Twila A. Jackson

DNA profiling analysis of endometrial and ovarian cell lines reveals misidentification, redundancy and contamination

OBJECTIVES Cell lines derived from human ovarian and endometrial cancers, and their immortalized non-malignant counterparts, are critical tools to investigate and characterize molecular mechanisms underlying gynecologic tumorigenesis, and facilitate development of novel therapeutics. To determine the extent of misidentification, contamination and redundancy, with evident consequences for the validity of research based upon these models, we undertook a systematic analysis and cataloging of endometrial and ovarian cell lines. METHODS Profiling of cell lines by analysis of DNA microsatellite short tandem repeats (STR), p53 nucleotide polymorphisms and microsatellite instability was performed. RESULTS Fifty-one ovarian cancer lines were profiled with ten found to be redundant and five (A2008, OV2008, C13, SK-OV-4 and SK-OV-6) identified as cervical cancer cells. Ten endometrial cell lines were analyzed, with RL-92, HEC-1A, HEC-1B, HEC-50, KLE, and AN3CA all exhibiting unique, uncontaminated STR profiles. Multiple variants of Ishikawa and ECC-1 endometrial cancer cell lines were genotyped and analyzed by sequencing of mutations in the p53 gene. The profile of ECC-1 cells did not match the EnCa-101 tumor, from which it was reportedly derived, and all ECC-1 isolates were genotyped as Ishikawa cells, MCF-7 breast cancer cells, or a combination thereof. Two normal, immortalized endometrial epithelial cell lines, HES cells and the hTERT-EEC line, were identified as HeLa cervical carcinoma and MCF-7 breast cancer cells, respectively. CONCLUSIONS Results demonstrate significant misidentification, duplication, and loss of integrity of endometrial and ovarian cancer cell lines. Authentication by STR DNA profiling is a simple and economical method to verify and validate studies undertaken with these models.

Tamoxifen resistant breast cancer: coregulators determine the direction of transcription by antagonist-occupied steroid receptors☆

Pharmacological antagonists of steroid receptor action had been thought to exert their effects by a passive mechanism driven principally by the ability of the antagonist to compete with agonist for the ligand binding site. However, recent analyses of antagonist-occupied receptor function suggest a more complex picture. Antagonists can be subdivided into two groups, type I, or pure antagonists, and type II, or mixed antagonists that can have variable transcriptional activity based upon differential dimerization and DNA binding properties. This led us to propose that receptor antagonism may not simply be a passive competition for the ligand binding site, but may, in some cases, involve active recruitment of corepressor or coactivator proteins to produce a mixed transcriptional phenotype. We used a yeast two-hybrid screen to identify proteins that interact specifically with antagonist-occupied receptors. Two proteins have been characterized: L7/SPA, a ribosome-associated protein that is localized in both the cytoplasm and nucleus, but with no known extranucleolar nuclear function; and hN-CoR, the human homolog of the mouse thyroid receptor corepressor mN-CoR. In in vivo transcription assays we show that L7/SPA enhances the partial agonist activity of type II mixed antagonists, and that N-CoR and the related corepressor, SMRT, suppresses it. The coregulators do not affect agonists or pure antagonists. Moreover, the net agonist activity seen with mixed antagonists is a function of the ratio of coactivator to corepressor. Based upon these results, we proposed that in breast tumors the inappropriate agonist activity seen with therapeutic antagonists such as tamoxifen is responsible for the hormone-resistant state. To confirm this, we are quantitating coactivator/corepressor ratios in breast tumor cells lines and clinical breast cancers. Results should provide new insights into the mechanisms underlying the progression of breast cancer to hormone resistance, and may suggest strategies for delaying or reversing this process.

Nuclear receptor conformation, coregulators, and tamoxifen-resistant breast cancer

The development of tamoxifen resistance and consequent disease progression are common occurrences in breast cancers, often despite the continuing expression of estrogen receptors (ER). Tamoxifen is a mixed antagonist, having both agonist and antagonist properties. We have suggested that the development of tamoxifen resistance is associated with an increase in its agonist-like properties, resulting in loss of antagonist effects or even inappropriate tumor stimulation. Nuclear receptor function is influenced by a family of transcriptional coregulators, that either enhance or suppress transcriptional activity. Using a mixed antagonist-biased two-hybrid screening strategy, we identified two such proteins: the human homolog of the nuclear receptor corepressor, N-CoR, and a novel coactivator, L7/SPA (Switch Protein for Antagonists). In transcriptional studies, N-CoR suppressed the agonist properties of tamoxifen and RU486, and L7/SPA increased agonist effects. We speculated that the relative levels of these coactivators and corepressors may determine the balance of agonist and antagonist properties of mixed antagonists, such as tamoxifen. Using quantitative RT-PCR, we, therefore, measured the levels of transcripts encoding these coregulators, as well as the corepressor SMRT, and the coactivator SRC-1, in a small cohort of tamoxifen-resistant and sensitive breast tumors. The results suggest that tumor sensitivity to mixed antagonists may be governed by a complex set of transcription factors, which we are only now beginning to understand.

Thoughts on tamoxifen resistant breast cancer. Are coregulators the answer or just a red herring

The antiestrogen tamoxifen is an effective treatment for estrogen receptor positive breast cancers, slowing tumor growth and preventing disease recurrence, with relatively few side effects. However, many patients who initially respond to treatment, later become resistant to treatment. Tamoxifen has both agonist and antagonist activities, which are manifested in a tissue-specific pattern. Development of tamoxifen resistance can be characterized by an increase in the partial agonist properties of the antiestrogen in the breast, resulting in loss of growth inhibition and even inappropriate tumor stimulation. Nuclear receptor function is modulated by transcriptional coregulators, which either enhance or repress receptor activity. Using a mixed antagonist-biased two-hybrid screening strategy, we identified two such proteins: the human homolog of the nuclear receptor corepressor, N-CoR, and a novel coactivator, L7/SPA (Switch Protein for Antagonists). In transcriptional studies N-CoR suppressed the agonist properties of tamoxifen and RU486, while L7/SPA increased agonist effects. We speculated that the relative level of these coactivators and corepressors might determine the balance of agonist and antagonist properties of mixed antagonists such as tamoxifen. Using quantitative RT-PCR we therefore measured the levels of transcripts encoding these coregulators, as well as the corepressor SMRT, and the coactivator SRC-1, in a small cohort of tamoxifen resistant and sensitive breast tumors. The results suggest that tumor sensitivity to mixed antagonists may be governed by a complex set of transcription factors, which we are only now beginning to understand.

GCUNC-45 is a novel regulator for the progesterone receptor/hsp90 chaperoning pathway.

ABSTRACT The hsp90 chaperoning pathway is a multiprotein system that is required for the production or activation of many cell regulatory proteins, including the progesterone receptor (PR). We report here the identity of GCUNC-45 as a novel modulator of PR chaperoning by hsp90. GCUNC-45, previously implicated in the activities of myosins, can interact in vivo and in vitro with both PR-A and PR-B and with hsp90. Overexpression and knockdown experiments show GCUNC-45 to be a positive factor in promoting PR function in the cell. GCUNC-45 binds to the ATP-binding domain of hsp90 to prevent the activation of its ATPase activity by the cochaperone Aha1. This effect limits PR chaperoning by hsp90, but this can be reversed by FKBP52, a cochaperone that is thought to act later in the pathway. These findings reveal a new cochaperone binding site near the N terminus of hsp90, add insight on the role of FKBP52, and identify GCUNC-45 as a novel regulator of the PR signaling pathway.

Endocrine disrupting activities of the flavonoid nutraceuticals luteolin and quercetin.

Dietary plant flavonoids have been proposed to contribute to cancer prevention, neuroprotection, and cardiovascular health through their anti-oxidant, anti-inflammatory, pro-apoptotic, and antiproliferative activities. As a consequence, flavonoid supplements are aggressively marketed by the nutraceutical industry for many purposes, including pediatric applications, despite inadequate understanding of their value and drawbacks. We show that two flavonoids, luteolin and quercetin, are promiscuous endocrine disruptors. These flavonoids display progesterone antagonist activity beneficial in a breast cancer model but deleterious in an endometrial cancer model. Concurrently, luteolin possesses potent estrogen agonist activity while quercetin is considerably less effective. These results highlight the promise and peril of flavonoid nutraceuticals and suggest caution in supplementation beyond levels attained in a healthy, plant-rich diet.

c-Jun N-terminal kinase regulates apoptosis in endometrial cancer cells

Abstractc-Jun N-terminal kinases (JNKs) are important regulators of cell proliferation and apoptosis that have been implicated in tumorigenesis. We investigated the role of JNKs in apoptotic responses in Ishikawa and HEC-50 cells, models of type I and type II endometrial cancer, respectively. Etoposide treatment or UV irradiation resulted in sustained activation of JNK, correlating with the induction of apoptosis. Inhibition of JNK, or MAP kinase kinase 4 (MKK4), selectively suppressed apoptotic responses in both Ishikawa and HEC-50 cells. Knockdown of protein kinase C δ (PKCδ) also attenuated apoptosis in endometrial cancer cells and inhibited the sustained, UV-mediated JNK activation in HEC-50, but not Ishikawa cells. Etoposide-induced JNK phosphorylation was unaffected by PKCδ knockdown, implying that JNK can regulate apoptosis by PKCδ-dependent and independent pathways, according to stimulus and cell type. Thus, expression and activity of JNK and PKCδ in endometrial cancer cells modulate apoptosis and sensitivity to chemotherapeutic agents and may function as tumor suppressors in the endometrium.

Differential regulation of cell growth and gene expression by FGF-2 and FGF-4 in pituitary lactotroph GH4 cells

Fibroblast growth factors, FGF-2 and FGF-4, are reported to play divergent roles in pituitary differentiation and tumor formation, stimulating cell differentiation or proliferation, respectively. However, mitogenic responses to FGFs have not been extensively characterized and little is known about the molecular mechanisms by which specific FGF isoforms may mediate distinct biological responses. Here we show that FGF-4 but not FGF-2 stimulated DNA synthesis and cell proliferation in GH4 cells. Microarray analyses revealed that FGF-4 induced expression of several oncogenes, growth factor receptors and cell cycle control proteins (e.g. cyclin D3/cdk4, N-myc, c-Raf, insulin and thyroid hormone receptors) while FGF-2 had no effect or down regulated these same genes. These transcriptional responses are consistent with a proliferative and/or tumorigenic role for FGF-4 versus a growth inhibitory effect of FGF-2. FGF-2 and FGF-4 also differentially regulated MAP kinase phosphorylation, which may underlie their isoform-specific effects on cell growth and gene expression.

Protein Kinase Cα Modulates Estrogen-Receptor-Dependent Transcription and Proliferation in Endometrial Cancer Cells

Endometrial cancer is the most common invasive gynecologic malignancy in developed countries. The most prevalent endometrioid tumors are linked to excessive estrogen exposure and hyperplasia. However, molecular mechanisms and signaling pathways underlying their etiology and pathophysiology remain poorly understood. We have shown that protein kinase Cα (PKCα) is aberrantly expressed in endometrioid tumors and is an important mediator of endometrial cancer cell survival, proliferation, and invasion. In this study, we demonstrate that expression of active, myristoylated PKCα conferred ligand-independent activation of estrogen-receptor- (ER-) dependent promoters and enhanced responses to estrogen. Conversely, knockdown of PKCα reduced ER-dependent gene expression and inhibited estrogen-induced proliferation of endometrial cancer cells. The ability of PKCα to potentiate estrogen activation of ER-dependent transcription was attenuated by inhibitors of phosphoinositide 3-kinase (PI3K) and Akt. Evidence suggests that PKCα and estrogen signal transduction pathways functionally interact, to modulate ER-dependent growth and transcription. Thus, PKCα signaling, via PI3K/Akt, may be a critical element of the hyperestrogenic environment and activation of ER that is thought to underlie the development of estrogen-dependent endometrial hyperplasia and malignancy. PKCα-dependent pathways may provide much needed prognostic markers of aggressive disease and novel therapeutic targets in ER positive tumors.

Rapid Estrogen Signaling Negatively Regulates PTEN Activity Through Phosphorylation in Endometrial Cancer Cells

Hyperestrogenicity is a risk factor for endometrial cancer. 17β-estradiol (E2) is known to stimulate both genomic and nongenomic estrogen receptor-α (ERα) actions in a number of reproductive tissues. However, the contributions of transcription-independent ERα signaling on normal and malignant endometrium are not fully understood. Phosphatase and tensin homolog (PTEN) is a tumor suppressor that decreases cellular mitosis primarily through negative regulation of the phosphoinositide 3-kinase/AKT signaling axis. PTEN levels are elevated during the E2 dominated, mitotically active, proliferative phase of the menstrual cycle, indicating possible hormonal regulation of PTEN in the uterus. In order to determine if rapid E2 signaling regulates PTEN, we used ERα-positive, PTEN-positive, endometrial cells. We show that cytosolic E2/ERα signaling leads to increased phosphorylation of PTEN at key regulatory residues. Importantly, E2 stimulation decreased PTEN lipid phosphatase activity and caused consequent increases in phospho-AKT. We further demonstrate that cytosolic ERα forms a complex with PTEN in an E2-dependent manner, and that ERα constitutively complexes with protein kinase2-α (CK2α), a kinase previously shown to phosphorylate the C-terminal tail of PTEN. These results provide mechanistic support for an E2-dependent, ERα cytosolic signaling complex that negatively regulates PTEN activity through carboxy terminus phosphorylation. Using an animal model, we show that sustained E2 signaling results in increased phospho-PTEN (S380, T382, and T383), total PTEN, and phospho-AKT (S473). Taken together, we provide a novel mechanism in which transcription-independent E2/ERα signaling may promote a pro-tumorigenic environment in the endometrium.