Andrew Skildum

Integration of progesterone receptor mediated rapid signaling and nuclear actions in breast cancer cell models: Role of mitogen-activated protein kinases and cell cycle regulators

Progesterone receptor (PR) isoforms are dual functioning steroid hormone receptors, capable of activation of target gene transcription, and rapid stimulation of membrane-initiated intracellular signaling cascades. Herein we provided a retrospective of our recent work investigating the role of progestin-activated intracellular signaling pathways on cell cycle progression in breast cancer cell models. We show that progestin-induced S-phase entry and upregulation of selected target genes, including cyclin D1, are MAPK-dependent events. Further experiments conducted with mutant PRs defective in either the transcriptional response (PR-S294A) or activation of c-Src-dependent intracellular signaling to MAPKs (PR-mPro) confirmed that the proliferative response of breast cancer cells to progestins is largely dependent on the ability of PR to rapidly activate Erk 1/2 MAPKs. During progestin-stimulated cell cycle progression, elevated cdk2 levels and activity target multiple phosphorylation sites on PR. Phosphorylation of Ser400 augments PR nuclear localization and mediates increased PR transcriptional activity in the absence of hormone, while the cdk inhibitor, p27, reversed these effects. Together, our data illustrate the versatility of PR as regulatory signaling molecules that also act as sensors for multiple kinase pathways, and suggest that progestins influence changes in breast cancer cell gene expression and proliferation via integration of PR functions as both ligand-activated transcription factors and rapid initiators of intracellular signaling pathways.

Mitochondrial remodeling in cancer metabolism and survival: potential for new therapies.

Mitochondria are semi-autonomous organelles that play essential roles in cellular metabolism and programmed cell death pathways. Genomic, functional and structural mitochondrial alterations have been associated with cancer. Some of those alterations may provide a selective advantage to cells, allowing them to survive and grow under stresses created by oncogenesis. Due to the specific alterations that occur in cancer cell mitochondria, these organelles may provide promising targets for cancer therapy. The development of drugs that specifically target metabolic and mitochondrial alterations in tumor cells has become a matter of interest in recent years, with several molecules undergoing clinical trials. This review focuses on the most relevant mitochondrial alterations found in tumor cells, their contribution to cancer progression and survival, and potential usefulness for stratification and therapy.

The cyclin-dependent kinase inhibitor p21WAF1/Cip1 is an antiestrogen-regulated inhibitor of Cdk4 in human breast cancer cells

The MCF-7 cell line is a model of estrogen-dependent, antiestrogen-sensitive human breast cancer. Antiestrogen treatment of MCF-7 cells causes dramatic decreases in both Cdk4 and Cdk2 activities, which leads to a G1phase cell cycle arrest. In this report, we investigate the mechanism(s) by which Cdk4 activity is regulated in MCF-7 cells. Through time course analysis, we demonstrate that changes in Cdk4 activity in response to estrogen or antiestrogen treatment do not correlate directly with cyclin D1 protein levels or association. In contrast, Cdk4 activity does correlate with changes in the level of the Cdk inhibitor p21WAF1/Cip1. Furthermore, we show that extracts of antiestrogen-treated cells contain a factor capable of inhibiting the Cdk4 activity present in extracts of estrogen-treated cells, and immunodepletion experiments identify this factor as p21WAF1/Cip1. These results identify p21WAF1/Cip1 as an important physiological regulator of Cdk4 complexes in human breast cancer cells.

Functional Ablation of pRb Activates Cdk2 and Causes Antiestrogen Resistance in Human Breast Cancer Cells

Estrogens are required for the proliferation of hormone dependent breast cancer cells, making estrogen receptor (ER) positive tumors amenable to endocrine therapies such as antiestrogens. However, resistance to these agents remains a significant cause of treatment failure. We previously demonstrated that inactivation of the retinoblastoma protein (pRb) family tumor suppressors causes antiestrogen resistance in MCF-7 cells, a widely studied model of estrogen responsive human breast cancers. In this study, we investigate the mechanism by which pRb inactivation leads to antiestrogen resistance. Cdk4 and cdk2 are two key cell cycle regulators that can phosphorylate and inactivate pRb, therefore we tested whether these kinases are required in cells lacking pRb function. pRb family members were inactivated in MCF-7 cells by expressing polyomavirus large tumor antigen (PyLT), and cdk activity was inhibited using the cdk inhibitors p16INK4A and p21Waf1/Cip1. Cdk4 activity was no longer required in cells lacking functional pRb, while cdk2 activity was required for proliferation in both the presence and absence of pRb function. Using inducible PyLT cell lines, we further demonstrated that pRb inactivation leads to increased cyclin A expression, cdk2 activation and proliferation in antiestrogen arrested cells. These results demonstrate that antiestrogens do not inhibit cdk2 activity or proliferation of MCF-7 cells in the absence of pRb family function, and suggest that antiestrogen resistant breast cancer cells resulting from pRb pathway inactivation would be susceptible to therapies that target cdk2.

Bioactive polybrominated diphenyl ethers from the marine sponge Dysidea sp.

A new polybrominated diphenyl ether ( 9), together with eight known compounds, were isolated from the crude organic extract of the marine sponge Dysidea sp. collected from the Federated States of Micronesia. Their structures were elucidated on the basis of various NMR spectroscopic data. These compounds exhibited inhibitory activities against Streptomyces 85E in the hyphae formation inhibition (HFI) assay and displayed antiproliferative activities against the human breast adenocarcinoma cancer cell line MCF-7. Compound 6 was selected for further evaluation in a cell cycle progression study.

Mitochondrial amplification selectively increases doxorubicin sensitivity in breast cancer cells with acquired antiestrogen resistance

The metabolic phenotype of cancer, characterized by uncoupled mitochondrial respiration and increased mitochondrial oxidative stress, is an attractive pharmacological target for sensitizing cancer cells to therapies that rely on oxidative stress for their tumor specific cytotoxicity. The identification of specific cancer sub-types for which metabolic priming of tumors prior to chemotherapy is beneficial is critical, particularly in heterogeneous diseases such as breast cancer. The effects of the thiazolidinedione drug troglitazone were examined in normal mammary epithelial cells and cancer cell lines representing three clinically relevant breast cancer phenotypes. Endpoints measured were PGC1α mRNA expression, proliferation, cell cycle phase distribution, mitochondrial capacity and superoxide generation, and sensitivity to the chemotherapy drug doxorubicin. Troglitazone increases expression of PGC1α, a key mediator of mitochondrial biogenesis, in normal mammary epithelial cells and in breast cancer cell lines. The induction of PGC1α mRNA is at least partially dependent on PPARγ activation. In estrogen receptor negative cells and cells with acquired antiestrogen resistance, troglitazone treatment increased mitochondrial superoxide production and mitochondrial capacity. At pharmacologically achievable doses, troglitazone pretreatment significantly enhanced the sensitivity of cancer cells to the chemotherapy agent doxorubicin. This effect was most dramatic in estrogen receptor positive cells with acquired antiestrogen resistance, in which troglitazone and doxorubicin combined had superadditive effects compared to treatment with either agent alone. In contrast, troglitazone treatment did not appreciably sensitize non-malignant mammary epithelial cells to doxorubicin induced cytotoxicity, despite increasing PGC1α mRNA. These data suggest that troglitazone or a similarly acting compound could be used to selectively prime tumor cells to the cytotoxic effects of anticancer agents such as doxorubicin and ionizing radiation. This novel treatment strategy may be most effective in women with antiestrogen insensitive tumors, a patient population with historically poor response to traditional therapies.

Stimulating basal mitochondrial respiration decreases doxorubicin apoptotic signaling in H9c2 cardiomyoblasts

Doxorubicin (DOX) is currently used in cancer chemotherapy, however, its use often results in adverse effects highlighted by the development of cardiomyopathy and ultimately heart failure. Interestingly, DOX cardiotoxicity is decreased by resveratrol or by physical activity, suggesting that increased mitochondrial activity may be protective. Conversely, recent studies showed that troglitazone, a PPARγ agonist, increases the cytotoxicity of DOX against breast cancer cells by up-regulating mitochondrial biogenesis. The hypothesis for the current investigation was that DOX cytotoxicity in H9c2 cardiomyoblasts is decreased when mitochondrial capacity is increased. We focused on several end-points for DOX cytotoxicity, including loss of cell mass, apoptotic signaling and alterations of autophagic-related proteins. Our results show that a galactose-based, modified cell culture medium increased H9c2 basal mitochondrial respiration, protein content, and mtDNA copy number without increasing maximal or spare respiratory capacity. H9c2 cardiomyoblasts cultured in the galactose-modified media showed lower DOX-induced activation of the apoptotic pathway, measured by decreased caspase-3 and -9 activation, and lower p53 expression, although ultimately loss of cells was not prevented. Treatment with the PPARγ agonist troglitazone had no effect on DOX toxicity in this cardiac cell line, which agrees with the fact that troglitazone did not increase mitochondrial DNA content or capacity at the concentrations and duration of exposure used in this investigation. Our results show that mitochondrial remodeling caused by stimulating basal rates of oxidative phosphorylation decreased DOX-induced apoptotic signaling and increased DOX-induced autophagy in H9c2 cardiomyoblasts. The differential effect on cytotoxicity in cardiac versus breast cancer cell lines suggests a possible overall improvement in the clinical efficacy for doxorubicin in treating cancer.

The deep end of the metabolite pool: influences on epigenetic regulatory mechanisms in cancer

Epigenetic control of gene expression is mediated by cytosine methylation/demethylation and histone modifications including methylation, acetylation and glycosylation. The epigenetic programme is corrupted in cancer cells to maintain a pattern of gene expression that leads to their de‐differentiated, rapidly proliferating phenotype. Enzymes responsible for modifying histones and cytosine are sensitive to the cellular metabolite pool and can be activated by an increase in their substrates or inhibited by an increase in their products or competitors for substrate binding.

Cross-talk between growth factor and progesterone receptor signaling pathways: implications for breast cancer cell growth.

Breast cancers often have increased mitogen-activated protein kinase (MAPK) activity; this pathway influences breast cancer cell growth in part by targeting steroid hormone receptors. Activation of p42 and p44 MAPKs increases progesterone receptor (PR) transcriptional activity in the presence of progestins, and triggers their rapid down-regulation by the ubiquitin-proteasome pathway. In turn, progestins increase the expression of type I growth factor receptor tyrosine kinases that feed into MAPK activation. Most recently, progestins have been shown to activate the p42/p44 MAPK module in a progesterone receptor (PR) dependent manner, but independently of their function as transcription factors. Indeed, mechanisms of bi-directional cross-talk between these two pathways are becoming well-documented. In this reveiw we provide an overview of the primary ways in which steroid hormone receptor and growth factor cross-talk occurs, using examples from our work and others with human PR as a model receptor. We highlight the regulation of PR by phosphorylation and the role of intracellular protein kinases as key mediators of PR action. Cross-talk between growth factor and PR-mediated signaling events is an important means by which growth regulatory genes may be coordinately regulated, and may contribute to the growth and development of hormonally responsive normal breast tissue and to breast cancer progression.

Altered regulation of PDK4 expression promotes antiestrogen resistance in human breast cancer cells

Acquired or de novo resistance to the selective estrogen receptor modulators tamoxifen and fulvestrant (ICI) is a major barrier to successful treatment of breast cancer. Gene expression patterns in tamoxifen resistant (TamR-MCF-7) cells were compared to their parental cells (MCF-7L) to identify an aberrantly regulated metabolic pathway. TamR-MCF-7 cells are cross resistant to ICI and doxorubicin, and have increased mitochondrial DNA. A small subset of genes had altered expression in TamR-MCF-7 relative to MCF-7L cells. One of the genes, pyruvate dehydrogenase kinase-4 (PDK4), phosphorylates pyruvate dehydrogenase (PDH). PDK4 expression was elevated in TamR-MCF-7 cells; this result was also observed in a second model of acquired antiestrogen resistance. PDK4 expression is controlled in part by glucocorticoid response elements in the PDK4 gene promoter. In MCF-7L cells, PDK4 mRNA expression was insensitive to glucocorticoid receptor agonists, while dexamethasone dramatically increased PDK4 expression in TamR-MCF-7 cells. Using siRNA to knock down PDK4 expression increased TamR-MCF-7 sensitivity to ICI; in contrast adapting cells to growth in glucose depleted media did not affect ICI sensitivity. Despite TamR-MCF-7 cells high levels of PDK4 mRNA relative to MCF-7L, TamR-MCF-7 cells have increased PDH activity. Wild type MCF-7 cells are reported to be heterozygous for a G to A mutation that results in a substitution of threonine for alanine near PDK4′s catalytic site. We found loss of heterozygosity in TamR-MCF-7 cells; TamR-MCF-7 are homozygous for the wild type allele. These data support a role for altered regulation of PDH by PDK4 and altered substrate utilization in the development of drug resistance in human breast cancer cells.