TNF and TNF receptor expression and insulin sensitivity in human omental and subcutaneous adipose tissue--influence of BMI and adipose distribution

Tumour necrosis factor (TNF)alpha is implicated in the relationship between obesity and insulin resistance/ type 2 diabetes. In an effort to understand this association better we (i) profiled gene expression patterns of TNF, TNFR1 and TNFR2 and (ii) investigated the effects of TNF on glucose uptake in isolated adipocytes and adipose tissue explants from omental and subcutaneous depots from lean, overweight and obese individuals. TNF expression correlated with expression of TNFR2, but not TNFR1, and TNF and TNFR2 expression increased in obesity. TNFR1 expression was higher in omental than in subcutaneous adipocytes. Expression levels of TNF or either receptor did not differ between adipocytes from individuals with central and peripheral obesity. TNF only suppressed glucose uptake in insulin-stimulated subcutaneous tissue and this suppression was only observed in tissue from lean subjects. These data support a relationship between the TNF system and body mass index (BMI), but not fat distribution, and suggest depot specificity of the TNF effect on glucose uptake. Furthermore, adipose tissue from obese subjects already appears insulin 'resistant' and this may be a result of the increased TNF levels.

Integrin αvβ3 mediates upregulation of epidermal growth-factor receptor expression and activity in human ovarian cancer cells

Upon overexpression of integrin αvβ3 and its engagement by vitronectin, we previously showed enhanced adhesion, proliferation, and motility of human ovarian cancer cells. By studying differential expression of genes possibly related to these tumor biological events, we identified the epidermal growth-factor receptor (EGF-R) to be under control of αvβ3 expression levels. Thus in the present study we characterized αvβ3-dependent changes of EGF-R and found significant upregulation of its expression and activity which was reflected by prominent changes of EGF-R promoter activity. Upon disruption of DNA-binding motifs for the transcription factors p53, ETF, the repressor ETR, p50, and c-rel, respectively, we sought to identify DNA elements contributing to αvβ3-mediated EGF-R promoter induction. Both, the p53- and ETF-mutant, while exhibiting considerably lower EGF-R promoter activity than the wild type promoter, retained inducibility by αvβ3. Mutation of the repressor motif ETR, as expected, enhanced EGF-R promoter activity with a further moderate increase upon αvβ3 elevation. The p50-mutant displayed EGF-R promoter activity almost comparable to that of the wild type promoter with no impairment of induction by αvβ3. However, the activity of an EGF-R promoter mutant displaying a disrupted c-rel-binding motif did not only prominently decline, but, moreover, was not longer responsive to enhanced αvβ3, involving this DNA element in αvβ3-dependent EGF-R upregulation. Moreover, αvβ3 did not only increase the EGF-R but, moreover, also led to obvious co-clustering on the cancer cell surface. By studying αvβ3/EGF-R-effects on the focal adhesion kinase (FAK) and the mitogen activated protein kinases (MAPK) p44/42 (erk−1/erk−2), having important functions in synergistic crosstalk between integrins and growth-factor receptors, we found for both significant enhancement of expression and activity upon αvβ3/VN interaction and cell stimulation by EGF. Upregulation of the EGF-R by integrin αvβ3, both receptor molecules with a well-defined role as targets for cancer treatment, might represent an additional mechanism to adapt synergistic receptor signaling and crosstalk in response to an altered tumor cell microenvironment during ovarian cancer progression.

Ghrelin and the brain-gut axis as a pharmacological target for appetite control

Appetite regulation is highly complex and involves a large number of orexigenic and anorexigenic peptide hormones. These are small, processed, secreted peptides derived from larger prepropeptide precursors. These peptides are important targets for the development of therapeutics for obesity, a global health epidemic. As a case study, we consider the ghrelin axis. The ghrelin axis is likely to be a particularly useful drug target, as it also plays a role in energy homeostasis, adipogenesis, insulin regulation and reward associated with food intake. Ghrelin is the only known circulating gut orexigenic peptide hormone. As it appears to play a role in diet-induced obesity, blocking the action of ghrelin is likely to be effective for treating and preventing obesity. The ghrelin peptide has been targeted using a number of approaches, with ghrelin mirror-image oligonucleotides (Spiegelmers) and immunotherapy showing some promise. The ghrelin receptor, the growth hormone secretagogue receptor, may also provide a useful target and a number of antagonists and inverse agonists have been developed. A particularly promising new target is the enzyme which octanoylates ghrelin, ghrelin O-acyltransferase (GOAT), and drugs that inhibit GOAT are likely to circumvent pharmacological issues associated with approaches that directly target ghrelin or its receptor.

Expression and in vitro functions of the ghrelin axis in endometrial cancer

Ghrelin is a peptide hormone produced in the stomach and a range of other tissues, where it has endocrine, paracrine and autocrine roles in both normal and disease states. Ghrelin has been shown to be an important growth factor for a number of tumours, including prostate and breast cancers. In this study, we examined the expression of the ghrelin axis (ghrelin and its receptor, the growth hormone secretagogue receptor, GHSR) in endometrial cancer. Ghrelin is expressed in a range of endometrial cancer tissues, while its cognate receptor, GHSR1a, is expressed in a small subset of normal and cancer tissues. Low to moderately invasive endometrial cancer cell lines were examined by RT-PCR and immunoblotting, demonstrating that ghrelin axis mRNA and protein expression correlate with differentiation status of Ishikawa, HEC1B and KLE endometrial cancer cell lines. Moreover, treatment with ghrelin potently stimulated cell proliferation and inhibited cell death. Taken together, these data indicate that ghrelin promotes the progression of endometrial cancer cells in vitro, and may contribute to endometrial cancer pathogenesis and represent a novel treatment target.

Ghrelin receptor (GHS-R1A) antagonism suppresses both operant alcohol self-administration and high alcohol consumption in rats

The mechanisms involved in alcohol use disorders are complex. It has been shown that ghrelin is an important signal for the control of body weight homeostasis, preferably by interacting with hypothalamic circuits, as well as for drug reward by activating the mesolimbic dopamine system. The ghrelin receptor (GHS-R1A) has been shown to be required for alcohol-induced reward. Additionally, ghrelin increases and GHR-R1A antagonists reduce moderate alcohol consumption in mice, and a single nucleotide polymorphism in the GHS-R1A gene has been associated with high alcohol consumption in humans. However, the role of central ghrelin signaling in high alcohol consumption is not known. Therefore, the role of GHS-R1A in operant self-administration of alcohol in rats as well as for high alcohol consumption in Long-Evans rats and in alcohol preferring [Alko alcohol (AA)] rats was studied here. In the present study, the GHS-R1A antagonist, JMV2959, was found to reduce the operant self-administration of alcohol in rats and to decrease high alcohol intake in Long-Evans rats as well as in AA rats. These results suggest that the ghrelin receptor signaling system, specifically GHS-R1A, is required for operant self-administration of alcohol and for high alcohol intake in rats. Therefore, the GHS-R1A may be a therapeutic target for treatment of addictive behaviors, such as alcohol dependence.

Calcitonin receptor plays a physiological role to protect against hypercalcemia in mice

It is well established that calcitonin is a potent inhibitor of bone resorption; however, a physiological role for calcitonin acting through its cognate receptor, the calcitonin receptor (CTR), has not been identified. Data from previous genetically modified animal models have recognized a possible role for calcitonin and the CTR in controlling bone formation; however, interpretation of these data are complicated, in part because of their mixed genetic background. Therefore, to elucidate the physiological role of the CTR in calcium and bone metabolism, we generated a viable global CTR knockout (KO) mouse model using the Cre/loxP system, in which the CTR is globally deleted by >94% but <100%. Global CTRKOs displayed normal serum ultrafiltrable calcium levels and a mild increase in bone formation in males, showing that the CTR plays a modest physiological role in the regulation of bone and calcium homeostasis in the basal state in mice. Furthermore, the peak in serum total calcium after calcitriol [1,25(OH)2D3]-induced hypercalcemia was substantially greater in global CTRKOs compared with controls. These data provide strong evidence for a biological role of the CTR in regulating calcium homeostasis in states of calcium stress.

A soluble activin Type IIA receptor induces bone formation and improves skeletal integrity

Diseases that affect the regulation of bone turnover can lead to skeletal fragility and increased fracture risk. Members of the TGF-superfamily have been shown to be involved in the regulation of bone mass. Activin A, a TGF-� signaling ligand, is present at high levels in bone and may play a role in the regulation of bone metabolism. Here we demonstrate that pharmacological blockade of ligand signaling through the high affinity receptor for activin, type II activin receptor (ActRIIA), by administration of the soluble extracellular domain of ActRIIA fused to a murine IgG2a-Fc, increases bone formation, bone mass, and bone strength in normal mice and in ovariectomized mice with established bone loss. These observations support the development of this pharmacological strategy for the treatment of diseases with skeletal fragility.

Intermittent Fugu parathyroid hormone 1 (1–34) is an anabolic bone agent in young male rats and osteopenic ovariectomized rats

Human parathyroid hormone (hPTH) is currently the only treatment for osteoporosis that forms new bone. Previously we described a fish equivalent, Fugu parathyroid hormone 1 (fPth1) which has hPTH-like biological activity in vitro despite fPth1(1–34) sharing only 53% identity with hPTH(1–34). Here we demonstrate the in vivo actions of fPth1(1–34) on bone. In study 1, young male rats were injected intermittently for 30 days with fPth1 [30 μg–1000 μg/kg body weight (b.w.), (30fPth1–1000fPth1)] or hPTH [30 μg–100 μg/kg b.w. (30hPTH–100hPTH)]. In proximal tibiae at low doses, the fPth1 was positively correlated with trabecular bone volume/total volume (TbBV/TV) while hPTH increased TbBV/TV, trabecular thickness (TbTh) and trabecular number (TbN). 500fPth1 and 1000fPth1 increased TbBV/TV, TbTh, TbN, mineral apposition rate (MAR) and bone formation rate/bone surface (BFR/BS) with a concomitant decrease in osteoclast surface and number. In study 2 ovariectomized (OVX), osteopenic rats and sham operated (SHAM) rats were injected intermittently with 500 μg/kg b.w. of fPth1 (500fPth1) for 11 weeks. 500fPth1 treatment resulted in increased TbBV/TV (151%) and TbTh (96%) in the proximal tibiae due to increased bone formation as assessed by BFR/BS (490%) and MAR (131%). The effect was restoration of TbBV/TV to SHAM levels without any effect on bone resorption. 500fPth1 also increased TbBV/TV and TbTh in the vertebrae (L6) and cortical thickness in the mid-femora increasing bone strength at these sites. fPth1 was similarly effective in SHAM rats. Notwithstanding the low amino acid sequence homology with hPTH (1–34), we have clearly established the efficacy of fPth1 (1–34) as an anabolic bone agent.

Hormonal carcinogenesis in breast cancer : cellular and molecular studies of malignant progression

We have established and characterized a series of variant cell lines in which to identify the critical factors associated with E2-induced malignant progression, and the acquisition to tamoxifen resistance in human breast cancer. Sublines of the hormone-dependent MCF-7 cell line (MCF7/MIII and MCF7/LCC1) form stable, invasive, estrogen independent tumors in the mammary fat pads of ovariectomized athymic nude mice. These cells retain expression of both estrogen (ER) and progesterone receptors (PGR), but retain sensitivity to each of the major structural classes of antiestrogens. The tamoxifen-resistant MCF7/LCC2 cells retain sensitivity to the inhibitory effects of the steroidal antiestrogen ICI 182780. By comparing the parental hormone-dependent and variant hormone-independent cells, we have demonstrated an altered expression of some estrogen regulated genes (PGR, pS2, cathepsin D) in the hormone-independent variants. Other genes remain normally estrogen regulated (ER, laminin receptor, EGF-receptor). These data strongly implicate the altered regulation of a specific subset or network of estrogen regulated genes in the malignant progression of human breast cancer. Some of the primary response genes in this network may exhibit dose-response and induction kinetics similar to pS2, which is constitutively upregulated in the MCF7/MIII, MCF7/LCC1 and MCF7/LCC2 cells.

Hormone resistance, invasiveness, and metastatic potential in breast cancer

Critical phenotypic changes that occur during the progression of breast cancer include the loss of hormone-dependence, acquired resistance to systemic therapies, and increased metastatic potential. We have isolated a series of MCF-7 human breast cancer variants which exhibit hormone-independent growth, antiestrogen resistance, and increased metastatic potential. Analysis of the phenotypes of these variants strongly suggests that changes in the expression of specific genes may be critical to the generation of phenotypic diversity in the process of malignant progression in breast cancer. Epigenetic changes may contribute significantly to the generation of these phenotypic changes observed during breast cancer progression. Many of the characteristics of the progressed phenotypes appear to have arisen in response to appropriate selective pressures (growth in ovariectomized nude mice; growth in the presence of antiestrogens). These observations are consistent with the concept of clonal selection and expansion in the process of malignant progression.

MCF7/LCC2: A 4-hydroxytamoxifen resistant human breast cancer variant that retains sensitivity to the steroidal antiestrogen ICI 182,780

The development of resistance to the antiestrogen tamoxifen occurs in a high percentage of initially responsive patients. We have developed a new model in which to investigate acquired resistance to triphenylethylenes. A stepwise in vitro selection of the hormone-independent human breast cancer variant MCF-7/LCC1 against 4-hydroxytamoxifen produced a stable resistant population designated MCF7/LCC2. MCF7/LCC2 cells retain levels of estrogen receptor expression comparable to the parental MCF7/LCC1 and MCF-7 cells. Progesterone receptor expression remains estrogen inducible in MCF7/LCC2 cells, although to levels significantly lower than observed in MCF-7 and MCF7/LCC1 cells. MCF7/ LCC2 cells form tumors in ovariectomized nude mice without estrogen supplementation, and these tumors are tamoxifen resistant but can be tstrogen stimulated. Significantly, MCF7/LCC2 cells have retained sensitivity to the steroidal antiestrogen ICI 182,780. These data suggest that some breast cancer patients who acquire resistance to tamoxifen may not develop cross-resistance to treatment with steroidal antiestrogens.

Mesenchymal-to-epithelial transition facilitates bladder cancer metastasis : role of fibroblast growth factor receptor-2

Epithelial-to-mesenchymal transition (EMT) increases cell migration and invasion, and facilitates metastasis in multiple carcinoma types, but belies epithelial similarities between primary and secondary tumors. This study addresses the importance of mesenchymal-to-epithelial transition (MET) in the formation of clinically significant metastasis. The previously described bladder carcinoma TSU-Pr1 (T24) progression series of cell lines selected in vivo for increasing metastatic ability following systemic seeding was used in this study. It was found that the more metastatic sublines had acquired epithelial characteristics. Epithelial and mesenchymal phenotypes were confirmed in the TSU-Pr1 series by cytoskeletal and morphologic analysis, and by performance in a panel of in vitro assays. Metastatic ability was examined following inoculation at various sites. Epithelial characteristics associated with dramatically increased bone and soft tissue colonization after intracardiac or intratibial injection. In contrast, the more epithelial sublines showed decreased lung metastases following orthotopic inoculation, supporting the concept that EMT is important for the escape of tumor cells from the primary tumor. We confirmed the overexpression of the IIIc subtype of multiple fibroblast growth factor receptors (FGFR) through the TSU-Pr1 series, and targeted abrogation of FGFR2IIIc reversed the MET and associated functionality in this system and increased survival following in vivo inoculation in severe combined immunodeficient mice. This model is the first to specifically model steps of the latter part of the metastatic cascade in isogenic cell lines, and confirms the suspected role of MET in secondary tumor growth.

Activation of matrix metalloproteinase-2 (MMP-2) by membrane type 1 matrix metalloproteinase through an artificial receptor for ProMMP-2 generates active MMP-2

The suggested model for pro-matrix metalloproteinase-2 (proMMP-2) activation by membrane type 1 MMP (MT1-MMP) implicates the complex between MT1-MMP and tissue inhibitor of MMP-2 (TIMP-2) as a receptor for proMMP-2. To dissect this model and assess the pathologic significance of MMP-2 activation, an artificial receptor for proMMP-2 was created by replacing the signal sequence of TIMP-2 with cytoplasmic/transmembrane domain of type II transmembrane mosaic serine protease (MSP-T2). Unlike TIMP-2, MSP-T2 served as a receptor for proMMP-2 without inhibiting MT1-MMP, and generated TIMP-2-free active MMP-2 even at a low level of MT1-MMP. Thus, MSP-T2 did not affect direct cleavage of the substrate testican-1 by MT1-MMP, whereas TIMP-2 inhibited it even at the level that stimulates proMMP-2 processing. Expression of MSP-T2 in HT1080 cells enhanced MMP-2 activation by endogenous MT1-MMP and caused intensive hydrolysis of collagen gel. Expression of MSP-T2 in U87 glioma cells, which express a trace level of endogenous MT1-MMP, induced MMP-2 activation and enhanced cell-associated protease activity, activation of extracellular signal-regulated kinase, and metastatic ability into chick embryonic liver and lung. MT1-MMP can exert both maximum MMP-2 activation and direct cleavage of substrates with MSP-T2, which cannot be achieved with TIMP-2. These results suggest that MMP-2 activation by MT1-MMP potentially amplifies protease activity, and combination with direct cleavage of substrate causes effective tissue degradation and enhances tumor invasion and metastasis, which highlights the complex role of TIMP-2. MSP-T2 is a unique tool to analyze physiologic and pathologic roles of MMP-2 and MT1-MMP in comparison with TIMP-2.

Expression of 67 kDa laminin receptor in human breast cancer cells : regulation by progestins

The level of 67 kDa laminin receptor (67LR) expression on breast and colon tumor cell surfaces was previously shown to be correlated with the capacity of tumor cells to metastasize. In the present work we investigate the effects of progestins and estrogen on the expression of 67LR in two sublines of the T47D human breast cancer cells: weakly tumorigenic, poorly invasive parental T47D cells and a highly tumorigenic, more invasive T47Dco subclone. Inmmunoblotting with an affinity purified antibody directed against a synthetic peptide recognizes the 67LR in these cells. 67LR expression in the T47Dco subclone is 5,5-fold higher than in their parental T47D cells. Treatment of T47D cells with 1 nM of the synthetic progestin R5020 results in a 4-fold increase in 67LR protein expression. Estrogen also induced 67LR expression, but only by 1.5-fold. The progestin-stimulated expression of the 67LR correlates with a 4.3-fold increase in attachment of T47D cells to laminin. A monoclonal antibody, mAb 13, directed against β1 integrin, completely blocks the attachment of T47D cells to fibronectin, only partially inhibits the attachment of T47D cells to laminin, and appears not to affect the progestin-stimulated laminin attachment of T47D cells. A new antiprogestin, ZK 112.993, significantly inhibits both progestin-stimulated 67LR expression and the increased attachment to laminin. These results suggest a possible role for progestin in mediating one of the multiple events thought to be important in metastasis of steroid receptor positive human breast cancer cells.

Remodeling of purinergic receptor-mediated Ca 2+ signaling as a consequence of EGF-induced epithelial-mesenchymal transition in breast cancer cells

Background The microenvironment plays a pivotal role in tumor cell proliferation, survival and migration. Invasive cancer cells face a new set of environmental challenges as they breach the basement membrane and colonize distant organs during the process of metastasis. Phenotypic switching, such as that which occurs during epithelial-mesenchymal transition (EMT), may be associated with a remodeling of cell surface receptors and thus altered responses to signals from the tumor microenvironment. Methodology/Principal Findings We assessed changes in intracellular Ca 2+ in cells loaded with Fluo-4 AM using a fluorometric imaging plate reader (FLIPR TETRA) and observed significant changes in the potency of ATP (EC 50 0.175 μM (-EGF) versus 1.731 μM (+EGF), P<0.05), and the nature of the ATP-induced Ca 2+ transient, corresponding with a 10-fold increase in the mesenchymal marker vimentin (P<0.05). We observed no change in the sensitivity to PAR2-mediated Ca 2+ signaling, indicating that these alterations are not simply a consequence of changes in global Ca 2+ homeostasis. To determine whether changes in ATP-mediated Ca 2+ signaling are preceded by alterations in the transcriptional profile of purinergic receptors, we analyzed the expression of a panel of P2X ionotropic and P2Y metabotropic purinergic receptors using real-time RT-PCR and found significant and specific alterations in the suite of ATP-activated purinergic receptors during EGF-induced EMT in breast cancer cells. Our studies are the first to show that P2X 5 ionotropic receptors are enriched in the mesenchymal phenotype and that silencing of P2X 5 leads to a significant reduction (25%, P<0.05) in EGF-induced vimentin protein expression. Conclusions The acquisition of a new suite of cell surface purinergic receptors is a feature of EGF-mediated EMT in MDA-MB-468 breast cancer cells. Such changes may impart advantageous phenotypic traits and represent a novel mechanism for the targeting of cancer metastasis.