7/ADR, but estradiol did not induce Akt activity in these cells. Transient transfection of ERalpha into these cells restored Akt activation by estradiol, suggesting that estradiol activation of Akt requires the ERalpha. Estradiol did not activate Akt in MCF-7 cells stably transfected with an anti-ErbB2-targeted ribozyme, further confirming a role for ErbB2. In vitro kinase assays using immunoprecipitation and anti-Akt1, -Akt2, and -Akt3-specific antibodies demonstrated that Akt1 is activated by estradiol in MCF-7 cells whereas Akt3 is the activated isoform in ER-negative MDA-MB231 cells, implying that selective activation of Akt subtypes plays a role in the actions of estradiol. Taken together, our data suggest that estradiol, bound to membrane ERalpha, interacts with and activates an ErbB dimer containing ErbB2, inducing activation of PI 3-K/Akt.

Pleiotrophin is a heparin-binding growth factor involved in the differentiation and proliferation of neuronal tissue during embryogenesis, and also secreted by melanoma and breast carcinoma cells. Pleiotrophin exhibits mitogenic and angiogenic properties and has been shown to influence the vascular supply, expansion and metastasis of tumour cells. Our aim was to study the serum and plasma concentrations of pleiotrophin and the classical angiogenic growth factor vascular endothelial growth factor. Using a specific ELISA-test we studied patients with small cell lung cancer (n=63), and patients with non-small cell lung cancer (n=22) in comparison to healthy control subjects (n=41). In most of the lung cancer patients (81%), we found serum levels of pleiotrophin above those of control subjects (P

We have demonstrated earlier that a secreted fibroblast growth factor-binding protein (FGF-BP) can enhance angiogenesis and promote tumor growth in vivo. Furthermore, we found that FGF-BP expression in squamous cell carcinoma (SCC) is reduced by concentrations of retinoids that are effective in the treatment of SCC and that this repression can occur at the transcriptional and post-transcriptional level. To further examine the mechanism of regulation of FGF-BP by retinoids and the role played by retinoid receptor subtypes, we utilized retinoic acid receptor (RAR)-selective (TTNPB) and retinoid X receptor (RXR)-selective (LG100268) ligands. In ME-180 SCC cells, FGF-BP mRNA was down-regulated by TTNPB with an ic 50 value of 1 nM, whereas transcription was only repressed at 10,000-fold higher concentrations ( ic 50 > 10 μM). This suggests that the major effects of retinoids on FGF-BP occur at the post-transcriptional level. In four additional SCC cell lines, FGF-BP was also down-regulated by TTNPB with ic 50 values of ≤ 1 nM, demonstrating that RAR receptors can modulate FGF-BP mRNA levels very effectively in SCC cells. The RXR-selective ligand on its own was only effective in two of the five cell lines ( ic 50 of ≈ 1 nM). In all of the SCC cell lines, a low concentration of RAR sensitized FGF-BP mRNA to treatment with the RXR ligand and the combination of the RXR and RAR ligands enhanced the efficacy beyond that of the individual ligands. We conclude that RAR receptors are major regulators of FGF-BP mRNA at the post-transcriptional level and propose that an RAR-induced gene product mediates the RXR effects on FGF-BP mRNA.

We showed previously that a secreted fibroblast growth factor-binding protein (FGF-BP) can mobilize and bioactivate locally-stored FGFs from the extracellular matrix. This FGF-BP is upregulated in various cancers and plays a rate limiting role as an angiogenic switch molecule during tumor growth. In this paper, we describe the cloning and sequence analysis of the rat homologue of FGF-BP and show its expression pattern and retinoid-mediated downregulation in normal adult rat tissues. The rat FGF-BP amino acid sequence is 91% and 70% homologous to mouse and human, respectively, and contains 10 cysteine residues whose position is conserved across species. In Northern blots, FGF-BP mRNA was detected in the gut, eye, thymus, skin, lung and tongue. Immunohistochemistry confirmed this tissue distribution with cerebellar Purkinje cells, the cerebral chorioid plexus and the eye showing the most distinctive staining patterns. Oral treatment of animals with all-trans-retinoic acid for one and two days induced a significant decrease of FGF-BP protein in tissues from stomach, eye and lung suggesting that regulation of FGF-BP can be one effector mechanism through which retinoids affect normal and pathological processes.

Amplification and overexpression of theHER-2/neu proto-oncogene frequently coincide with an aggressive clinical course of certain human adenocarcinomas. To assess whether HER-2/neu plays a rate-limiting role in ovarian cancer, we used human SK-OV-3 ovarian cancer cells as a model. We applied a conditional mRNA depletion strategy of HER-2/neu with anti-HER-2/neu-targeted hammerhead ribozymes expressed under the control of a tetracycline-regulated promoter system. In these ovarian cancer cells, we reduced HER-2/neu mRNA, protein expression, and tumor growth in nude mice by transfection with HER-2/neu-targeted ribozymes and generated cell lines expressing different levels of HER-2/neu. Expression of the most effective ribozyme (Rz3) quenched HER-2/neu mRNA levels by >90%. Concomitantly, fluorescence-activated cell sorting analysis revealed that expression of the HER-2/neu-encoded surface glycoprotein was almost completely abrogated. In nude mice, tumor growth was dramatically inhibited in the HER-2/neu-depleted Rz3-expressing SK-OV-3 cells. Furthermore, already established tumors started to regress when Rz3 expression was activated midstream by withdrawal of the tetracycline treatment. This study supports the thesis that HER-2/neu can be rate-limiting for the malignant phenotype of ovarian cancer in a gene dose-dependent manner.

Pleiotrophin (PTN) is a developmentally regulated protein which exhibits neurite-outgrowth, mitogenic, and angiogenic properties. It has also been shown to be involved in tumor growth and metastasis. Here we used primary BEL (bovine epithelial lens) cells to investigate the signal transduction pathways involved in the mitogenic activity of recombinant PTN. PTN was purified from conditioned media of SW-13 cells transfected with the human PTN cDNA. We show that inhibitors of tyrosine kinase, mitogen-activated protein kinase, or phosphoinositide (PI) 3-kinase inhibit DNA synthesis stimulated by PTN. Analysis of tyrosine-phosphorylated proteins following PTN stimulation showed phosphorylation of two novel 190- and 215-kDa proteins in addition to SHC, ERK1, and ERK2. A mobility shift of phosphorylated ERK1 and ERK2 was detected with a panERK antibody confirming the phosphorylation of the two ERKs. Furthermore, in vitroimmunocomplex kinase assay with Akt1, a natural substrate of PI 3-kinase, showed an activation of the kinase following PTN stimulation and a reversal by the PI 3-kinase inhibitor wortmannin. We conclude that the mitogenic activity of PTN is dependent on tyrosine kinase activation and utilizes the mitogen-activated protein kinase and the PI 3-kinase pathways to transduce a mitogenic signal.

Retinoids are potent regulators of growth and differentiation and have shown promise as chemotherapeutic agents against selected cancers in particular squamous cell carcinoma (SCC). Earlier studies from our laboratory showed that a secreted binding protein for fibroblast growth factors (BP) is expressed at high levels in SCC cell lines and tissue samples. Here we investigate whether retinoids affect BP gene expression in SCC. In six different human SCC cell lines, we found that all-trans-retinoic acid (tRA) down-regulated BP mRNA by 39-89% within 24 h. From this group of cell lines, we selected the ME-180 cell line for more detailed studies of the mechanisms of this regulation. tRA down-regulated BP mRNA in a time- and dose-dependent manner. The effect of tRA was reversible, and BP mRNA returned to control levels within 24 h after removal of tRA. We also measured BP mRNA half-life and performed nuclear run-on experiments to study if tRA down-regulates BP by destabilizing the mRNA and/or by decreasing the rate of transcription. BP mRNA in ME-180 cells is very stable with a half-life of >16 h, and tRA decreased BP mRNA with a half-time of 5 h. Actinomycin D and cycloheximide blocked the tRA effect, suggesting that transcriptional regulation as well as de novo protein synthesis contribute to this post-transcriptional regulation of BP mRNA levels. In addition, tRA decreased the rate of BP gene transcription by 2- to 3-fold within 1 h. We conclude that retinoids down-regulate BP gene expression by post-transcriptional as well as by transcriptional mechanisms.

9-cis retinoic acid (9-cis RA) is a retinoid receptor pan-agonist that binds with high affinity to both retinoic acid receptors (RARs) and retinoid X receptors (RXRs). Using a variety of in vivo and in vitro cancer models, we present experimental data that 9-cis RA has activity as a potential chemotherapeutic agent. Treatment of the human promyelocytic leukemia cell line HL-60 with 9-cis RA decreases cell proliferation, increases cell differentiation, and increases apoptosis. Induction of apoptosis correlates with an increase in tissue transglutaminase (type II) activity. In vivo, 9-cis RA induces complete tumor regression of an early passage human lip squamous cell carcinoma xenograft. Finally, 9-cis RA inhibits the anchorage-independent growth of the human breast cancer cell lines MCF-7 and LY2 (an antiestrogen-resistant MCF-7 variant). Transient co-transfection assays indicate that 9-cis RA inhibits estrogen receptor transcription of an ERE-tk-LUC reporter through RAR or RXR receptors. These data suggest that retinoid receptors can antagonize estrogen-dependent transcription and provides one possible mechanism for the inhibition of cell growth by 9-cis RA in breast cancer cell lines. In summary, these findings present evidence that 9-cis RA has a wide range of activities in human cancer models.

Fibroblast growth factors (FGFs) play a crucial role during embryonic development, tissue homeostasis, wound healing and angiogenesis. FGF-binding protein 1 (FGFBP1) is a secreted protein that binds FGFs stored in the extracellular matrix and transports them to their receptor, therefore they can modulate FGF signaling. FGFBP1 contributes to the progression of many cancers including colon, pancreas and squamous cell cancer. It acts as an angiogenic switch during tumor development. E0771 cells, a murine mammary carcinoma cell line, was subcutaneously injected into syngeneic C57BL/6 mice. While these cells form aggressive tumors within two weeks and metastasize to the lungs in wild type FGFBP1 (FGFBP1+/+) mice, there was no tumor growth or metastasis in FGFBP1 knock out (FGFBP1-/-) mice. A genome-wide shRNA library screen revealed a novel role for structural maintenance of the chromosome 2 (SMC2) in compensating for the loss of FGFBP1 in the stroma of FGFBP1-/- mice. E0771 cells with SMC2 knock down (KD) or CRISPR targeting SMC2 formed aggressive tumors in FGFBP1-/- mice and metastasized to the lungs. Surprisingly, FGFBP1 expression level was 32 folds lower in SMC2 KD E0771 cells yet FGF2 was 16 folds up in the tumors from SMC2 KD E0771 cells. Tumor histology shows impaired and leaky angiogenesis in FGFBP1-/- compared to FGFBP1+/+ mice. Furthermore, higher levels of GM-CSF and G-CSF were detected in the conditioned media (CM) of SMC2 KD E0771 cells compared to control cells suggesting differential immune cell recruitment. Also, an endothelial monolayer exposed to CM from SMC2 KD E0771 cells showed delayed wound closure and loose junctions suggesting a tendency for endothelium disintegration and initiation of blood vessel formation. Interestingly, the CM effect was blocked using PD173074, a pan FGFR inhibitor. In conclusion, SMC2 demonstrate a role in regulating FGF signaling which affects the course of angiogenesis during tumor development. Note: This abstract was not presented at the conference. Citation Format: Ghada M. Sharif, Marcel O. Schmidt, Casey Shuptrine, Weiner M. Louis, Anna T. Riegel, Anton Wellstein. SMC2 role in regulating tumor angiogenesis via FGF signaling. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr A15.

s: AACR Special Conference on Tumor Metastasis; November 30-December 3, 2015; Austin, TX Cancer cell vascular invasion is a crucial step in the malignant progression towards metastasis. Here we used a genome-wide RNAi screen with E0771 mammary cancer cells to uncover drivers of endothelial monolayer invasion. We identified keratin-associated protein 5-5 (Krtap5-5) as a candidate. Krtap5-5 belongs to large family of cysteine-rich proteins that is implicated in crosslinking keratin intermediate filaments during hair formation. To date, no role of these proteins in cancer has been reported. We found that depletion of Krtap5-5 from cancer cells led to cell blebbing, a loss of keratins 14 and 18 in addition to the upregulation of vimentin intermediate filaments. Krtap5-5 depletion did not impact cell viability but reduced cell motility and extravasation of cancer cells into tissues in a zebrafish model of extravasation. We conclude that Krtap5-5 is a previously unknown regulator of cytoskeletal function in cancer cells that modulates cell motility and vascular invasion. Citation Format: Eric B. Berens, Ghada M. Sharif, Marcel O. Schmidt, Casey W. Shuptrine, Louis M. Weiner, Eric Glasgow, Anna T. Riegel, Anton Wellstein. Keratin-associated protein 5-5 controls cytoskeletal function and cancer cell vascular invasion. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Metastasis; 2015 Nov 30-Dec 3; Austin, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(7 Suppl):Abstract nr B44.

TPS773 Background: Treatment with Regorafenib (REGO) has shown significant clinical benefits in metastatic colorectal cancer (mCRC) patients (pts) as demonstrated in the CORRECT and CONCUR trials. Results from both studies suggest that subgroups have differential responses. Further research to identify these subgroups through the identification of molecular biomarkers is needed. Methods: Forty pts with refractory mCRC are being enrolled in this study. The primary objective is to prospectively identify tissue and serum-based biomarkers that can predict response to REGO. Secondary objectives are to determine molecular mechanisms by which REGO controls refractory mCRC, as well as molecular pathways involved in the acquisition of resistance. Tumor and blood samples are obtained prior to and 2 weeks after starting REGO. Blood samples are collected on day 1 of each cycle thereafter. Pts will receive 160 mg REGO daily for 3 weeks of each 4-week cycle until disease progression or unacceptable toxicity. Multi-omic based biomarker discovery approaches will be used to uncover predictive marker candidates with special attention to the tumor microenvironment. Laser capture microdissection will be used on tumor tissue to procure highly enriched populations of pt-matched epithelial and stromal/immune cell infiltrates. Each of these entities will be analyzed for RNA expression changes and protein signaling/drug target activation mapping. Protein signaling analysis will be performed by reverse phase protein array of key REGO-related proteins and phosphoproteins (e.g. phosphoVEGFR, Tie2, phosphoRET), as well as broad-scale mapping of mitogenic, survival, autophagy, inflammatory, motility, and signaling networks. Tumor profiling will include next-generation sequencing for 592 genes with 53 selected gene fusions, and IHC and FISH/CISH for selected biomarkers, including PDL1, HER2, MSI, TS, ERCC1, and TOPO1. Blood samples will undergo protein, miRNA, and mutated DNA analysis, as well as exosomal signature study via a proprietary synthetic polyligand multiplexed aptamer-based assay. Exploratory analysis of biomarkers will be used to determine correlations between the presence of, or change in, biomarker levels and clinical response. Clinical trial information: NCT01949194.

Basic fibroblast growth factor (bFGF) is a strong inducer of angiogenesis and thus may play an important role in the growth of solid tumors. However, bFGF is usually found immobilized on the extracellular matrix, and it is only partly understood how it is solubilized to reach and activate its extracellular receptors. We studied the potential contribution to this process by a secreted binding protein (BP) with high affinity for FGFs. An expression vector for BP was transfected into a human cell line (SW-13) that contains constitutively high levels of bFGF. The BP-expressing cells began to grow colonies in soft agar due to their autocrine stimulation by bFGF and released biologically active bFGF into their media. Furthermore, they grew into well vascularized tumors in athymic nude mice. In addition, we found the BP mRNA expressed at high levels in squamous cell carcinoma (SCC) tissues from patients and in SCC cell lines of different origin as well as in immortalized keratinocytes. However, we failed to detect BP mRNA in normal adult tissues or in a number of non-SCC tumor cell lines. Expression of the secreted BP appears to be a mechanism through which immobilized FGF can be activated to support tumor growth and angiogenesis.

Reactive oxygen species enhance or impair autoregulation. Because superoxide is a vasoconstrictor, we tested the hypothesis that stretch generates superoxide that mediates myogenic responses. Increasing perfusion pressure of mouse isolated perfused renal afferent arterioles from 40 to 80 mm Hg reduced their diameter by 13.3±1.8% ( P P P l -nitroarginine methylester, or Ca 2+ -free medium, relating it to Ca 2+ -independent vascular superoxide. Compared with vehicle, basal tone and myogenic contractions were reduced significantly ( P l -Nitroarginine methylester enhanced basal tone, but neither it (15.8±3.3%) nor endothelial NO synthase knockout (10.2±1.8%) significantly changed myogenic contractions. Tempol had no further effect after superoxide dismutase but remained effective after catalase. H 2 O 2 >50 μmol/L caused contractions but at 25 μmol/L inhibited myogenic responses (7.4±0.8%; P 2+ -independent increased vascular superoxide production from nicotinamide adenine dinucleotide phosphate oxidase, which enhanced myogenic contractions largely independent of NO, whereas H 2 O 2 impaired pressure-induced contractions but was not implicated in the normal myogenic response.

AIB1 (amplified in breast cancer 1), also called SRC-3 and NCoA-3, is a member of the p160 nuclear receptor co-activator family and is considered an important oncogene in breast cancer. Increased AIB1 levels in human breast cancer have been correlated with poor clinical prognosis. Overexpression of AIB1 in conjunction with members of the epidermal growth factor receptor (EGF/HER) tyrosine kinase family, such as HER2, is associated with resistance to tamoxifen therapy and decreased disease-free survival. A number of functional studies in cell culture and in rodents indicate that AIB1 has a pleiotropic role in breast cancer. Initially AIB1 was shown to have a role in the estrogen-dependent proliferation of breast epithelial cells. However, AIB1 also affects the growth of hormone-independent breast cancer and AIB1 levels are limiting for IGF-1-, EGF- and heregulin-stimulated biological responses in breast cancer cells and consequently the PI3 K/Akt/mTOR and other EGFR/HER2 signaling pathways are controlled by changes in AIB1 protein levels. The cellular levels and activity of AIB1 are in turn regulated at the levels of transcription, mRNA stability, post-translational modification, and by a complex control of protein half life. In particular, AIB1 activity as well as its half-life is modulated through a number of post-translational modifications including serine, threonine and tyrosine phosphorylation via kinases that are components of multiple signal transduction pathways. This review summarizes the possible mechanisms of how dysregulation of AIB1 at multiple levels can lead to the initiation and progression of breast cancer as well as its role as a predictor of response to breast cancer therapy, and as a possible therapeutic target.

Effects of AF-DX 116, a cardioselective antagonist, on M cholinergic receptors (M-ChR) were studied in healthy volunteers. Occupancy of M-ChR subtypes by drug present in plasma samples (radioreceptor assay) was compared with these effects. After an intravenous dose of AF-DX 116 saturating greater than 90% of cardiac M2-ChR, an increase in heart rate by 25 beats/min was observed. This cardiac receptor occupancy and effect wore off with a parallel time course within 10 hours. No inhibition of salivary flow was observed, coinciding with a lack of M3-ChR blockade in the radioreceptor assay. Beta-adrenergic receptor blockade by propranolol did not affect either of the effects. No indication for active metabolites or stereoselective drug metabolism was found comparing HPLC and receptor assay for drug concentrations in plasma. We conclude that AF-DX 116 may be a useful drug for the treatment of bradycardia. Its lack of troublesome side effects is the result of its selectivity for cardiac M-ChR.

The region -15 to -3 of the pro-opiomelanocortin (POMC) gene promoter specifically binds a transcription factor previously designated PO-B. This region of the POMC gene is involved in the control of constitutive POMC gene expression since mutation of the PO-B DNA-binding site severely reduces transcription from the POMC promoter both in vivo and in vitro (Riegel, A. T., Remenick, J., Wolford, R., Berard, D., and Hager, G. (1990) Nucleic Acids Res. 18, 4513-4521). We have now purified PO-B from HeLa cells approximately 25,000-fold to greater than 90% homogeneity by a combination of ion exchange and reversed phase chromatography. In addition we have studied post-translational modifications that alter the affinity of purified PO-B for its cognate DNA binding site. In Southwestern analysis of column fractions, two bands of apparent molecular masses of 54 and 56 kDa bound specifically to the PO-B recognition sequence. The two copurified components have indistinguishable amino acid composition, are highly hydrophobic, and are heat and acid stable. DNA-binding specificity studies suggest that PO-B does not represent any previously described transcription factor. In addition, dephosphorylation of both species with acid phosphatase induced an about 30-fold increase in DNA binding but failed to produce any significant change in electrophoretic mobility. We conclude that the purified PO-B species represent products of the same gene and suggest that the in vivo function of PO-B may be regulated by its phosphorylation status.

Overexpression and activation of the steroid receptor coactivator amplified in breast cancer 1 (AIB1)/steroid receptor coactivator-3 (SRC-3) have been shown to have a critical role in oncogenesis and are required for both steroid and growth factor signaling in epithelial tumors. Here, we report a new mechanism for activation of SRC coactivators. We demonstrate regulated tyrosine phosphorylation of AIB1/SRC-3 at a C-terminal tyrosine residue (Y1357) that is phosphorylated after insulin-like growth factor 1, epidermal growth factor, or estrogen treatment of breast cancer cells. Phosphorylated Y1357 is increased in HER2/neu (v-erb-b2 erythroblastic leukemia viral oncogene homolog 2) mammary tumor epithelia and is required to modulate AIB1/SRC-3 coactivation of estrogen receptor alpha (ERalpha), progesterone receptor B, NF-kappaB, and AP-1-dependent promoters. c-Abl (v-Abl Abelson murine leukemia viral oncogene homolog 1) tyrosine kinase directly phosphorylates AIB1/SRC-3 at Y1357 and modulates the association of AIB1 with c-Abl, ERalpha, the transcriptional cofactor p300, and the methyltransferase coactivator-associated arginine methyltransferase 1, CARM1. AIB1/SRC-3-dependent transcription and phenotypic changes, such as cell growth and focus formation, can be reversed by an Abl kinase inhibitor, imatinib. Thus, the phosphorylation state of Y1357 can function as a molecular on/off switch and facilitates the cross talk between hormone, growth factor, and intracellular kinase signaling pathways in cancer.

The steroid receptor coactivator amplified in breast cancer 1 (AIB1) as well as epidermal growth factor receptor (EGFR) family members are frequently overexpressed in epithelial tumors, and their expression is associated with poor prognosis. However, a direct role of AIB1 in EGF signaling has not been determined. To address this, we reduced endogenous AIB1 levels using RNA interference in lung, breast, and pancreatic cancer cell lines. We found that a knockdown of AIB1 levels resulted in a loss of the growth response of these cell lines to EGF. Further analysis revealed that the depletion of AIB1 reduced tyrosine phosphorylation of EGFR at multiple residues both at autophosphorylation and Src kinase phosphorylation sites. AIB1 knockdown did not affect tyrosine phosphorylation of the receptor tyrosine kinases, platelet-derived growth factor receptor and HER3, or overall tyrosine phosphorylation of cellular proteins. However, EGF-dependent phosphorylation of HER2 was decreased. EGFR levels and membrane trafficking were not changed by AIB1 depletion, but there was less recruitment of Src homology 2 domain-containing proteins to the EGFR. This led to a substantial reduction in EGF-induced phosphorylation of signal transducers and activators of transcription 5 and c-Jun NH2-terminal kinase but no significant change in the activation of AKT. Vanadate treatment of cells revealed that the reduction in EGFR tyrosine phosphorylation is dependent in part on changes in cellular phosphatase activity. We propose that a portion of the oncogenic effect of AIB1 could be through control of EGFR and HER2 activity and subsequent modulation of cellular signaling pathways. [Cancer Res 2007;67(15):7256–65]

The secreted growth factor pleiotrophin (PTN) can induce mitogenesis in cells that express the receptor for this growth factor, anaplastic lymphoma kinase (ALK). Here we examine the ability of PTN to produce anti-apoptotic signals. We demonstrate that PTN is a survival factor for SW-13 epithelial cells and show that ribozyme-mediated depletion of ALK from SW-13 cells abolishes this effect of PTN. Furthermore, in serum-starved NIH3T3 fibroblasts PTN prevents apoptosis (measured by annexin V staining) with an EC(50) of 0.2 ng/ml and induces cell growth at higher concentrations of PTN. A polyclonal antibody against the PTN ligand-binding domain of the ALK receptor (alpha-LBD) was a partial agonist for ALK in NIH3T3 cells. This alpha-LBD antibody showed high agonist activity for anti-apoptosis (56 +/- 9% relative to PTN), low agonist activity for cell growth (21 +/- 1% relative to PTN), and was an antagonist of PTN-induced cell growth (61 +/- 2% inhibition). Both MAP kinase and phosphatidylinositol (PI) 3-kinase cascades in NIH3T3 cells were activated by PTN, and this effect persisted for up to 3 h. Surprisingly, the anti-apoptotic effect of PTN was completely blocked by the MAP kinase inhibitor UO126, but was not affected by the PI 3-kinase inhibitor LY294002. In contrast, PTN-dependent cell growth required both MAPK and PI 3-kinase activity. We conclude that anti-apoptotic signaling of PTN through ALK in NIH3T3 fibroblasts is via the MAP kinase pathway.

Up-regulation of a fibroblast growth factor binding protein in children with renal diseases. Background Basic fibroblast growth factor (bFGF) is an angiogenic growth factor that is involved in renal growth and the pathogenesis of renal diseases. We have detected high levels of bFGF accumulated in the kidney of HIV-transgenic mice and in children with HIV-associated renal diseases and the hemolytic uremic syndrome (HUS). However, the mechanism modulating the activity of bFGF under these circumstances is poorly understood. We carried out experiments to determine whether a secreted binding protein (FGF-BP) that modulates the activity of bFGF during the process of tumor growth was expressed in pediatric kidneys and to define whether the expression of FGF-BP was altered in pediatric renal diseases associated with high levels of bFGF. Methods Immunohistochemistry and in situ hybridization studies were done in 41 renal sections from children with HIV nephropathies, HUS, other pediatric renal diseases, controls, and fetal kidneys. Western blots and reverse transcriptase-polymerase chain reaction studies were done in selected urine samples and cultured renal cells. Recombinant FGF-BP was produced to study the mitogenic activity of FGF-BP in cultured human renal proximal tubular epithelial cells (RPTEcs). Results The expression of FGF-BP was up-regulated predominately in renal tubular epithelial cells in children with renal tubular injury, HIV-associated nephropathy (HIVAN), and HUS, and FGF-BP was secreted in the urine of these patients. FGF-BP was also abundantly expressed in developing fetal renal tubules. Recombinant FGF-BP enhanced the mitogenic effects of bFGF in cultured human RPTEcs. Conclusions The localization of FGF-BP in renal tubular epithelial cells could provide a mechanism by which the activity of bFGF is modulated in developing and regenerating renal tubules of children.

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.

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. Clinical Pharmacology & Therapeutics (1995) 58, 210–220; doi

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. Clinical Pharmacology and Therapeutics (1993) 53, 436–442; doi:10.1038/clpt.1993.48

We have purified and characterized a novel 30-kDa glycoprotein (gp30) with TGF alpha-like properties secreted from the estrogen receptor negative breast cancer cell line MDA-MB-231. This factor was immunoprecipitated by an anti-TGF alpha polyclonal antibody and also had TGF alpha-like biological activity, as assayed by EGF radioreceptor assay and anchorage-independent assays. In addition, the novel growth factor stimulated phosphorylation of the EGF receptor and erbB-2 receptor. However, the novel growth factor, unlike EGF and TGF alpha, bound to heparin-Sepharose. Purification of gp30 was obtained to apparent homogeneity by heparin affinity chromatography and subsequent reversed-phase chromatography. Tunicamycin treatment in vivo or N-glycanase deglycosylation in vitro revealed a putative precursor of approximately 22 kDa molecular mass in contrast to the "normal" 16-kDa precursor species for TGF alpha. In vitro translation of total mRNA from MDA-MB-231 cells confirmed the size of the putative precursor. Biochemical characterization of gp30 was begun by V8 protease digestion of the deglycosylated polypeptide and the translated products. Peptide mapping of V8-digested, immunoprecipitated material suggests that the amino acid sequence of this unique protein is distinct from mature TGF alpha and not the result of a posttranslational modification of the precursor. We conclude that this TGF alpha-like (gp30) polypeptide is a novel growth factor with agonistic activity for both EGF and erbB-2 receptors.

The potency order of the catecholamines (-)-isoprenaline (Iso), (-)-noradrenaline (NA), and (-)-adrenaline (Adr) in competition for radiolabelled sites is used for their pharmacological classification. It is shown that the radioligand 3H-CGP 12177 exclusively labels beta 1-adrenoceptors in rat salivary gland membranes (Iso greater than NA greater than Adr), and beta 2-adrenoceptors in rat reticulocytes (Iso greater than Adr greater than or equal to NA). These models are then used to derive the subtype-selectivity of the classical beta-adrenoceptor antagonists (+/-)-propranolol (prop; twofold beta 2-selective) and (+/-)-atenolol (aten; 35-fold beta 1-selective), as well as of the newer antagonists (+/-)-betaxolol and (+/-)-bisoprolol (betax and biso; 35-fold and 75-fold beta 1-selective, respectively). The ligand with the highest selectivity is ICI 118,551 (ICI), with a 300-fold beta 2-subtype selectivity. For comparison with antagonistic effects in humans at given plasma concentrations, the equilibrium dissociation constants of the ligands are measured in the presence of native human plasma and yield values for the relative selectively labelled subtype in the mean (Ki-values in nmol/l): prop: 20, aten: 250, biso: 24, betax: 23, and ICI: 2.5.