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.

Role of the insulin-like growth factor system on an estrogen-dependent cancer phenotype in the MCF-7 human breast cancer cell line.

We previously established that exposure of the estrogen receptor (ER) alpha positive MCF-7 human breast cancer cell line to 17-beta-estradiol (E2) results in the post-confluent development of multilayered cellular aggregates (foci) which is consistent with the in vivo cancer phenotype of uncontrolled cellular proliferation. In this investigation, the interaction between the insulin-like growth factor receptor (IGF-IR) and ER-signaling systems in regard to post-confluent focus development was studied. We demonstrated that focus development requires the presence of E2 and insulin-like growth factor I (IGF-I) or insulin-like growth factor II (IGF-II), as well as intact ER and IGF-IR. Focus development in MCF-7 cultures, which occurs only after formation of a confluent monolayer, coincides with E2 regulation of key members of the IGF-signaling system such as IGF-IR, IGF-II, insulin receptor substrate 1 (IRS-1), and insulin-like growth factor binding protein 3 (IGFBP-3), as demonstrated by real-time polymerase chain reaction (PCR). To establish the relevancy of an intact IGF-signaling system for foci formation, we generated stable clones from MCF-7 with IGF-IR suppressed by siRNA. Results from these studies implicate signaling through the IGF-IR to be an integral requirement for E2-dependent post-confluent proliferation and focus formation. In summary, these studies establish the interactive roles of IGFs and E2 in the post-confluent development of foci, and will allow subsequent identification of targets for therapeutic intervention in the control and treatment of estrogen-dependent breast cancer.

Differential expression of estrogen receptor alpha (ERalpha) protein in MCF-7 breast cancer cells chronically exposed to TCDD.

Estrogens play a key role in the development and evolution of breast cancer tumors. Estrogen receptor alpha (ERalpha) mediates many of the biological activities of estrogens, and its expression is associated with low invasiveness and good prognosis. Recent epidemiological reports suggest that long-term exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is implicated in the increased incidence of breast cancer in exposed women. TCDD interferes with the expression of some ERalpha-dependent genes and inhibits estradiol (E2)- dependent growth of breast cancer cells in vitro. However, E2-dependent xenographs of MCF-7 human breast cancer cells resumed growth after a 2-week exposure to TCDD. The mechanisms involved in the resumption of cell growth are not completely understood. In this study, we show that short term-exposure (16 days) to 1 nM TCDD results in the suppression of ERalpha protein expression, while chronic exposure for more than 1 year (LTDX cells) results in the partial re-expression of the receptor. Immunocytochemistry studies showed that re-expression of ERalpha in LTDX cells occurred in some of the cells. Analysis by Western immunoblots indicated that four out of five LTDX clones expressed ERalpha at levels comparable to those in unexposed MCF-7 cells. Removal of TCDD treatment for 16 days restored the expression of ERalpha in the ERalpha-negative clonal cells. These results suggest that MCF-7 cells chronically exposed to TCDD contain at least two cell subpopulations that may respond differently to the ERalpha-mediated effects of TCDD.

Inhibition of MCF-7 breast cancer cell proliferation by MCF-10A breast epithelial cells in coculture.

A coculture system was developed to investigate the interactions between MCF-10A breast epithelial cells and MCF-7 breast cancer cells stably expressing the green fluorescent protein (MCF-7-GFP). Studies with this MCF-10A/MCF-7-GFP coculture system on microtiter plates and on reconstituted basement membrane (Matrigel), revealed paracrine inhibition of MCF-7-GFP cell proliferation. Epidermal growth factor, which in monocultures modestly enhanced MCF-7-GFP and markedly increased MCF-10A cell proliferation, greatly inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures. 17beta-Estradiol, which stimulated MCF-7-GFP but not MCF-10A cell proliferation in monoculture, inhibited MCF-7-GFP cell proliferation in MCF-10A/MCF-7-GFP cocultures, an effect that was blocked by the antiestrogen, ICI 182,780. On Matrigel, complex MCF-10A/MCF-7-GFP cellular interactions were observed in real time that resulted in the formation of acinus-like structures. These results indicate a role of normal epithelial cells in inhibiting tumor-cell proliferation and demonstrate the utility of this coculture system as a model of early paracrine control of breast cancer.

Methyl mercury influences growth-related signaling in MCF-7 breast cancer cells.

Environmental contaminants have been shown to alter growth-regulating signaling pathways through molecular mechanisms that are mainly unclear. Here we report that within a narrow concentration range (0.5-1 microM) methyl mercury (MeHg) significantly stimulated growth of MCF-7 cells, induced Ca(2+) mobilization, and activated extracellular signal-regulated kinase (1/2) (Erk1/2). MeHg modulated E(2)-dependent stimulation of growth in a dose-dependent manner, although MeHg neither suppresses nor increases constitutive E(2) metabolism. MeHg demonstrated weak estrogen receptor (ER)-binding ability. However, long preincubation with antiestrogens LY(156,758) and ICI(164,384) decreased MeHg-induced foci formation, Ca(2+) mobilization, and Erk1/2 activation, confirming involvement of ERs. The MeHg-induced increase in [Ca(2+)](i) was observed to coincide with enhanced Erk1/2 phosphorylation. These data suggest that MeHg can significantly modulate the intracellular signaling environment in MCF-7 cells, resulting in a dose-dependent alteration of ER-mediated estrogenic capacity and therefore should be considered as a potential estrogen-disrupting compound.

Gene-environment interaction signatures by quantitative mRNA profiling in exfoliated buccal mucosal cells.

Exfoliated cytologic specimens from mouth (buccal) epithelium may contain viable cells, permitting assay of gene expression for direct and noninvasive measurement of gene-environment interactions, such as for inhalation (e.g., tobacco smoke) exposures. We determined specific mRNA levels in exfoliated buccal cells collected by cytologic brush, using a recently developed RNA-specific real-time quantitative reverse transcription-PCR strategy. In a pilot study, metabolic activity of exfoliated buccal cells was verified by 3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium assay in vitro. Transcriptional activity was observed, after timed in vivo exposure to mainstream tobacco smoke resulted in induction of CYP1B1 in serially collected buccal samples from the one subject examined. For a set of 11 subjects, mRNA expression of nine genes encoding carcinogen- and oxidant-metabolizing enzymes qualitatively detected in buccal cells was then shown to correlate with that in laser-microdissected lung from the same individuals (Chi2 = 52.91, P < 0.001). Finally, quantitative real-time reverse transcription-PCR assays for seven target gene (AhR, CYP1A1, CYP1B1, GSTM1, GSTM3, GSTP1, and GSTT1) and three reference gene [glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-actin, and 36B4] transcripts were performed on buccal specimens from 42 subjects. In multivariate analyses, gender, tobacco smoke exposure, and other factors were associated with the level of expression of CYP1B1, GSTP1, and other transcripts on a gene-specific basis, but substantial interindividual variability in mRNA expression remained unexplained. Within the power limits of this pilot study, gene expression signature was not clearly predictive of lung cancer case or control status. This noninvasive and quantitative method may be incorporated into high-throughput human applications for probing gene-environment interactions associated with cancer.

Phenotypic changes in MCF-7 cells during prolonged exposure to tamoxifen.

MCF-7 breast tumor cells form multicellular nodules (foci) over a confluent monolayer in an estradiol (E2)-dependent, antiestrogen-sensitive reaction. A cell line cloned from MCF-7 that displays these phenotypes was probed to determine the effects of long term exposure to tamoxifen on the growth of foci, estrogen receptor alpha (ERalpha) status, and gene responsiveness to E2. In one of two experiments, a heterogeneous cell population emerged (TMX2) that over-expressed estrogen receptor alpha wild type mRNA (ERalpha mRNA) (approximately 20-fold) missing exon 3 (ERDelta3 mRNA) and its corresponding protein (ERDelta3P). On a per mRNA to protein basis, ERDelta3P and wild-type ERalpha were equivalently expressed. Return of the TMX2 population to medium without tamoxifen eventually selected for a population that expressed predominately wild-type ERalpha, whereas TMX2 clones over expressing ERDelta3 mRNA and ERDelta3P retained this phenotype in tamoxifen-free media. In both experiments, expression of all ERalpha mRNAs and proteins declined to barely detectable levels during 6-12 months exposure, concomitant with a progressive increase in the ability of the cells to form foci independently of E2 or tamoxifen. Selection for these various populations suggests that tamoxifen can induce and/or support certain cellular changes that lead to altered ERalpha expression, E2-independent cell growth and resistance to antiestrogens.

Testing for endocrine disruption: how much is enough?

The article highlighted in this issue is "Comparison of the Developmental and Reproductive Toxicity of Diethylstilbestrol Administered to Rats in Utero, Lactationally, Preweaning or from Weaning" by J. Odum, P. A. Lefevre, H. Tinwell, J. P. Van Miller, R. L. Joiner, R. E. Chapin, N. T. Wallis and J. Ashby (pp. 147-163).

A peptide derived from alpha-fetoprotein prevents the growth of estrogen-dependent human breast cancers sensitive and resistant to tamoxifen.

An 8-mer peptide (EMTOVNOG) derived from alpha-fetoprotein was compared with tamoxifen for activity against growth of human breast cancer xenografts implanted in immune-deficient mice. Both peptide and tamoxifen prevented growth of estrogen-receptor-positive MCF-7 and T47D human breast cancer xenografts. A subline of MCF-7, made resistant to tamoxifen by a 6-month exposure to this drug in culture, was found to be resistant to tamoxifen in vivo. Peptide completely prevented the xenograft growth of this tamoxifen-resistant subline of MCF-7. Neither peptide nor tamoxifen was effective in slowing the xenograft growth of the estrogen-receptor-negative MDA-MB-231 human breast cancer. A worrisome side effect of tamoxifen is its hypertrophic effect on the uterus. In this study, tamoxifen was shown to stimulate the growth of the immature mouse uterus in vivo, and the peptide significantly inhibited tamoxifen's uterotrophic effect. The mechanism of action of peptide is different from that of tamoxifen in that the peptide does not interfere with the binding of [(3)H]estradiol to the estrogen receptor. In conclusion, alpha-fetoprotein-derived peptide appears to be a novel agent that interferes with the growth of tamoxifen-sensitive as well as tamoxifen-resistant estrogen-receptor-positive human breast cancers; it inhibits the uterotrophic side effect of tamoxifen and, thus, it may be useful in combination with or in place of tamoxifen for treatment of estrogen-receptor-positive human breast cancers.