Anna T. Riegel

ROCK inhibitor and feeder cells induce the conditional reprogramming of epithelial cells.

We demonstrate that a Rho kinase inhibitor (Y-27632), in combination with fibroblast feeder cells, induces normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of exogenous viral or cellular genes. Primary prostate and mammary cells, for example, are reprogrammed toward a basaloid, stem-like phenotype and form well-organized prostaspheres and mammospheres in Matrigel. However, in contrast to the selection of rare stem-like cells, the described growth conditions can generate 2 × 10(6) cells in 5 to 6 days from needle biopsies, and can generate cultures from cryopreserved tissue and from fewer than four viable cells. Continued cell proliferation is dependent on both feeder cells and Y-27632, and the conditionally reprogrammed cells (CRCs) retain a normal karyotype and remain nontumorigenic. This technique also efficiently establishes cell cultures from human and rodent tumors. For example, CRCs established from human prostate adenocarcinoma displayed instability of chromosome 13, proliferated abnormally in Matrigel, and formed tumors in mice with severe combined immunodeficiency. The ability to rapidly generate many tumor cells from small biopsy specimens and frozen tissue provides significant opportunities for cell-based diagnostics and therapeutics (including chemosensitivity testing) and greatly expands the value of biobanking. In addition, the CRC method allows for the genetic manipulation of epithelial cells ex vivo and their subsequent evaluation in vivo in the same host.

Expression of the nuclear coactivator AIB1 in normal and malignant breast tissue

The gene of the nuclear receptor coactivator AIB1 (amplified in breast cancer 1) is amplified in breast cancer cell lines as well as in breast tumor tissues. AIB1 mRNA is often highly expressed (>60) in primary breast tumors and it has been shown that AIB1 enhances estrogen and progesterone dependent transcription invitro. Therefore, it has been postulated that AIB1 contributes to the development of breast cancer. However, to date, it has not been shown that AIB1 amplification and overexpression correlates with elevated protein levels in breast cancer tissues. In this study we analyzed protein levels of AIB1 in normal and breast tumor tissues by immunohistochemistry. We compared 41 human breast tumor tissues with 24 normal breast tissue samples and found that AIB1 stained in the nuclei of approximately 46% of the tumors and 30% of the normal tissues. Overall, AIB1 protein levels were significantly higher in tumor tissue than in normal tissue and the highest levels of nuclear staining were found exclusively in breast tumor tissues in 9.8% of the cases. These data suggest that increased AIB1 mRNA expression does not always translate into elevated protein levels and that AIB1 most likely will be relevant to the etiology of a subset of about 10% of breast carcinomas.

Ribozyme Targeting Demonstrates That the Nuclear Receptor Coactivator AIB1 Is a Rate-limiting Factor for Estrogen-dependent Growth of Human MCF-7 Breast Cancer Cells

Human breast tumorigenesis is promoted by the estrogen receptor pathway, and nuclear receptor coactivators are thought to participate in this process. Here we studied whether one of these coactivators, AIB1 (amplified inbreast cancer 1), was rate-limiting for hormone-dependent growth of human MCF-7 breast cancer cells. We developed MCF-7 breast cancer cell lines in which the expression of AIB1 can be modulated by regulatable ribozymes directed against AIB1 mRNA. We found that depletion of endogenous AIB1 levels reduced steroid hormone signaling via the estrogen receptor α or progesterone receptor β on transiently transfected reporter templates. Down-regulation of AIB1 levels in MCF-7 cells did not affect estrogen-stimulated cell cycle progression but reduced estrogen-mediated inhibition of apoptosis and cell growth. Finally, upon reduction of endogenous AIB1 expression, estrogen-dependent colony formation in soft agar and tumor growth of MCF-7 cells in nude mice was decreased. From these findings we conclude that, despite the presence of different estrogen receptor coactivators in breast cancer cells, AIB1 exerts a rate-limiting role for hormone-dependent human breast tumor growth.

Overexpression of the Nuclear Receptor Coactivator AIB1 (SRC-3) during Progression of Pancreatic Adenocarcinoma

Purpose: The nuclear receptor coactivator amplified in breast cancer 1 (AIB1) was found to be amplified and overexpressed in breast and some other epithelial tumors. We have reported that expression of AIB1 is rate limiting for growth factor, as well as hormone signaling. Here, we assess the involvement of AIB1 in the development of pancreatic adenocarcinoma. Experimental Design: We investigated expression levels of AIB1 protein and mRNA in pancreatic cancer cell lines and in a series of archival pancreatic adenocarcinoma (n = 78), pancreatic intraepithelial neoplasia (n = 93), pancreatitis (n = 28), and normal pancreas tissues (n = 52). We also determined AIB1 gene copy numbers by fluorescence in situ hybridization in a subset of cases. Results: In normal pancreas ducts, we rarely found detectable levels of AIB1 mRNA or protein (<6% of the samples). In pancreatitis and low-grade intraepithelial neoplasia, we found an increased frequency of AIB1 expression (>14 and >23%, respectively) relative to normal tissues (P < 0.01). Adenocarcinoma, as well as high-grade intraepithelial neoplasia, showed increased levels as well as the highest frequency of AIB1 expression with >65% of samples positive for mRNA and protein (P < 0.0001 relative to the other groups). An increased copy number of the AIB1 gene, observed in 37% of cancers, may account for a portion of the increase in expression. Conclusions: AIB1 overexpression is frequent in pancreatic adenocarcinoma and its precursor lesions. On the basis of its rate-limiting role for the modulation of growth factor signals, we propose a major role of AIB1 in the multistage progression of pancreatic cancer.

The nuclear receptor coactivator AIB1 mediates insulin-like growth factor I-induced phenotypic changes in human breast cancer cells.

The nuclear receptor coactivator AIB1 (amplified in breast cancer 1) is overexpressed in human breast cancers and is required for estrogen signaling. However, the role of AIB1 in breast cancer etiology is not known. Here, we show that AIB1 is rate-limiting for insulin-like growth factor I (IGF-I)-dependent phenotypic changes and gene expression in human breast cancer cells. Reduction of endogenous AIB1 levels by small interfering RNA in MCF-7 breast cancer cells prevented IGF-I–stimulated anchorage-independent growth by reducing IGF-I–dependent anti-anoikis. cDNA array and immunoblot analysis of gene expression revealed that reduction in AIB1 levels led to a significant decrease in the expression of several genes controlling the cell cycle and apoptosis. These AIB1-dependent changes were also observed in the presence of estrogen antagonist and were corroborated in the estrogen receptor-negative cell line MDA MB-231. AIB1 reduction decreased the expression of the IGF-I receptor and IRS-1 in MCF-7 but not in MDA MB-231 cells. IGF-I–stimulated activation of AKT was reduced by AIB1 small interfering RNA treatment, whereas mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) activation by IGF-I was unaffected. We conclude that AIB1 is required for IGF-I–induced proliferation, signaling, cell survival, and gene expression in human breast cancer cells, independent of its role in estrogen receptor signaling.

The Nuclear Receptor Coactivator Amplified in Breast Cancer-1 Is Required for Neu (ErbB2/HER2) Activation, Signaling, and Mammary Tumorigenesis in Mice

Overexpression of the oncogene amplified in breast cancer 1 (AIB1)/steroid receptor coactivator-3 (SRC-3) induces mammary tumorigenesis in mice. In breast cancer, high levels of AIB1/SRC-3 and the growth factor receptor HER2/neu predict resistance to endocrine therapy and poor outcome. However, a mechanistic relationship between AIB1/SRC-3 and HER2/neu in the development of breast cancer has not been shown. Here, we show that deletion of one allele of SRC-3 significantly delays Neu-induced mammary tumor development in mice. Homozygous deletion of SRC-3 in mice completely prevents Neu-induced tumor formation. By ages 3 to 4 months, Neu/SRC-3(+/-) mice exhibit a noticeable reduction in lateral side-bud formation, accompanied by reduced cellular levels of phosphorylated Neu compared with Neu/SRC-3(wt) mice. In Neu-induced tumors, high levels of SRC-3, phosphorylated Neu, cyclin D1, cyclin E, and proliferating cell nuclear antigen expression are observed, accompanied by activation of the AKT and c-Jun NH(2) kinase (JNK) signaling pathways. In comparison, phosphorylated Neu, cyclin D1, and cyclin E are significantly decreased in Neu/SRC-3(+/-) tumors, proliferation is reduced, and AKT and JNK activation is barely detectable. Our data indicate that AIB1/SRC-3 is required for HER2/neu oncogenic activity and for the phosphorylation and activation of the HER2/neu receptor. We predict that reducing AIB1/SRC-3 levels or activity in the mammary epithelium could potentiate therapies aimed at inhibiting HER2/neu signaling in breast cancer.

Tissue and Serum microRNAs in the KrasG12D Transgenic Animal Model and in Patients with Pancreatic Cancer

microRNAs (miRs) modulate the expression levels of mRNAs and proteins and can thus contribute to cancer initiation and progression. In addition to their intracelluar function, miRs are released from cells and shed into the circulation. We postulated that circulating miRs could provide insight into pathways altered during cancer progression and may indicate responses to treatment. Here we focus on pancreatic cancer malignant progression. We report that changes in miR expression patterns during progression of normal tissues to invasive pancreatic adenocarcinoma in the p48-Cre/LSL-KrasG12D mouse model mirrors the miR changes observed in human pancreatic cancer tissues. miR-148a/b and miR-375 expression were found decreased whereas miR-10, miR-21, miR-100 and miR-155 were increased when comparing normal tissues, premalignant lesions and invasive carcinoma in the mouse model. Predicted target mRNAs FGFR1 (miR-10) and MLH1 (miR-155) were found downregulated. Quantitation of nine microRNAs in plasma samples from patients distinguished pancreatic cancers from other cancers as well as non-cancerous pancreatic disease. Finally, gemcitabine treatment of control animals and p48-Cre/LSL-KrasG12D animals with pancreatic cancer caused distinct and up to 60-fold changes in circulating miRs that indicate differential drug effects on normal and cancer tissues. These findings support the significance of detecting miRs in the circulation and suggests that circulating miRs could serve as indicators of drug response.

Signal Transduction Pathways Involved in the Mitogenic Activity of Pleiotrophin IMPLICATION OF MITOGEN-ACTIVATED PROTEIN KINASE AND PHOSPHOINOSITIDE 3-KINASE PATHWAYS

Pleiotrophin (PTN) is a developmentally regulated protein which exhibits neurite-outgrowth, mitogenic, and angiogenic properties. It has also been shown to be involved in tumor growth and metastasis. Here we used primary BEL (bovine epithelial lens) cells to investigate the signal transduction pathways involved in the mitogenic activity of recombinant PTN. PTN was purified from conditioned media of SW-13 cells transfected with the human PTN cDNA. We show that inhibitors of tyrosine kinase, mitogen-activated protein kinase, or phosphoinositide (PI) 3-kinase inhibit DNA synthesis stimulated by PTN. Analysis of tyrosine-phosphorylated proteins following PTN stimulation showed phosphorylation of two novel 190- and 215-kDa proteins in addition to SHC, ERK1, and ERK2. A mobility shift of phosphorylated ERK1 and ERK2 was detected with a panERK antibody confirming the phosphorylation of the two ERKs. Furthermore, in vitroimmunocomplex kinase assay with Akt1, a natural substrate of PI 3-kinase, showed an activation of the kinase following PTN stimulation and a reversal by the PI 3-kinase inhibitor wortmannin. We conclude that the mitogenic activity of PTN is dependent on tyrosine kinase activation and utilizes the mitogen-activated protein kinase and the PI 3-kinase pathways to transduce a mitogenic signal.

E6AP Mediates Regulated Proteasomal Degradation of the Nuclear Receptor Coactivator Amplified in Breast Cancer 1 in Immortalized Cells

The steroid receptor coactivator oncogene, amplified in breast cancer 1 (AIB1; also known as ACTR/RAC-3/TRAM-1/SRC-3/p/CIP), is amplified and overexpressed in a variety of epithelial tumors. AIB1 has been reported to have roles in both steroid-dependent and steroid-independent transcription during tumor progression. In this report, we describe that the cellular levels of AIB1 are controlled through regulated proteasomal degradation. We found that serum withdrawal or growth in high cell density caused rapid degradation of AIB1 protein, but not mRNA, in immortalized cell lines. Proteasome inhibitors prevented this process, and high molecular weight ubiquitylated species of AIB1 were detected. Nuclear export was required for proteasomal degradation of AIB1 and involved the ubiquitin ligase, E6AP. AIB1/E6AP complexes were detected in cellular extracts, and reduction of cellular E6AP levels with E6AP short interfering RNA prevented proteasomal degradation of AIB1. Conversely, overexpression of E6AP promoted AIB1 degradation. The COOH terminus of AIB1 interacted with E6AP in vitro and deletion of this region in AIB1 rendered it resistant to degradation in cells. From our results, we propose a model whereby signals promoted by changes in the cellular milieu initiate E6AP-mediated proteasomal degradation of AIB1 and thus contribute to the control of steady-state levels of this protein.

A role for TGF-β in estrogen and retinoid mediated regulation of the nuclear receptor coactivator AIB1 in MCF-7 breast cancer cells

AIB1 (amplified in breast cancer 1) is a nuclear receptor coactivator gene amplified and overexpressed in breast cancer. However, the mechanisms by which AIB1 is regulated are unclear. Here we show that 17β-estradiol represses AIB1 mRNA and protein expression in MCF-7 human breast cancer cells primarily by suppressing AIB1 gene transcription. Estrogen levels present in fetal calf serum are sufficient to maintain AIB1 mRNA and protein at low basal levels, and this repression is reversed by the addition of antiestrogens or all-trans retinoic acid. Interestingly, cycloheximide inhibition experiments revealed that secondary protein synthesis was necessary to induce AIB1 expression by antiestrogens and retinoids. Experiments with TGF-β and TGF-β blocking antibodies demonstrated that this growth factor modulates AIB1 expression and showed that the antiestrogen and retinoid induction of AIB1 gene expression is mediated at least in part through TGF-β. These data reveal a mechanism of estrogen-induced down-modulation of the overall hormone sensitivity of cells through feedback inhibition of coactivator gene expression. These data also suggest that antiestrogens can shift the sensitivity of cells to non-estrogenic proliferative signaling by increasing cellular levels of AIB1. This effect may play a role in breast cancer progression and resistance to drug treatment.