BP1 mRNA Research Articles

Abstract 3947: BP1 protein, a transcription factor, is secreted by breast cancer cells

Abstract Background. BP1 is a homeobox gene which is normally expressed during early hematopoiesis. Homeotic proteins regulate the expression of multiple genes involved in development and differentiation. We have previously shown that BP1 mRNA and protein levels are elevated in 80% of women with invasive ductal breast cancer. Furthermore, BP1 protein (pBP1) overexpression is associated with larger and more aggressive breast tumors. In cultured breast cancer cell lines overexpressing pBP1, we observe decreased cell death and increased proliferation. BP1 overexpression has also been found to stimulate known oncogenes including BCL2 and c-MYC. Methods. Levels of pBP1 were detected in cell extracts (CE) and conditioned media (CM) using Western Blot analysis. Depleted media (DM), in which the pBP1 was removed, and recombinant BP1 protein (rpBP1) were used as controls. Cell viability was assessed using MTT assays. Real-time qPCR was used to measure expression of selected oncogenes. Results. Our results show that: (a) pBP1 is secreted from three breast cancer cell lines but not from two normal breast epithelial cell lines. (b) pBP1 exhibits mitogenic activity towards breast cancer cells and normal breast cells, whether cells are grown in media containing 10x conditioned medium (CM) from cells secreting pBP1 or in media containing purified recombinant pBP1 (rpBP1). (c) Growth of breast cancer cells or normal breast epithelial cells in CM or rpBP1 leads to up-regulation of selected oncogenes known to be targets of BP1. These oncogenes include BCL2 and MET. BP1 itself was stimulated, but only in normal breast epithelial cells. The mechanism of secretion is under investigation. Conclusions. Our data suggest that secreted pBP1 can stimulate cell proliferation and gene expression in tumor cells as well as in nearby normal cells. Citation Format: Jinguen Rheey, Anna Yakovleva, Kellie-Ann Yamane, Patricia E. Berg. BP1 protein, a transcription factor, is secreted by 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 3947. doi:10.1158/1538-7445.AM2015-3947

Epigenetic inactivation of DLX4 is associated with disease progression in chronic myeloid leukemia

Aberrant DNA methylation of various genes has been identified to be associated with disease progression in chronic myeloid leukemia (CML). Our study was intended to investigate DLX4 methylation pattern in different clinical stages of CML and further determine its role in regulating DLX4 expression. Real-time quantitative methylation-specific PCR and bisulfite sequencing PCR were applied to detect DLX4 methylation. 5-aza-2′-deoxycytidine (5-aza-dC) was used for demethylation studies. DLX4 was significantly hypermethylated in CML patients (P = 0.002) especially in blastic phase (BC) stage (P < 0.001) as compared with controls. Moreover, DLX4 methylation level in BC stage was significantly higher than in chronic phase (CP) stage (P < 0.001). DLX4 methylation density was significantly increased during the progression of CML among the tested two patients (P < 0.001). DLX4 hypermethylation occurred with the highest incidence in BC stage (83%), lower incidence in acute phase (AP) stage (43%), and the lowest incidence in CP stage (26%) (P = 0.001). Moreover, t(9; 22) with additional alteration cases had significantly higher frequency of DLX4 hypermethylation compared with the other cytogenetics (P = 0.010). Significantly negative correlation was observed between DLX4 methylation and DLX4-TV2 (the shorter DLX4 isoform) expression (R = −0.382, P = 0.001, n = 78) but not between DLX4 methylation and BP1 (the longer DLX4 isoform) expression (R = 0.134, P = 0.244, n = 78) in CML patients. Both DLX4-TV2 and BP1 mRNA were significantly increased after 5-aza-dC treatment in K562 cell line (P < 0.001). Our study indicated that hypermethylation of DLX4 correlated with disease progression of CML. Moreover, DLX4 expression was regulated by its methylation in CML.

Abstract P5-09-11: BP1, a homeoprotein, regulates estrogen receptor alpha and induces estrogen independence

Abstract Introduction: BP1 is a member of the homeobox gene family of transcription factors. Our recent studies have shown that BP1 may play a role in breast cancer cell survival, aggressiveness and metastasis. BP1 protein (pBP1) is expressed in 80% of invasive ductal breast tumors. Moreover, 100% of inflammatory breast tumors are BP1 positive. These data led us to define the mechanism of BP1-related tumorigenesis and aggressiveness in breast cancer. Materials and Methods: MCF-7/O1, O2 and O4 cells overexpressing BP1 and control V1 and V2 cells were tested for growth in estrogen free media, malignant potential and invasiveness using cell viability assays, soft agar assays and matrigel assays, respectively. To determine the influence of BP1 overexpression on tumor characteristics, empty vector cells (V1) and overexpressor cells (O2 and O4) were injected into the fat pads of athymic nude mice. Mice were supplemented with estrogen pellets or were unsupplemented. Chromatin immunoprecipitation assays (ChIP) were used to validate the binding of pBP1 to ERa and EP300. The effects of BP1 expression on ERα and EP300 were investigated using immunoblotting and qRT-PCR. Effects of BP1 overexpression on tamoxifen sensitivity were measured using the MTT assay. Results: Cells overexpressing BP1 showed higher viability (p&amp;lt;0.05) when grown in the absence of serum, greater invasive potential (p&amp;lt;0.05) and formed larger and more rapidly growing colonies (p&amp;lt;0.0001) compared with cells containing the empty vector. Tumors were larger in mice receiving O1, O2 or O4 cells than in mice receiving V1 or V2 cells (p&amp;lt;0.0001). There was also a positive correlation between BP1 mRNA levels and tumor size in patients (p = 0.01). 20% of mice injected with O2 or O4 developed tumors in the absence of estrogen, in contrast to control mice which did not develop tumors. Several mechanisms of estrogen independence related to BP1 were established: (1) an indirect mechanism, in which BP1 stabilizes ERα protein by transcriptional activation of p300, and (2) a direct mechanism, in which BP1 binds to the first intron of ERα and upregulates ERα RNA and protein expression. In addition, breast cancer anti-estrogen resistance 1 (BCAR1) RNA expression was higher in O2 as compared to V1 cells. Consistent with these findings, O2 cells exhibited increased proliferation when treated with tamoxifen, while V1 cells showed growth inhibition. Conclusion: High BP1 levels can lead to estrogen independence in ER positive breast cancer cells and tumors in mice by at least two mechanisms, indirect and direct, and are associated with resistance to tamoxifen. These results suggest that BP1 may be an important therapeutic target in ER positive breast cancer. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P5-09-11.

Abstract 1812: The Role of the BP1 homeobox gene in triple negative breast cancer .

Abstract Background: Triple negative breast cancers (TNBC) lack the expression of ER, PR and HER2 receptors, thus targeted treatments against these receptors are not effective. We are studying BETA PROTEIN 1, a member of homeobox gene family, which we cloned and showed is activated in 80% of invasive breast cancers; its expression correlates with breast cancer progression and invasion. The BP1 gene is located on 17q21, a region that is reported to be altered in 30% of breast tumors. Since BP1 mRNA and protein are highly expressed in ER and PR negative and clinically aggressive tumors, we hypothesize that BP1 may play a relevant role in TNBC. Our main objective was to evaluate the gene copy number and mRNA/protein expression status of BP1 in TNBC, in both clinical cases and established TNBC cell lines. Methods: We have characterized seven cell lines (5 TNBC and 2 normal breast cell lines) for the expression of BP1 using qPCR and Western blot analysis. To delineate pathways in TNBC cells that are dysregulated by BP1 expression, we studied both the seven parental cell lines and a stably overexpressed BP1 TNBC cell line, Hs578T-OE. Microarray analysis was performed in all these cell lines and the data obtained was verified using qPCR and ELISA. Formalin-Fixed, Paraffin-Embedded tissues of TNBC were analyzed by array-CGH and confirmed by Taqman copy number (using a BP1-TAM specific probe) and FISH (using our constructed BP1-BAC probe) assays. Results: All of the parental TNBC cells had at least 2.8-fold more pBP1 than the normal breast cell lines suggesting that TNBC tumors have higher levels of pBP1 than non-TNBC tumors. Gene expression microarrays in the TNBC cell lines revealed up/down regulation of several genes in comparison to the normal breast cell lines, including the EGFR. Interestingly, the IL-6 and IL-8 genes were significantly upregulated in TNBC cells overexpressing BP1. ELISA assays confirmed a correlation between elevated levels of IL-6 and IL-8 production and BP1 in the conditioned media from Hs578T cells overexpressing BP1 compared with the empty vector. Studies are underway to establish the mechanism by which these pathways are regulated by BP1. BP1 increase copy number was observed in 37% (25/67) of the cases by array-CGH. Confirmation of these changes by FISH and TaqMan copy number assays revealed increased copy number in 20% (13/66) and 23% (9/38) of the cases, respectively. Protein expression analysis by IHC is currently been conducted in these cases to determine if BP1 increase DNA copy number can be one of the mechanisms that leads to pBP1overexpression. Conclusions: Both BP1 copy number and protein expression are dysregulated in TNBC. Microarray analysis in TNBC cell lines have revealed important cancer related genes and oncogenic pathways that may be upregulate by BP1, suggesting that BP1 could be a valuable molecular marker and therapeutic target in this aggressive form of breast cancer. Citation Format: Mandeep Gill, Saurab Kirolikar, Svetlana Ghimbovschi, Luciane R. Cavalli, Patricia E. Berg. The Role of the BP1 homeobox gene in triple negative breast cancer . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1812. doi:10.1158/1538-7445.AM2013-1812

Prognostic significance of BP1 mRNA expression level in patients with non-small cell lung cancer

Objectives To examine the association of BP1 mRNA level with tumor characteristics and clinical prognosis in non-small cell lung cancer (NSCLC) patients. Design and methods Tumor specimens from 98 NSCLC patients who underwent surgical resection were quantitatively determined for BP1 mRNA expression by real-time RT-PCR. Results BP1 mRNA was expressed at significantly higher levels in tumors than in adjacent nontumorous tissues and normal lung samples. The level of BP1 transcript was significantly associated with tumor histological type and cell differentiation grade, but not related with other clinicopathological factors and p53 mutations. Patients with high BP1 mRNA expression had a poorer prognosis in terms of both disease-free survival (DFS) and overall survival (OS) rates. Additionally, BP1 mRNA expression level was an independent prognostic factor for DFS. Conclusions BP1 may be part of a pathway contributing to NSCLC development and/or progression. BP1 mRNA level could be a novel prognostic marker for NSCLC.

The expression of BP1 mRNA and its clinical significance

有学者发现BP1在80%的乳腺癌中有表达,且与ER的表达和人种具有相关性.我们应用逆转录聚合酶链反应(RT-PCR)方法检测了BP1 mRNA在乳腺癌中的表达,并探讨其与临床病理指标和相关免疫组织化学指标的关系。

Correlation of expression of BP1, a homeobox gene, with estrogen receptor status in breast cancer.

BackgroundBP1 is a novel homeobox gene cloned in our laboratory. Our previous studies in leukemia demonstrated that BP1 has oncogenic properties, including as a modulator of cell survival. Here BP1 expression was examined in breast cancer, and the relationship between BP1 expression and clinicopathological data was determined.MethodsTotal RNA was isolated from cell lines, tumors, and matched normal adjacent tissue or tissue from autopsy. Reverse transcription polymerase chain reaction was performed to evaluate BP1 expression. Statistical analysis was accomplished with SAS.ResultsAnalysis of 46 invasive ductal breast tumors demonstrated BP1 expression in 80% of them, compared with a lack of expression in six normal breast tissues and low-level expression in one normal breast tissue. Remarkably, 100% of tumors that were negative for the estrogen receptor (ER) were BP1-positive, whereas 73% of ER-positive tumors expressed BP1 (P = 0.03). BP1 expression was also associated with race: 89% of the tumors of African American women were BP1-positive, whereas 57% of those from Caucasian women expressed BP1 (P = 0.04). However, there was no significant difference in BP1 expression between grades I, II, and III tumors. Interestingly, BP1 mRNA expression was correlated with the ability of malignant cell lines to cause breast cancer in mice.ConclusionBecause BP1 is expressed abnormally in breast tumors, it could provide a useful target for therapy, particularly in patients with ER-negative tumors. The frequent expression of BP1 in all tumor grades suggests that activation of BP1 is an early event.

Increased serum corticosterone and glucose in offspring of chromium(III)-treated male mice.

Preconceptional carcinogenesis occurs in animals and is suspected for humans--for example, after occupational metals exposure. Several characteristics in animal models, including high frequency and non-Mendelian inheritance patterns, have suggested an epigenetic mechanism, possibly involving hormone changes in offspring. To test this hypothesis, we treated male mice with chromium(III) chloride, a preconceptional carcinogen, 2 weeks before mating, in two separate experiments. Their 10-week-old offspring showed highly significant increases in average serum corticosterone and glucose, compared with control offspring. Average serum levels of insulin-like growth factor 1 (IGF1) showed more modest possible increases. A previous microarray experiment identified hepatic insulin-like growth factor binding protein 1 (IGF BP1) gene expression as consistently changed in correlation with serum corticosterone levels. In the present study, hepatic IGF BP1 mRNA correlated with serum IGF1 in male offspring of chromium-treated fathers, but not in controls; serum glucose correlated positively with hepatic IGF BP1 in chromium-group offspring but negatively in controls. These results support the hypothesis that preconceptional exposure effects may alter hormones, metabolism, and control of tissue gene expression, probably through epigenetic mechanisms. Risk of neoplasia may be influenced by these changes.

Hormonal regulation of expression of messenger RNA encoding insulin-like growth factor binding proteins in human endometrial stromal cells cultured in vitro.

To investigate the presence of messenger RNA (mRNA) encoding insulin-like growth factor binding proteins (IGFBP) in human secretory endometrial stromal cells cultured in vitro, total cellular mRNA and protein extracted from cells treated with various hormones were detected and identified by Northern and Western blotting techniques respectively. Northern blot analysis detected 1.4 and 2.5 kilobase (kb) mRNA transcripts for IGFBP-2 and IGFBP-3 respectively, in both control and progestin-treated human endometrial stromal cells in vitro. However, the 1.5 kb mRNA transcript of IGFBP-1 was detected only in progestin-treated cells but not in the controls. Progestin alone markedly stimulated cellular BP-1 protein and mRNA, but only moderately stimulated cellular IGFBP-2 and IGFBP-3 protein mRNA in a dose-dependent fashion. Adding relaxin at the same time as progestin further enhanced the stimulatory effects of progesterone. Oestradiol had a stimulatory effect on cellular IGFBP-2 mRNA, but had an inhibitory effect on protein and mRNA of IGFBP-3, also in a dose-dependent fashion. In general, for each specific binding protein, the amount of cellular mRNA correlated well with the amount of cellular protein. Therefore, IGFBP protein and mRNA transcript in human secretory endometrial stromal cells appears to be under hormonal influence. These hormones may control the synthesis of IGFBPs at the transcription rather than the translation level.

Mouse thymus dendritic cells: kinetics of development and changes in surface markers during maturation

The early thymus precursor population of adult mice has the capacity to generate T cells, B cells and dendritic cells (DC). These precursors were injected into the thymus of irradiated recipients in order to follow the kinetics of thymic DC development. The resultant cohort of T-lineage cells developing in the thymus was accompanied by a parallel cohort of DC, present at 10(3)-fold lower frequency. The intrathymic lifespan of these DC was as short as that of T-lineage thymocytes. As the thymic DC matured, some markers characteristic of the original precursor population gradually declined (Ly-5, c-kit, Sca-2) whereas markers characteristic of thymic DC appeared and were maintained (major histocompatibility complex class II, CD11c, NLDC-145 and CD8 alpha). Some thymic DC expressed the early B-cell marker BP-1, and BP-1 mRNA, throughout their maturation. The surface markers on thymic DC could be divided into two groups. Some markers, including class I and class II MHC, CD8 alpha and BP-1, appeared to be integral components of the DC surface. In contrast, other markers, including Thy-1, CD4 and CD8 beta, had probably been picked up from associated thymocytes.

Widespread tissue distribution of aminopeptidase A, an evolutionarily conserved ectoenzyme recognized by the BP-1 antibody.

Early B-lineage cells in mice express a cell surface glycoprotein, recognized by the BP-1 and 6C3 monoclonal antibodies, that has been identified as aminopeptidase A (APA E.C.3.4.11.7). In the present studies we obtained evidence by DNA "zoo-blot" analysis that the APA gene is highly conserved. This ectoenzyme catalyzes the removal of N-terminal Glu- and Asp-residues to convert angiotensin II to angiotensin III, a degradation step important in local regulation of blood pressure in mammals. To gain further insight into the physiology of this molecule, which is shared between immune and vascular systems, we examined the tissue distribution of BP-1 mRNA using a cDNA probe and of the protein antigen using the BP-1 antibody for immunohistology. APA transcripts were present in all tissues examined. Abundant BP-1/APA was found in the intestinal brush border of the small intestine, renal glomeruli, proximal renal tubules, pulmonary alveolar walls and vascular endothelium in many organs. Other tissues containing the BP-1 antigen included stromal cells in the thymus cortex, bile canaliculi in liver, gall bladder epithelium, interlobular ducts in pancreas, the ovarian theca interna, basement membrane of the epididymis and the splanchnopleure in placenta. APA enzyme activities have been identified in most of these locations, in keeping with identification of the BP-1/6C3 antigen as APA. The data suggest this ectopeptidase may serve diverse physiologic roles in a broad spectrum of tissues.

Nutritional regulation of insulin-like growth factor-binding protein gene expression in the ovine fetus and pregnant ewe.

The factors controlling the synthesis and degradation of the insulin-like growth factor-binding proteins (IGFBPs) during pregnancy are poorly understood. To clarify the roles of nutritional factors in the regulation of fetal and maternal IGFBP production, we examined the effects of fasting, refeeding, and glucose administration on plasma IGFBP concentrations and hepatic IGFBP mRNA levels in fetal lambs and pregnant ewes (n = 24). Maternal fasting for 3 days in late gestation stimulated a 50-100% increase in maternal plasma BP-1 concentrations (P < 0.05) and a 2- to 3-fold increase in fetal plasma BP-1 (P < 0.05), as determined by densitometric analysis of Western ligand blots. Fasting also stimulated a 40-70% increase in maternal plasma BP-2 concentrations (P < 0.05), but had no significant effect on fetal plasma BP-2 levels. Levels of hepatic BP-1 mRNA in the fetus and pregnant ewe during fasting paralleled plasma BP-1 levels, suggesting that fasting modulates fetal and maternal plasma BP concentrations at least in part through effects on hepatic gene expression. The effects of fasting on both mRNA and plasma levels of BP-1 and BP-2 were reversed by 3 days of refeeding and were prevented by glucose infusion during fasting. When ewes were made hyperglycemic by the infusion of hypertonic glucose, plasma BP-1 and BP-2 concentrations varied inversely with blood glucose concentrations. In addition, hyperglycemia reduced maternal liver BP-1 and BP-2 mRNA levels and fetal BP-1 mRNA levels by 50-65%. Direct administration of hypertonic glucose to the fetus decreased fetal plasma BP-1 levels acutely and reduced fetal BP-1 mRNA levels by 57%, but had no effect on fetal plasma BP-2 or fetal hepatic BP-2 mRNA levels. These findings indicate that glucose and other nutritional factors regulate gene expression and plasma levels of BP-1 and BP-2 in the pregnant ewe and BP-1 in the fetal lamb. The changes in expression of these IGFBPs during fasting and hyperglycemia may play roles in adaptation of the pregnant mother and fetus to metabolic stress.

Expression of hepatic insulin-like growth factor-I and insulin-like growth factor-binding protein-1 genes is transcriptionally regulated in streptozotocin-diabetic rats.

Insulin-like growth factor-I (IGF-I) and IGF-binding protein-1 (BP-1) are critical cell regulators, with regulation and action in endocrine, paracrine, and autocrine modes. Although IGF-I and BP-1 are thought to be modulated mainly at the level of synthesis, underlying molecular mechanisms are poorly understood. To examine regulation by insulin, we used run-on assays to measure IGF-I and BP-1 gene transcription rates in nuclei isolated from the livers of normal and diabetic rats. Streptozotocin (STZ)-treated rats exhibited 20-25% weight loss, a 2.5- to 3-fold increase in serum glucose, and a 50-60% fall in circulating IGF-I levels (all P less than 0.001). Diabetic animals also had a 45% reduction in hepatic IGF-I mRNA and over 400% increases in BP-1 mRNA (both P less than 0.005); all parameters were restored toward normal after treatment with insulin. Metabolically responsive IGF-I gene transcription was evaluated effectively with a 3.2-kilobase BglII/EcoRI genomic probe located down-stream from all initiation sites in exon 1, while BP-1 gene transcription was studied with a cDNA probe. Animals treated with 144 mg/kg STZ exhibited 50-97% decreases in IGF-I gene transcription (P less than 0.05), while insulin treatment raised IGF-I gene transcription to control levels (P less than 0.02). IGF-I gene transcription appeared to be more sensitive to metabolic status than IGF-I mRNA levels, resulting in a modest correlation between transcription rates and mRNA levels (r = 0.68; P less than 0.001). In contrast, changes in BP-1 mRNA and gene transcription appeared to be exquisitely sensitive to metabolic status.(ABSTRACT TRUNCATED AT 250 WORDS)