Brian T. Pentecost

Role of thrombin receptor in breast cancer invasiveness

SummaryInvasion, the ability of an epithelial cancer cell to detach from and move through a basement membrane, is a central process in tumour metastasis. Two components of invasion are proteolysis of extracellular matrix and cellular movement through it. A potential promoter of these two processes is thrombin, the serine proteinase derived from the ubiquitous plasma protein prothrombin. Thrombin promotes the invasion of MDA-MB231 breast tumour cells (a highly aggressive cell line) in an in vitro assay. Invasion by MDA-MB436 and MCF-7 cells, less aggressive cell lines, is not promoted by thrombin. Thrombin, added to the cells, is a stimulator of cellular movement; fibroblast-conditioned medium is the chemotaxin. Thrombin-promoted invasion is inhibited by hirudin. Stimulation of invasion is a receptor-mediated process that is mimicked by a thrombin receptor-activating peptide. Thrombin has no effect on chemotaxis in vitro. Thrombin receptor is detectable on the surface of MDA-MB231 cells, but not on the other two cell lines. Introduction of oestrogen receptors into MDA-MB231 cells by transfection with pHEO had no effect on thrombin receptor expression, in the presence or absence of oestradiol. This paper demonstrates that thrombin increases invasion by the aggressive breast cancer cell line MDA-MB231 by a thrombin receptor-dependent mechanism.

Use of an Aggressive MCF-7 Cell Line Variant, TMX2-28, to Study Cell Invasion in Breast Cancer

An estrogen receptor–negative variant of the MCF-7 breast cancer cell line, TMX2-28, was used as a model in which to study breast cancer cell invasion. Using a reconstituted basement membrane (Matrigel) assay to evaluate cell invasion, we determined that TMX2-28 cells are more invasive than MCF-7 cells and that the invasiveness of TMX2-28 is similar to that of the aggressive MDA-MB-231 breast cancer cell line. TMX2-28 cells displayed a rounded, epithelial cell–like morphology, suggesting an amoeboid mode of cell invasion, in contrast to the mesenchymal mode of invasion characteristic of spindle-shaped, fibroblast-like MDA-MB-231 cells. Using real-time reverse transcription-PCR, we found that mitogen-inducible gene 2 (MIG2) is expressed at a 17-fold higher level in TMX2-28 cells than in nonaggressive MCF-7 cells and that MIG2 mRNA levels are low in the nontumorigenic human mammary epithelial cell line, 184. We determined that MIG2 plays a role in cell invasion by using small interfering RNA (siRNA) to suppress the expression of MIG2 mRNA levels in TMX2-28 cells. TMX2-28 cell invasion was reduced by 48% when the cells were transfected with siRNAs targeting MIG2, relative to cells transfected with siRNAs against glyceraldehyde-3-phosphate dehydrogenase. Finally, MIG2 expression was evaluated in reductive mammoplasty and breast tumor tissue. Although all 21 normal tissues from reduction mammoplasty showed immunoreactivity for MIG2, ranging from weak (62%) to strong (24%), only half of the 34 formalin-fixed breast tumors showed immunoreactivity for MIG2. Of these 17 positive cases, 10 were considered to overexpress MIG2 (moderate to strong staining). Examination of 30 frozen breast tumors supported the finding that MIG2 is overexpressed in a subset of breast cancers. We suggest that MIG2s normal regulation and function are disrupted in breast cancer. (Mol Cancer Res 2006;4(12):905–13)

Estrogen receptor α increases basal and cigarette smoke extract-induced expression of CYP1A1 and CYP1B1, but not GSTP1, in normal human bronchial epithelial cells

Gender‐specific estrogen receptor α (ERα) expression may plausibly influence lung carcinogenesis in females. Initial genome‐wide microarray studies confirmed that carcinogen metabolism genes (CYP1A1, CYP1B1) were those most responsive to cigarette smoke extract (CSE) in normal bronchial epithelial (NHBE) cells. These two genes encoding phase I bioactivating enzymes and the GSTP1 gene encoding a phase II deactivating enzyme were then tested for induction by ERα. NHBE cells (native ERα−) were transfected with wild‐type ERα‐adenoviral constructs, and then exposed to CSE, 17β‐estradiol (E2), and/or the ERα inhibitor, ICI 182,780. The expression levels of CYP1A1, CYP1B1, and GSTP1 were then determined by RNA‐specific quantitative RT‐PCR and immunoassay. ERα increased the basal expression of CYP1B1 4.04‐fold (P < 0.01) at the mRNA level and 6.5‐fold at the protein level. ERα also increased the CSE‐induced mRNA expression of CYP1B1 2.26‐fold (P < 0.01), but not the protein expression. ERα did not alter the CYP1A1 mRNA levels, but did increase protein expression 2.0‐fold (P < 0.01) on CSE exposure, and 6.2‐fold (P < 0.01) upon E2 exposure. These effects could be inhibited by ICI 182,780. ERα did not alter the expression of GSTP1. Chromatin immunoprecipitation assay (ChIP) assay confirmed ERα binding to CYP1B1 promoter near the transcription start site. These results suggest that ERα regulates the CYP1B1 expression at a transcriptional level, and CYP1A1 expression at a translational level. These data raise the possibility that inter‐gender differences in expression of ERα that are known to exist in human lung may contribute to inter‐individual expression differences in CYP1A1 and CYP1B1, and to differences in carcinogen metabolism and mutation.

Effects of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin, 12‐O‐tetradecanoylphorbol‐13‐acetate and 17β‐estradiol on estrogen receptor regulation in MCF‐7 human breast cancer cells

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD) exhibits remarkably potent antiestrogenic activity. To further elucidate the role of estrogen receptor (ER) regulation in this response, we examined the effects of exposure to TCDD in MCF‐7 human breast cancer cells on ER mRNA levels by using an RNase protection assay, on ER accumulation by using an ER immunocytochemical essay (ER‐ICA), and on ER function by competitive binding assays under conditions of saturating 17β‐estradiol (E2). Comparative studies were conducted with E2 and 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), as both compounds are known to suppress ER expression. Our results indicate that 1 nM E2 and 100 nM TPA both suppress ER mRNA levels as early as 4 h after exposure and to 33.6% and 16.5% of control levels, respectively, after 72 h. In contrast, no significant effect on ER mRNA levels was attributed to exposure to 10 nM TCDD. A greater than 50% reduction in positive staining was observed by ER‐ICA after 72 h exposure to 1 nM E2 and to 100 nM TPA, while only an 11% reduction in positive staining was observed with 10 nM TCDD. Specific binding of [3H]E2 under saturating conditions (10 nM E2) in whole cells was reduced by 50% in cultures exposed to 100 nM TPA, although no effect on binding was observed with exposure to 10 nM TCDD. In contrast, specific binding using subsaturating 1 nM [3H]E2 was depressed by 49% in MCF‐7 cells exposed to 10 nM TCDD for 72 h. This depression was inhibited by a 1‐h treatment with 5 μM α‐naphthoflavone, which inhibits TCDD‐induced, P450‐mediated, E2 metabolism, and subsequent E2 depletion. In conclusion, while TPA and E2 effectively down‐regulate ER expression, TCDD, under antiestrogenic conditions, has little if any effect on total ER levels in MCF‐7 cells, and thus ER modulation is probably not necessary for the suppression of estrogenic activity in MCF‐7 cells by TCDD.

PLD1 is overexpressed in an ER-negative MCF-7 cell line variant and a subset of phospho-Akt-negative breast carcinomas

We have used a novel variant of the human oestrogen receptor (ER)-positive MCF-7 cell line, TMX2-28, as a model to study breast cancer. TMX2-28 cells show no detectable levels of mRNA or protein expression for the ER and express basal cytokeratins (CKs) 5, 14, and 17. cDNA microarray comparison between TMX2-28 and its parent cell line, MCF-7, identified 1402 differentially expressed transcripts, one of which was, phospholipase D1 (PLD1). Using real-time RT–PCR, we confirmed that PLD1 mRNA levels are 10-fold higher in TMX2-28 cells than in MCF-7 cells. We next examined PLD1 expression in human breast carcinomas. Phospholipase D1 mRNA levels were higher in breast tumours that expressed high-mRNA levels of basal CKs 5 and/or 17, but PLD1 mRNA levels were not significantly higher in ER-negative tumours. Phospholipase D1 protein was overexpressed in 10 of 42 (24%) breast tumours examined by IHC. Phospholipase D1 was overexpressed in 6 of 31 ER-positive tumours and 4 of 11 ER-negative tumours. Phospholipase D1 was overexpressed in three of the four tumours that showed high CK5/17 expression. Five PLD1-positive tumours were negative for phospho-Akt expression, but positive for phospho-mammalian target of rapamycin (mTOR) expression. The other five PLD1-positive breast tumours showed positive expression for phospho-Akt; however, only two of these cases were positive for phospho-mTOR. In this study, we report that PLD1 and phospho-mTOR are coexpressed in a subset of phospho-Akt-negative breast carcinomas.

The RhoA pathway mediates MMP-2 and MMP-9-independent invasive behavior in a triple-negative breast cancer cell line.

Breast cancer is a heterogeneous disease that varies in its biology and response to therapy. A foremost threat to patients is tumor invasion and metastasis, with the greatest risk among patients diagnosed with triple‐negative and/or basal‐like breast cancers. A greater understanding of the molecular mechanisms underlying cancer cell spreading is needed as 90% of cancer‐associated deaths result from metastasis. We previously demonstrated that the Tamoxifen‐selected, MCF‐7 derivative, TMX2‐28, lacks expression of estrogen receptor α (ERα) and is highly invasive, yet maintains an epithelial morphology. The present study was designed to further characterize TMX2‐28 cells and elucidate their invasion mechanism. We found that TMX2‐28 cells do not express human epidermal growth factor receptor 2 (HER2) and progesterone receptor (PR), in addition to lacking ERα, making the cells triple‐negative. We then determined that TMX2‐28 cells lack expression of active matrix metalloproteinases (MMPs)‐1, MMP‐2, MMP‐9, and other genes involved in epithelial–mesenchymal transition (EMT) suggesting that TMX2‐28 may not utilize mesenchymal invasion. In contrast, TMX2‐28 cells have high expression of Ras Homolog Gene Family Member, A (RhoA), a protein known to play a critical role in amoeboid invasion. Blocking RhoA activity with the RhoA pathway specific inhibitor H‐1152, or a RhoA specific siRNA, resulted in inhibition of invasive behavior. Collectively, these results suggest that TMX2‐28 breast cancer cells exploit a RhoA‐dependent, proteolytic‐independent invasion mechanism. Targeting the RhoA pathway in triple‐negative, basal‐like breast cancers that have a proteolytic‐independent invasion mechanism may provide therapeutic strategies for the treatment of patients with increased risk of metastasis. J. Cell. Biochem. 114: 1385–1394, 2013.

Transcriptional Regulation of Rat CYP2A3 by Nuclear Factor 1 IDENTIFICATION OF A NOVEL NFI-A ISOFORM, AND EVIDENCE FOR TISSUE-SELECTIVE INTERACTION OF NFI WITH THE CYP2A3 PROMOTER IN VIVO

Rat CYP2A3 and its mouse and human orthologs are expressed preferentially in the olfactory mucosa. We found previously that an element in the proximal promoter region of CYP2A3 (the nasal predominant transcriptional activating (NPTA) element), which is similar to a nuclear factor 1 (NFI)-binding site, is critical for transcriptional activation of CYP2A3 in vitro. We proposed that this element might be important for tissue-selective CYP2A3 expression. The goals of the present study were to characterize NPTA-binding proteins and to obtain more definitive evidence for the role of NFI in the transcriptional activation of CYP2A3. The NPTA-binding proteins were isolated by DNA-affinity purification from rat olfactory mucosa. Mass spectral analysis indicated that isoforms corresponding to all four NFI genes were present in the purified NPTA-binding fraction. Further analysis of NPTA-binding proteins led to the identification of a novel NFI-A isoform, NFI-A-short, which was derived from alternative splicing of the NFI-A transcript. Transient transfection assay showed that NFI-A2, an NFI isoform previously identified in the olfactory mucosa, transactivated the CYP2A3 promoter, whereas NFI-A-short, which lacks the transactivation domain, counteracted the activation. Chromatin immunoprecipitation assays indicated that NFI proteins are associated with the CYP2A3 promoter in vivo, in rat olfactory mucosa, but essentially not in the liver where the CYP2A3 promoter is hypermethylated and CYP2A3 is not expressed. These data strongly support a role for NFI transcription factors in the transcriptional activation of CYP2A3.

Functional evaluation of novel single nucleotide polymorphisms and haplotypes in the promoter regions of CYP1B1 and CYP1A1 genes

Interindividual variation in the expression of the carcinogen‐ and estrogen‐metabolizing enzymes cytochrome P4501B1 and 1A1 (CYP1B1 and CYP1A1) has been detected in human lung. To search for polymorphisms with functional consequences for CYP1B1 and CYP1A1 gene expression, we examined 1.5 kb of the promoter region of each gene. Genomic DNA from 21 Caucasian individuals was amplified by polymerase chain reaction (PCR) for direct cycle sequencing. Eight single nucleotide polymorphisms (SNPs) for CYP1B1 and 13 SNPs for CYP1A1 were found. The majority of polymorphisms occurred as multiSNP combinations for individual subjects. The wild‐type sequences were cloned into a luciferase reporter construct. The most frequent polymorphisms were then recreated by iterative site‐directed mutagenesis, replicating single polymorphisms and multiSNP combinations. These wild‐type and variant constructs were functionally evaluated in transient transfection experiments employing exposures to either the index polycyclic aromatic hydrocarbon (PAH) inducer benzo[a]pyrene (B[a]P), a composite mixture of cigarette smoke extract (CSE), or the repressor chemopreventive agent trans‐3,4,5‐trihydroxystilbene (reseveratrol). Results indicated that all wild‐type and variant constructs responded in qualitatively concordant fashion to the inducers and to the repressor. The CYP1B1 haplotypes and the majority of CYP1A1 haplotypes were shown to have no functional consequence, as compared to those of the wild‐type promoter sequences. Two constructs of composite polymorphisms of CYP1A1 appeared to result in a statistically significant increase in basal promoter activity (1.38‐ and 1.50‐fold, respectively), but the degree of functional impact was judged unlikely to be biologically important in vivo. We conclude that the observed promoter region polymorphisms in these genes are common, but are of unclear functional consequence.

High-density array analysis of DNA methylation in Tamoxifen-resistant breast cancer cell lines

Roughly two-thirds of all breast cancers are ERα-positive and can be treated with the antiestrogen, Tamoxifen, however resistance occurs in 33% of women who take the drug for more than 5 y. Aberrant DNA methylation, an epigenetic mechanism that alters gene expression in cancer, is thought to play a role in this resistance. To develop an understanding of Tamoxifen-resistance and identify novel pathways and targets of aberrant methylation, DNA from MCF-7 breast cancer cells and Tamoxifen-resistant derivatives, TMX2–11 and TMX2–28, were analyzed using the Illumina HumanMethylation450 BeadChip. Normalizing against MCF-7 values, ERα-positive TMX2–11 had 4000 hypermethylated sites and ERα-negative TMX2–28 had over 33 000. Analysis of CpG sites altered in both TMX2–11 and TMX2–28 revealed that the Tamoxifen-resistant cell lines share 3000 hypermethylated and 200 hypomethylated CpGs. ZNF350 and MAGED1, two genes hypermethylated in both cell lines, were examined in greater detail. Treatment with 5-aza-2′deoxycitidine caused a significant reduction in promoter methylation of both ZNF350 and MAGED1 and a corresponding increase in expression in TMX2–28. A similar relationship between methylation and expression was not detected in TMX2–11. Our findings are indicative of the variable responses to methylation-targeted breast cancer therapy and highlight the need for biomarkers that accurately predict treatment outcome.

SKP2 Overexpression Is Associated With Increased Serine 10 Phosphorylation of p27 (pSer10p27) in Triple‐Negative Breast Cancer

S‐phase kinase‐associated protein 2 (SKP2) is an important cell cycle regulator, targeting the cyclin‐dependent kinase (CDK) inhibitor p27 for degradation, and is frequently overexpressed in breast cancer. p27 regulates G1/S transition by abrogating the activity of cyclin/CDK complexes. p27 can undergo phosphorylation at serine 10 (pSer10p27). This phosphorylation event is associated with increased cell proliferation and poor prognosis in patients with glioma. The relationship between SKP2 and pSer10p27 in breast cancer has not been previously investigated. Immunohistochemistry (IHC) of SKP2, p27, pSer10p27, and other genes involved in this pathway, was analyzed in 188 breast tumors and 50 benign reduction mammoplasty samples. IHC showed SKP2 to be more highly expressed in estrogen receptor α (ERα)‐negative breast cancers and demonstrated that triple‐negative tumors were more likely to have high expression of SKP2 than were non‐triple negative, ERα‐negative tumors. A significant positive relationship was discovered for SKP2 and pSer10p27. High levels of SKP2 and pSer10p27 were observed significantly more often in ERα‐negative and triple‐negative than in ERα‐positive breast cancers. Use of the triple‐negative TMX2‐28 breast cancer cell line to address the role of SKP2 in cell cycle progression confirmed that SKP2 contributes to a more rapid cell cycle progression and may regulates pSer10p27 levels. Together, the results indicate that presence of high SKP2 plus high pSer10p27 levels in triple‐negative breast cancers is associated with aggressive growth, and highlight the validity of using SKP2 inhibitors as a therapeutic approach for treating this subset of breast cancers. J. Cell. Physiol. 229: 1160–1169, 2014.