Zenaida P. Lopez-Dee

Thrombospondin-1: Multiple Paths to Inflammation

Inflammation is a defensive process against tissue injury. Once this self-protective strategy is initiated, an effective resolution of the process is crucial to avoid major and unnecessary tissue damage. If the underlying event inducing inflammation is not addressed and homeostasis is not restored, this process can become chronic and lead to angiogenesis and carcinogenesis. Thrombospondin-1 (TSP-1) is a matricellular protein involved in angiogenesis, cancer, and inflammation. The effects of TSP-1 have been studied in many preclinical tumor models, and mimetic peptides are being tested in cancer clinical trials. However, the molecular mechanisms explaining its role in inflammatory processes are not well understood. This paper will discuss the role of TSP-1 in inflammation and its interaction with key receptors that may explain its functions in that process. Recent literature will be reviewed showing novel mechanisms by which this multifaceted protein could modulate the inflammatory process and impact its resolution.

Activation of NFkB is a novel mechanism of pro-survival activity of glucocorticoids in breast cancer cells.

Glucocorticoids (GCs) are pro-apoptotic as a co-medication to treat leukemia and lymphoma. However, the effects in breast cancer (BC) are diverse with mechanisms less understood. In this study using BC model cell MCF7, we found that dexamethasone (Dex) promotes cell proliferation. Gene expression analysis identified that c-Myc was enhanced by Dex, providing an important link to the pro-survival effect of GCs in BC. Dex treatment promoted NFkB transcriptional activity leading to the up-regulation of c-Myc. RelA was activated by Dex but with decreased interaction with GR, thus identifying a new pattern of regulation of NFkB by GC/GR in BC cells.

Thrombospondin-1 Type 1 Repeats in a Model of Inflammatory Bowel Disease: Transcript Profile and Therapeutic Effects

Thrombospondin-1 (TSP-1) is a matricellular protein with regulatory functions in inflammation and cancer. The type 1 repeats (TSR) domains of TSP-1 have been shown to interact with a wide range of proteins that result in the anti-angiogenic and anti-tumor properties of TSP-1. To ascertain possible functions and evaluate potential therapeutic effects of TSRs in inflammatory bowel disease, we conducted clinical, histological and microarray analyses on a mouse model of induced colitis. We used dextran sulfate sodium (DSS) to induce colitis in wild-type (WT) mice for 7 days. Simultaneously, mice were injected with either saline or one form of TSP-1 derived recombinant proteins, containing either (1) the three type 1 repeats of the TSP-1 (3TSR), (2) the second type 1 repeat (TSR2), or (3) TSR2 with the RFK sequence (TSR2+RFK). Total RNA isolated from the mice colons were processed and hybridized to mouse arrays. Array data were validated by real-time qPCR and immunohistochemistry. Histological and disease indices reveal that the mice treated with the TSRs show different patterns of leukocytic infiltration and that 3TSR treatment was the most effective in decreasing inflammation in DSS-induced colitis. Transcriptional profiling revealed differentially expressed (DE) genes, with the 3TSR-treated mice showing the least deviation from the WT-water controls. In conclusion, this study shows that 3TSR treatment is effective in attenuating the inflammatory response to DSS injury. In addition, the transcriptomics work unveils novel genetic data that suggest beneficial application of the TSR domains in inflammatory bowel disease.

Thrombospondin-1 in a Murine Model of Colorectal Carcinogenesis

Colorectal Cancer (CRC) is one of the late complications observed in patients suffering from inflammatory bowel diseases (IBD). Carcinogenesis is promoted by persistent chronic inflammation occurring in IBD. Understanding the mechanisms involved is essential in order to ameliorate inflammation and prevent CRC. Thrombospondin 1 (TSP-1) is a multidomain glycoprotein with important roles in angiogenesis. The effects of TSP-1 in colonic tumor formation and growth were analyzed in a model of inflammation-induced carcinogenesis. WT and TSP-1 deficient mice (TSP-1-/-) of the C57BL/6 strain received a single injection of azoxymethane (AOM) and multiple cycles of dextran sodium sulfate (DSS) to induce chronic inflammation-related cancers. Proliferation and angiogenesis were histologically analyzed in tumors. The intestinal transcriptome was also analyzed using a gene microarray approach. When the area containing tumors was compared with the entire colonic area of each mouse, the tumor burden was decreased in AOM/DSS-treated TSP-1-/- versus wild type (WT) mice. However, these lesions displayed more angiogenesis and proliferation rates when compared with the WT tumors. AOM-DSS treatment of TSP-1-/- mice resulted in significant deregulation of genes involved in transcription, canonical Wnt signaling, transport, defense response, regulation of epithelial cell proliferation and metabolism. Microarray analyses of these tumors showed down-regulation of 18 microRNAs in TSP-1-/- tumors. These results contribute new insights on the controversial role of TSP-1 in cancer and offer a better understanding of the genetics and pathogenesis of CRC.

Defective lipid metabolism associated with mutation in klf-2 and klf-3 : important roles of essential dietary salts in fat storage

BackgroundDietary salts are important factors in metabolic disorders. They are vital components of enzymes, vitamins, hormones, and signal transduction that act synergistically to regulate lipid metabolism. Our previous studies have identified that Krüppel-like factor −3 (KLF-3) is an essential regulator of lipid metabolism. However, it is not known if KLF-2 also regulates lipid metabolism and whether KLF-2 and −3 mediate the effects of dietary salts on lipid metabolism.MethodsIn this study, we used klf mutants [homozygous klf-2 (ok1043) V and klf-3 (ok1975) II mutants] to investigate the role of dietary salts in lipid metabolism. All gene expression was quantified by qRT-PCR. Localization of KLF-2 was analyzed by the expression of klf-2::gfp (in pPD95.75 vector) using a fluorescent microscope. Fat storage was measured by Oil Red O staining.ResultsKlf-2 was identified to express in the intestine during all stages of Caenorhabditis elegans development with peak expression at L3 stage. Mutation of klf-2 increased fat accumulation. Under regular growth media free of Ca2+, the expression of both klf-2 and −3 was inhibited slightly; further their expression reduced significantly in WT worms fed on 10X Ca2+ diet. When klf-3 was mutated, the expression of klf-2 increased under 10X Ca2+ diet; but when klf-2 was mutated, the expression of klf-3 was not altered under 10X Ca2+ diet. Overall, Mg2+ and K+ were less effective on the gene expression of klfs. KLF target gene Ce-C/EBP-2 showed elevated expression in WT and klf-3 (ok1975) worms with changed Ca2+ concentrations but not in klf-2 (ok1043) worms. However, high Ca+2 diet exhibited inhibitory effect on Ce-SREBP expression in WT worms.ConclusionDietary Ca2+ is most effective on fat storage and klf-2 expression, wherein high Ca2+ diet decreased klf-2 expression and reduced fat buildup. Mechanistic study identified Ce-C/EBP (C48E7.3; lpd-2) and Ce-SREBP (Y47D3B.7; lpd-1) as the target genes of klf-2 and/or klf-3 to mediate lipid metabolism. This study identifies a new function of klf-2 in inhibiting fat buildup and reveals the interplay between dietary salts and klf-2 and klf-3 in lipid metabolism.

Regulation of mRNA Translation Is a Novel Mechanism for Phthalate Toxicity

Phthalates are a group of plasticizers that are widely used in many consumer products and medical devices, thus generating a huge burden to human health. Phthalates have been known to cause a number of developmental and reproductive disorders functioning as endocrine modulators. They are also involved in carcinogenesis with mechanisms less understood. To further understand the molecular mechanisms of phthalate toxicity, in this study we reported a new effect of phthalates on mRNA translation/protein synthesis, a key regulatory step of gene expression. Butyl benzyl phthalate (BBP) was found to directly inhibit mRNA translation in vitro but showed a complicated pattern of affecting mRNA translation in cells. In human kidney embryonic cell (HEK-293T), BBP increased cap-dependent mRNA translation at lower concentrations but showed inhibitory effect at higher concentrations. Cap-independent translation was not affected. On the other hand, mono (2-ethylhexyl) phthalate (MEHP) as a major metabolite of another important phthalate di (2-ethylhexyl) phthalate (DEHP) inhibited both can-dependent and -independent mRNA translation in vivo. In contrast, BBP and MEHP exhibited an overall promoting effect on mRNA translation in cancer cells. Mechanistic studies identified that the level and phosphorylation of eIF4E-BP (eIF4E binding protein) and the amount of eIF4GI in eIF4F complex were altered in accordance with the effect of BBP on translation. BBP was also identified to directly bind to eIF4E, providing a further mechanism underlying the regulation of mRNA by phthalate. At the cellular level BBP inhibited normal cell growth but slightly promoted cancer cells (HT29) growth. Overall, this study provides the first evidence that phthalates can directly regulate mRNA translation as a novel mechanism to mediate their biological toxicities.

Abstract 1981: Regulation of NF-kB signaling pathway by glucocorticoid in breast cancer cells

Steroid hormone signaling through nuclear receptors plays critical roles in every stage of breast cancer development. Although estrogen receptor (ER) and progesterone receptor (PR) are well studied, due to the high heterogeneity of breast cancer, therapies targeting these nuclear receptors are still not effective enough or generate inconsistent and even controversial results. In this study, we focused on the glucocorticoid (GC)/ glucocorticoid receptor (GR) signaling to investigate how it regulates NFkB activity that leads to differential outcomes in different subtypes of breast cancer. A qRT-PCR array containing a curated NF-kB responsive gene set (84 genes) was utilized in this study to analyze the effect of GC/GR on basal and luminal subtypes of breast cancer cells. It was identified that MDA-MB-231 cells (basal subtype) were more responsive than MCF7 cells (luminal subtype) to GC treatment as the former showed more down- or up-regulated NF-kB target genes. Negative regulation of the NF-kB target genes was more prevalent in both cells, which is consistent with the general rule of trans-repression of NF-kB by GR. However, there were also a number of NF-kB target genes up-regulated by GC in both subtypes of breast cancer cells, suggesting that the trans-activation of NF-kB by GR may also play an important role in breast cancer cell growth and pathological behaviors. Among the differentially expressed genes in both cell types, genes with functions in cell migration and adhesion are highly regulated, such as CXCL1, CCL2, ICAM1, VCAM1, BIRC3 and SNAP25, implying that GC may mainly regulate the invasion and metastasis of breast cancer cells via NF-kB signaling. Biochemical studies identified that GR was activated by GC differently in MDA-MB-231 and MCF7 cells; the interaction between GR and various NF-kB subunits were also different in these cells, thus elucidating part of mechanisms underlying the differential regulation of NF-kB by GC in different subtypes of breast cancer cells. Citation Format: Zenaida Lopez-Dee, Jun Ling. Regulation of NF-kB signaling pathway by glucocorticoid in breast cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1981. doi:10.1158/1538-7445.AM2015-1981

Abstract 3305: Glucocorticoid differentially regulates gene expression in luminal and basal subtypes of breast cancer

Steroid hormone receptors (SHRs), such as estrogen- and progesterone- receptors, are well-validated drug targets and prognostic indicators for breast cancer (BC). When targeting SHRs for BC therapy, the major challenge is how to increase the selectivity of drugs against SHRs in specific cell/tissue. While estrogen- and progesterone- receptors are well studied in BC, glucocorticoid (GC) as a ubiquitous stress activated steroid hormone is less investigated. However, the elevated GC circadian dynamics is associated with poor prognosis, and GC receptor (GR) is highly expressed in metastatic BC. GCs are used successfully as an adjuvant therapy to induce apoptosis of leukemia and lymphoma. However, when applied to BC, the results are controversial with most GCs showing either pro-survival or no effects. To analyze the underlying mechanisms, gene expression profiling was performed in this study using qRT-PCR array that covers the genes as the drivers of BC. Our results showed that compared to MCF7 (luminal subtype) cells, MDA-MB-231(basal B subtype) cells are more responsive to Dexamethasone (Dex; a synthetic GC) treatment with the higher number of genes that were either up- or down-regulated. Shared by both MCF7 and MDA-MB-231 cells, the Dex-regulated genes are highly related to cell adhesion and epithelial-mesenchymal transition (EMT), followed by the genes involved in signal transduction, cell division and apoptosis, including SERPIN-E1, CDKN2A, c-Myc, THBS1, Snai2 and E-cadherin. However, the up- and down-regulation patterns are different between MDA-MB-231 and MCF7 cells. Using a real-time label-free cell assay, we identified that Dex promotes MDA-MB-231 cell adhesion and migration but only the spreading for MCF7 cells, suggesting that there are cell-specific effects of GC/GR in different subtypes of breast cancer. Citation Format: Jun Ling, Zenaida Lopez-Dee, Shagufta H. Khan, Raj Kumar. Glucocorticoid differentially regulates gene expression in luminal and basal subtypes of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3305. doi:10.1158/1538-7445.AM2014-3305