Bruce A. White

Coordinate regulation of FOXO1 by miR-27a, miR-96, and miR-182 in breast cancer cells

The FOXO1 transcription factor orchestrates the regulation of genes involved in the apoptotic response, cell cycle checkpoints, and cellular metabolism. FOXO1 is a putative tumor suppressor, and the expression of this gene is dysregulated in some cancers, including prostate and endometrial cancers. However, the molecular mechanism resulting in aberrant expression of human FOXO1 in cancer cells is poorly understood. We show here that FOXO1 mRNA is down-regulated in breast tumor samples as compared with normal breast tissue. Silencing of the microRNA processing enzymes, Drosha and Dicer, led to an increase in FOXO1 expression. We also identified functional and specific microRNA target sites in the FOXO1 3′-untranslated region for miR-27a, miR-96, and miR-182, microRNAs that have previously been linked to oncogenic transformation. The three microRNAs, miR-27a, miR-96 and miR-182, were observed to be highly expressed in MCF-7 breast cancer cells, in which the level of FOXO1 protein is very low. Antisense inhibitors to each of these microRNAs led to a significant increase in endogenous FOXO1 expression and to a decrease in cell number in a manner that was blocked by FOXO1 siRNA. Overexpression of FOXO1 resulted in decreased cell viability because of inhibition of cell cycle traverse and induction of cell death. We have identified a novel mechanism of FOXO1 regulation, and targeting of FOXO1 by microRNAs may contribute to transformation or maintenance of an oncogenic state in breast cancer cells.

Argonaute-2 Expression Is Regulated by Epidermal Growth Factor Receptor and Mitogen-Activated Protein Kinase Signaling and Correlates with a Transformed Phenotype in Breast Cancer Cells

Argonaute (Ago) 2 is the catalytic engine of mammalian RNA interference, but little is known concerning the regulation of Ago2 by cell-signaling pathways. In this study we show that expression of Ago2, but not Ago1, Ago3, or Ago4, is elevated in estrogen receptor (ER) alpha-negative (ERalpha(-)) vs. ERalpha-positive (ERalpha+) breast cancer cell lines, and in ERalpha(-) breast tumors. In MCF-7 cells the low level of Ago2 was found to be dependent upon active ERalpha/estrogen signaling. Interestingly, the high expression of Ago2 in ERalpha(-) cells was severely blunted by inhibition of the epidermal growth factor (EGF) receptor/MAPK signaling pathway, using either a pharmacological MAPK kinase inhibitor, U0126, or a small interfering RNA directed against EGF receptor. Half-life studies using cycloheximide indicated that EGF enhanced, whereas U0126 decreased, Ago2 protein stability. Furthermore, a proteosome inhibitor, MG132, blocked Ago2 protein turnover. The functional consequences of elevated Ago2 levels were examined by stable transfection of ERalpha+ MCF-7 cells with full-length and truncated forms of Ago2. The full-length Ago2 transfectants displayed enhanced proliferation, reduced cell-cell adhesion, and increased migratory ability, as shown by proliferation, homotypic aggregation, and wound healing assays, respectively. Overexpression of full-length Ago2, but not truncated forms of Ago2 or an empty vector control, reduced the levels of E-cadherin, beta-catenin, and beta-actin, as well as enhanced endogenous miR-206 activity. These data indicate that Ago2 is regulated at both the transcriptional and posttranslational level, and also implicate Ago2 and enhanced micro-RNA activity in the tumorigenic progression of breast cancer cell lines.

ERα, microRNAs, and the epithelial–mesenchymal transition in breast cancer

The most common form of breast cancer, luminal A, is estrogen receptor α (ERα)-positive and epithelial, but nevertheless can metastasize. The process of epithelial-mesenchymal transition (EMT) is probably the first step in the metastasis of epithelial cancers. We discuss the characteristics of EMT, including factors that induce EMT, and the relationship of EMT to cancer stem cells (CSCs). Estrogen/ERα signaling maintains an epithelial phenotype and suppresses EMT. An overview of microRNAs in breast cancer is presented, including how microRNA biogenesis is altered in cancer and regulated by ERα. We also discuss the role of the miR-200 family in opposing EMT. Finally, we discuss specific microRNAs that target ERα and regulate EMT in breast cancer, and the role of these microRNAs in breast cancer progression.

Characterization of a Putative Membrane Receptor for Progesterone in Rat Granulosa Cells

Abstract Progesterone (P4) inhibits granulosa cell apoptosis in a steroid-specific, dose-dependent manner, but these cells do not express the classic nuclear P4 receptor. It has been proposed that P4 mediates its action through a 60-kDa protein that functions as a membrane receptor. The present studies were designed to determine the P4 binding characteristics of this protein. Western blot analysis using an antibody that recognizes the P4 binding site of the nuclear P4 receptor (C-262) confirmed that the 60-kDa protein was localized to the plasma membrane of both granulosa cells and spontaneously immortalized granulosa cells (SIGCs). To determine whether this protein binds P4, proteins were immunoprecipitated with the C-262 antibody, electrophoresed, transferred to nitrocellulose, and probed with a horseradish peroxidase-labeled P4 in the presence or absence of nonlabeled P4. This study demonstrated that the 60-kDa protein specifically binds P4. Scatchard plot analysis revealed that 3H-P4 binds to a single site (i.e., single protein), which is relatively abundant (200 pmol/mg) with a Kd of 360 nM. 3H-P4 binding was not reduced by dexamethasone, mifepristone (RU 486), or onapristone (ZK98299). Further studies with SIGCs showed that P4 inhibited apoptosis and mitogen-activated protein kinase kinase (MEK) activity, and maintained calcium homeostasis. These studies taken together support the concept that the 60-kDa P4 binding protein functions as a low-affinity, high-capacity membrane receptor for P4.

Quantitative measurement of mRNAs by polymerase chain reaction

Although polymerase chain reaction (PCR) has been used to detect the presence of specific mRNA species, there are no reports indicating that PCR can be used as a reliable, reproducible assay to quantify the relative level of an mRNA. In this study we examined the enzymatic steps (reverse transcription and PCR) required to analyze RNA by PCR and determined the conditions under which the product obtained reproducibly reflects the relative amounts of amplified species in the starting material. Aliquots of total RNA from rat ovaries and GH3 pituitary cells were used to prepare cDNAs for PCR amplification of beta-actin and prolactin (PRL) sequences, respectively. Assay of equivalent dilutions of ovarian cDNAs made from 10, 2, and 0.4 micrograms of RNA demonstrated that the amount of PCR product obtained was proportional to both the amount of cDNA amplified and the amount of RNA transcribed, with a relatively small variability for both reactions. cDNAs were also made against RNA prepared from GH3 cells cultured in the presence or absence of Ca2+, which induces PRL gene expression. Measurement of PRL mRNA by PCR gave results comparable to those obtained by Northern blot (4.7-fold induction vs. 5.9-fold), and again was highly reproducible. Additionally, PCR analysis of cDNA against GH3 nuclear RNA allowed us to detect an apparent splice variant of the PRL nuclear RNA that is also Ca2+ regulated. These results indicate the sensitivity and reliability of PCR as a quantitative assay for specific mRNAs, and demonstrate the possibilities for obtaining data not readily available by other means.

Membrane-Initiated Events Account for Progesterone's Ability to Regulate Intracellular Free Calcium Levels and Inhibit Rat Granulosa Cell Mitosis

Abstract It has been proposed that the antimitogenic action of progesterone (P4) is mediated through a membrane receptor that has GABAA receptor-like characteristics. To test this hypothesis, studies were designed to compare the antimitogenic effects of P4 with its gamma amino butyric acidA (GABAA) receptor-activating metabolite, 5α-pregnane-3α–21-diol-20-one (5α3α). These studies revealed that P4 was more effective than 5α3α in blocking mitogen-dependent mitosis of both small granulosa cells (GCs) and spontaneously immortalized granulosa cells (SIGCs). Ligand binding studies illustrated that P4 bound to SIGCs with an apparent dissociation constant (Kd) of 0.32 ± 0.09 μM, whereas 5α3α bound with an apparent Kd of 40 ± 19 μM. Further, the GABAA antagonist, bicuculline, did not attenuate P4s antimitotic action in SIGCs. Finally, reverse transcriptase-polymerase chain reaction (RT-PCR) studies demonstrated that none of the 6 known α chains of the GABAA receptors to which bicuculline binds were detected in SIGCs. Taken together, these studies suggest that P4 does not mediate its action via a GABAA-like receptor. Additional studies revealed that P4 regulated intracellular free calcium levels ([Ca2+]i) as part of its antimitotic action. Specifically, P4 maintained a basal [Ca2+]i level that was slightly lower than normal. Increasing extracellular calcium not only increased basal [Ca2+]i but also attenuated P4s antimitogenic effect. P4s actions appeared to be initiated at the membrane, since horseradish peroxidase conjugated-P4 (HP-P4), which is cell impermeable, was as effective in blocking mitosis as P4. Progesterone receptor (PR) mRNA was not detected in SIGCs by RT-PCR analysis, which is consistent with the findings in GCs. However, a 60-kDa protein was detected within crude membrane fractions of both GCs and SIGCs using an antibody directed against the ligand binding domain of the PR (C-262). This antibody was also used in immunocytochemical studies to detect a protein that was associated with the plasma membrane of SIGCs. It is proposed that this 60-kDa protein mediates P4s membrane-initiated actions.

Involvement of MicroRNAs in Breast Cancer

MicroRNAs regulate numerous aspects of normal and pathologic cellular processes, including cancer. Breast cancer is a heterogeneous form of cancer that is derived from mammary epithelial cells. This review discusses the involvement of microRNAs in the regulation of normal mammary epithelial stem cells, their differentiation into basal and luminal phenotypes, and their control of breast cancer stem cells, also referred to as tumor-initiating cells. In the second section, we summarize the findings of differential microRNA expression in normal versus breast tumor tissue and among the various subtypes of breast cancer (primarily luminal, basal-like, and HER2). In the third and fourth sections of the review, specific mRNA targets of microRNAs in breast cancer are discussed, including those encoding the estrogen receptor-alpha and epidermal growth factor receptor, as well as survival, tumor suppressor, and cell-cycle-related proteins. Finally, the involvement of microRNAs in the promotion and suppression of breast cancer metastasis is reviewed. The studies presented herein provide a rationale for the design of therapeutic agents that target specific microRNAs in the treatment of breast cancer. Hopefully, this review will provide an impetus for more studies on the role of microRNAs in the regulation of normal mammary gland development and function.

Epithelial–mesenchymal transition induces similar metabolic alterations in two independent breast cancer cell lines

Epithelial-mesenchymal transition (EMT) induces invasive properties in epithelial tumors and promotes metastasis. Although EMT-mediated cellular and molecular changes are well understood, very little is known about EMT-induced metabolic changes. HER2-positive BT-474 breast cancer cells were induced to undergo a stable EMT using mammosphere culture, as previously described by us for the ERα-positive MCF-7 breast cancer cells. Two epithelial breast cancer cell lines (BT-474 and MCF-7) were compared to their respective EMT-derived mesenchymal progeny (BT-474(EMT) and MCF-7(EMT)) for changes in metabolic pathways including glycolysis, glycogen metabolism, anabolic pathways and gluconeogenesis. Both EMT-derived cells displayed enhanced aerobic glycolysis along with the overexpression of specific glucose transporters, lactate dehydrogenase isoforms, monocarboxylate transporters and glycogen phosphorylase isoform. In contrast, both EMT-derived cells suppressed the expression of crucial enzymes in anabolic pathways and gluconeogenesis. STAT3, a transcription factor involved in tumor initiation and progression, plays a role in the EMT-related changes in the expression of specific enzymes and transporters. This study provides a broad overview of similar metabolic changes induced by EMT in two independent breast cancer cell lines. These metabolic changes may provide novel therapeutic targets for metastatic breast cancer.

Transcriptional and posttranscriptional regulation of the rat prolactin gene by calcium.

The rat prolactin gene is expressed at a high basal level in the pituitary tumor GH3 cell line. Culturing GH3 cells in a low-Ca2+, serum-free medium (SFM) depresses prolactin mRNA levels, and subsequent addition of Ca2+ to the SFM results in a specific, gradual, and sustained increase in prolactin mRNA levels. We have now examined whether the observed increase in prolactin mRNA levels can be attributed solely to an increase in the transcriptional rate of the prolactin gene. Treatment of GH3 cells in SFM with 0.4 mM CaCl2 for 24 to 48 h increased cytoplasmic prolactin mRNA levels by 5- to 10-fold, whereas the transcriptional rate of the prolactin gene was increased by less than twofold over values for SFM controls. Prolactin mRNA levels increased progressively during the 24-h period after Ca2+ addition, whereas prolactin gene transcription never exceeded a twofold increase over values for SFM controls. The activities of nuclear extracts from control and Ca2(+)-induced cells were examined in an in vitro transcription assay. The two extracts directed transcription from the prolactin promoter and the adenovirus major late promoter equally well. Cycloheximide had no effect on the ability of Ca2+ to increase or maintain prolactin mRNA levels. In dactinomycin mRNA clearance experiments, prolactin mRNA was cleared at the same rate in the absence and presence of Ca2+. These results demonstrate that although Ca2+ has a small effect on the transcriptional rate of the prolactin gene, Ca2+ produces a significant increase in prolactin mRNA levels by acting at a posttranscriptional site(s). Furthermore, Ca(2+) appears to increase prolactin mRNA levels by posttranslational modification of a stable protein, probably at a nuclear site.

Polymerase chain reaction : basic protocols.

The melding of a technique for repeated rounds of DNA synthesis with the discovery of a thermostable DNA polymerase has given scientists the very powerful technique known as polymerase chain reaction (PCR). PCR is based on three simple steps required for any DNA synthesis reaction: (1) denaturation of the template into single strands; (2) annealing of primers to each original strand for new strand synthesis; and (3) extension of the new DNA strands from the primers. These reactions may be carried out with any DNA polymerase and result in the synthesis of defined portions of the original DNA sequence. However, in order to achieve more than one round of synthesis, the templates must again be denatured, which requires temperatures well above those that inactivate most enzymes. Therefore, initial attempts at cyclic DNA synthesis were carried out by adding fresh polymerase after each denaturation step (1,2). The cost of such a protocol becomes rapidly prohibitive.