Mitochondrial activities play a pivotal role in regulating cell cycle in response to doxorubicin.

Doxorubicin induces both DNA damage and metabolic interference. How these effects interact to modulate cellular toxicity is not completely understood but important given the widespread use of doxorubicin in cancer treatment. This study tests the hypothesis that cell cycle arrest and survival are affected by distinct mitochondrial activities during doxorubicin exposure.Parental and mutant S. cerevisiae strains deficient in selected genes with mitochondrial function were treated with doxorubicin and assayed for changes in proliferation rates, cell survival and cell cycle arrest kinetics. Mitochondrial DNA content was estimated using quantitative PCR. Mitochondrial function was assessed by measuring oxygen consumption with and without an uncoupler.Parental cells growing in a non-fermentable carbon source medium and mutants lacking mitochondria and grown in glucose medium both show abrupt cell cycle and proliferation arrest during doxorubicin exposure compared to parental cells grown in glucose. Mitochondrial DNA increases during doxorubicin exposure in S. cerevisiae and in human breast cancer cells. Yeast strains deficient in TCA cycle activity or electron transport both show more abrupt cell cycle arrest than parental cells when exposed to doxorubicin. Concurrent treatment with the mitochondrial uncoupler dinitrophenol facilitates cell cycle progression and proliferation during doxorubicin exposure.Doxorubicin exposure induces mitochondrial DNA synthesis with TCA cycle and oxidative phosphorylation activity having opposing effects on cell proliferation, survival and cell cycle kinetics. TCA cycle activity provides biosynthetic substrates to support cell cycle progression and cell proliferation while electron transport and oxidative phosphorylation facilitate cell cycle arrest and possibly increased cytotoxicity.

A tRNA fragment, tRF5-Glu, regulates BCAR3 expression and proliferation in ovarian cancer cells.

Ovarian cancer is a complex disease marked by tumor heterogeneity, which contributes to difficulties in diagnosis and treatment. New molecular targets and better molecular profiles defining subsets of patients are needed. tRNA fragments (tRFs) offer a recently identified group of noncoding RNAs that are often as abundant as microRNAs in cancer cells. Initially their presence in deep sequencing data sets was attributed to the breakdown of mature tRNAs, however, it is now clear that they are actively generated and function in multiple regulatory events. One such tRF, a 5' fragment of tRNA-Glu-CTC (tRF5-Glu), is processed from the mature tRNA-Glu and is shown in this study to be expressed in ovarian cancer cells. We confirmed that tRF5-Glu binds directly to a site in the 3'UTR of the Breast Cancer Anti-Estrogen Resistance 3 (BCAR3) mRNA thereby down regulating its expression. BCAR3 has not previously been studied in ovarian cancer cells and our studies demonstrate that inhibiting BCAR3 expression suppresses ovarian cancer cell proliferation. Furthermore, mimics of tRF5-Glu were found to inhibit proliferation of ovarian cancer cells. In summary, BCAR3 and tRF5-Glu contribute to the complex tumor heterogeneity of ovarian cancer cells and may provide new targets for therapeutic intervention.

Development of an interdisciplinary pre-matriculation program designed to promote medical students' self efficacy.

BACKGROUND AND OBJECTIVES: A four-week interdisciplinary pre-matriculation program for Native American and rural medical students was created and its impact on students' transition to medical school was assessed. The program extends the goals of many pre-matriculation programs by aiming to increase not only students' understanding of basic science knowledge, but also to build student self-efficacy through practice with medical school curricular elements while developing their academic support networks. DESIGN: A mixed method evaluation was used to determine whether the goals of the program were achieved (n = 22). Student knowledge gains and retention of the microbiology content were assessed using a microbiology concept inventory. Students participated in focus groups to identify the benefits of participating in the program as well as the key components of the program that benefitted the students. RESULTS: Program participants showed retention of microbiology content and increased confidence about the overall medical school experience after participating in the summer program. CONCLUSIONS: By nurturing self-efficacy, participation in a pre-matriculation program supported medical students from Native American and rural backgrounds during their transition to medical school. ABBREVIATIONS: CAIMH: Center of American Indian and Minority Health; MCAT: Medical College Admission Test; PBL: Problem based learning; UM MSD: University of Minnesota Medical School Duluth.

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.

Aspartate facilitates mitochondrial function, growth arrest and survival during doxorubicin exposure.

Genomic screens of doxorubicin toxicity in S. cerevisiae have identified numerous mutants in amino acid and carbon metabolism which express increased doxorubicin sensitivity. This work examines the effect of amino acid metabolism on doxorubicin toxicity. S. cerevisiae were treated with doxorubicin in combination with a variety of amino acid supplements. Strains of S. cerevisiae with mutations in pathways utilizing aspartate and other metabolites were examined for sensitivity to doxorubicin. S. cerevisiae cultures exposed to doxorubicin in minimal media showed significantly more toxicity than cultures exposed in rich media. Supplementing minimal media with aspartate, glutamate or alanine reduced doxorubicin toxicity. Cell cycle response was assessed by examining the budding pattern of treated cells. Cultures exposed to doxorubicin in minimal media arrested growth with no apparent cell cycle progression. Aspartate supplementation allowed cultures exposed to doxorubicin in minimal media to arrest after one division with a budding pattern and survival comparable to cultures exposed in rich media. Aspartate provides less protection from doxorubicin in cells mutant in either mitochondrial citrate synthase (CIT1) or NADH oxidase (NDI1), suggesting aspartate reduces doxorubicin toxicity by facilitating mitochondrial function. These data suggest glycolysis becomes less active and mitochondrial respiration more active following doxorubicin exposure.

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.

BACKGROUND: 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. METHODS: This review is based on publications identified on PubMed using a literature search of cytosine methylation, histone methylation, acetylation and glycosylation. RESULTS: In cancer, changes in glycolytic enzymes lead to increased production of serine, increasing the pool of S-adenosylmethionine (the major methyl donor for methylation reactions) and UDP-N-acetylglucosamine (a substrate for O-linked glycosylation of histones and cytosine methyltransferases). Mutations in tricarboxylic acid cycle enzymes lead to accumulation of fumarate, succinate and hydroxyglutarate, all of which inhibit demethylation of cytosine and histones. In contrast, proline catabolism produces α-ketoglutarate and reactive oxygen, both of which promote the activity of enzymes that remove methyl groups from cytosine and histones, and the key enzyme in proline catabolism acts as a tumour suppressor. CONCLUSIONS: Our emerging understanding of how the epigenetic profiles are metabolically reprogrammed in cancer cells will lead to novel diagnostic and therapeutic targets for treatment of patients.

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.

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.

Short-term prophylactic tamoxifen reduces the incidence of antiestrogen-resistant/estrogen receptor-positive/progesterone receptor-negative mammary tumors.

Although many estrogen receptor-positive (ER+) breast cancers are effectively treated with selective estrogen receptor modulators and down-regulators (SERM/SERD), some are highly resistant. Resistance is more likely if primary cancers are devoid of progesterone receptors (PR-) or have high levels of growth factor activity. In this study, a transgenic mouse line that expresses transforming growth factor-alpha (NRL-TGFalpha mice) and that develops ER+/PR- mammary tumors was used to assess the possible effects of (a) therapeutic delivery of the SERM, tamoxifen, or SERD, ICI I82,780 (ICI), on the growth of established tumors and (b) short-term prophylactic tamoxifen administration on the initial development of new mammary tumors. To determine the therapeutic effects of tamoxifen and ICI on the growth of established tumors, mice were exposed to 3 weeks of drug treatment. Neither drug influenced tumor growth or glandular pathology. To determine if early prophylactic tamoxifen could alter tumorigenesis, a 60-day tamoxifen treatment was initiated in 8-week-old mice. Compared with placebo-treated mice, tamoxifen reduced tumor incidence by 50% and significantly decreased the degree of mammary hyperplasia. Prophylactic tamoxifen also significantly extended the life span of tumor-free mice. These data show that in this mouse model, established ER+/PR- mammary tumors are resistant to SERM/SERD treatment but the development of new mammary tumors can be prevented by an early course of tamoxifen. This study validates the utility of NRL-TGFalpha mice for (a) identifying candidate biomarkers of efficacious tamoxifen chemoprevention and (b) modeling the evolution of tamoxifen resistance.

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.

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 p16(INK4A) and p21(Waf1/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.

Linkage of progestin and epidermal growth factor signaling: phosphorylation of progesterone receptors mediates transcriptional hypersensitivity and increased ligand-independent breast cancer cell growth.

Progesterone receptor (PR) action is linked to epidermal growth factor (EGF) initiated signaling pathways at multiple levels; mitogen-activated protein kinases (MAPKs) are key mediators of this important cross-talk. Herein, we probed the effects of EGF on PR function and regulation of breast cancer cell growth. EGF stimulated rapid and transient phosphorylation of PR-B Ser294 relative to persistent phosphorylation of this site induced by the synthetic progestin, R5020. EGF induced nuclear translocation and DNA binding of unliganded wild-type, but not mutant PRs containing an Ala at position 294 (S294A). However, EGF alone induced little to no PR-B transcriptional activity; S294A PR-B was transcriptionally impaired. In contrast, pretreatment of cells with EGF (30min) significantly increased the potency and efficacy of wild-type, but not S294A PR transcriptional activity in response to progestin, and enhanced ligand-dependent downregulation of wild-type but not S294A PR. Replacement of Ser294 with aspartic acid (S294D) to mimic phosphorylation at this site decreased receptor stability and, as predicted, heightened progestin-induced transcription relative to wild-type PR-B. RT-PCR demonstrated the Ser294 phosphorylation-dependence of selected PR target genes (TGFalpha and HB-EGF). Surprisingly, PR-B expressing cells growing in soft agar were highly responsive to EGF or progestin, and this was further stimulated by the combination of both hormones. Cells expressing S294A PR exhibited reduced soft agar growth, and were also sensitive to R5020 alone, but failed to respond to EGF. These results suggest that PR Ser294 is an important "sensor" for growth factor inputs that affects PR function and breast cancer cell growth in the absence of progestin or in the presence of low or "sub-threshold" progestin concentrations. PR function likely contributes to breast cancer progression when EGFR family members or their ligands are overexpressed, a condition that predicts low abundance, but highly active and nuclear PR.

Progesterone receptors induce cell cycle progression via activation of mitogen-activated protein kinases.

Progestins induce proliferation of breast cancer cells and are implicated in the development of breast cancer. The effects of progestins are mediated by progesterone receptors (PRs), although it is unclear whether proliferative effects are delivered through activities as ligand-activated transcription factors or via activation of cytoplasmic kinases. We report that progestin induces S phase entry of T47D cells stably expressing either wild-type (wt) PR-B or a transcriptionally impaired PR-B harboring a point mutation at Ser294, a ligand-dependent and MAPK consensus phosphorylation site (S294A). Both wt and S294A PR are capable of activating p42/p44 MAPKs and promoting proliferation. However, cells expressing wt, but not S294A PR, exhibited enhanced proliferation in response to combined epidermal growth factor and progestin. S phase progression correlated with up-regulation of cyclin D1. The PR antagonist RU486 also induced MAPK activation, increased cyclin D1 expression, and stimulated S phase entry, which was blocked by inhibition of either p42/p44 or p38 MAPKs, whereas proliferation induced by R5020 was sensitive only to p42/p44 MAPK inhibition. MCF-7 cells stably expressing a mutant PR unable to bind c-Src and activate MAPK failed to support progestin-induced proliferation. These data suggest that PR mediate cell cycle progression primarily through activation of cytoplasmic kinases and independently of direct regulation of transcription, whereas the coordinate regulation of both aspects of PR action are required for enhanced proliferation in response to progestins in the presence of growth factors. Targeting the ability of steroid receptors to activate MAPKs may be beneficial for breast cancer patients.

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 G(1) phase 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 p21(WAF1/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 p21(WAF1/Cip1). These results identify p21(WAF1/Cip1) as an important physiological regulator of Cdk4 complexes in human breast cancer cells.