In breast cancer, nuclear receptors (NRs) play a prominent role in governing gene expression, have prognostic utility, and are therapeutic targets. We built a regulatory map for 24 NRs, six chromatin state markers, and 14 breast-cancer-associated transcription factors (TFs) that are expressed in the breast cancer cell line MCF-7. The resulting network reveals a highly interconnected regulatory matrix where extensive crosstalk occurs among NRs and other breast -cancer-associated TFs. We show that large numbers of factors are coordinately bound to highly occupied target regions throughout the genome, and these regions are associated with active chromatin state and hormone-responsive gene expression. This network also provides a framework for stratifying and predicting patient outcomes, and we use it to show that the peroxisome proliferator-activated receptor delta binds to a set of genes also regulated by the retinoic acid receptors and whose expression is associated with poor prognosis in breast cancer.

Calorie restriction (CR) improves obesity-related insulin resistance through undefined molecular mechanisms. Insulin receptor substrate (IRS)-1 serine/threonine kinases have been proposed to modulate insulin sensitivity through phosphorylation of IRS proteins. The aim of this study is to test the hypothesis that changes in the activity of IRS1 serine/threonine kinases may underlie the molecular mechanism of CR in improving insulin sensitivity. Obese and lean Zucker rats were subjected to 40% CR or allowed to feed ad libitum (AL) for 20 weeks; body weight and insulin sensitivity were monitored throughout this period. The activity of IRS1 serine/threonine kinases -including JNK, ERK, MTOR/p70 S6K (RPS6KB1 as listed in the MGI Database), glycogen synthase kinase 3b (GSK3B), AMPK (PRKAA1 as listed in the MGI Database), and protein kinase Cq (PRKCQ) in liver tissue extracts was measured by an in vitro kinase assay using various glutathione-S-transferase (GST)-IRS1 fragments as substrates, while phosphorylation of IRS1 and serine kinases was determined by western blotting using phosphospecific antibodies. CR in obese rats significantly reduced body weight and increased insulin sensitivity compared to AL controls. Serine kinase activity toward IRS1 S612 (corresponding to S616 in human IRS1) and IRS1 S632/635 (corresponding to S636/639 in human IRS1) was increased in obese rats compared to lean littermates, and was markedly decreased following CR. Concomitantly, obesity increased and CR decreased the activity of hepatic ERK and p70 S6K against IRS1. The close association between the activity of hepatic ERK and p70 S6K with insulin resistance suggests an important role for ERK and p70 S6K in the development of insulin resistance, presumably via phosphorylation of IRS proteins.

Protein phosphorylation is an important mechanism that controls many cellular activities. Phosphorylation of a given protein is precisely controlled by two opposing biochemical reactions catalyzed by protein kinases and protein phosphatases. How these two opposing processes are coordinated to achieve regulation of protein phosphorylation is unresolved. We have developed a novel experimental approach to directly study protein dephosphorylation in cells. We determined the kinetics of dephosphorylation of insulin receptor substrate-1/2, Akt, and ERK1/2, phosphoproteins involved in insulin receptor signaling. We found that insulin-induced ERK1/2 and Akt kinase activities were completely abolished 10 min after inhibition of the corresponding upstream kinases with PD98059 and LY294002, respectively. In parallel experiments, insulin-induced phosphorylation of Akt, ERK1/2, and insulin receptor substrate-1/2 was decreased and followed similar kinetics. Our findings suggest that these proteins are dephosphorylated by a default mechanism, presumably via constitutively active phosphatases. However, dephosphorylation of these proteins is overcome by activation of protein kinases following stimulation of the insulin receptor. We propose that, during acute insulin stimulation, the kinetics of protein phosphorylation is determined by the interplay between upstream kinase activity and dephosphorylation by default.The reversible phosphorylation of proteins has emerged as a major mechanism for the regulation of cellular signal transduction (1, 2). Disruption of protein phosphorylation/dephosphorylation has been linked to many pathological conditions, including cancer, neurodegeneration, diabetes, cystic fibrosis, asthma, and cardiovascular disease (3-5). Despite the critical importance of protein phosphorylation in signaling and disease, the underlying mechanisms that determine the kinetics of dephosphorylation remain unresolved. Two general models have been proposed to describe these mechanisms. One model posits that the phosphorylation level is determined by constitutively active phosphatases and transiently activated kinases (2, 6). This model was established based on results gathered using potent phosphatase inhibitors such as okadaic acid on intracellular proteins as well as in in vitro cell-free assays. Therefore, although it is an attractive explanation, it may not reflect mechanisms operative in intact living cells. A second model based on studies employing advanced molecular techniques conducted in both animals and genetically engineered cell lines suggests that both kinases and phosphatases are coordinately regulated (3, 7-10). However, most of these studies assessed protein dephosphorylation in the presence of upstream kinase activity; therefore, potential influences from constitutively active phosphatases may have been hidden and thus overlooked.Methods to directly measure protein dephosphorylation in vivo in the absence of upstream kinase activity are lacking. As a result, despite the aforementioned studies, we still ...

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Retinoblastoma gene (Rb1) loss of function mutations is commonly seen in osteosarcomas resulting in poorly differentiated tumors. In addition to cell cycle regulation, Rb1 function has been shown to be important in cell fate determination. It is known that loss of Rb1 function in mesenchymal stem cells inhibits osteoblast differentiation and predisposes them towards adipocytic lineage. Recent studies have suggested a role for Rb1 in cell adhesion but mechanistic details are missing. As cadherin 11 (Cad11) and gap junctional protein connexin43 (Cx43), play important roles in adhesion and communication in osteoblasts, we studied these proteins in MC3T3-E1 mouse calvaria osteoblast cell line. We compared osteoblasts with reduced Rb1 expression to vector-transfected osteoblasts to assess the relationship between Rb1, Cad11, and Cx43. Decreased RNA and protein expression of Cad11 were noted during differentiation of Rb1 deficient osteoblasts when compared to controls. Gap junctional intercellular communication was also studied and found to be reduced with Rb1 loss. Rb1 deficient cells had a higher steady-state level of adipocyte transcription factors and adipocyte markers when compared to control. Interestingly no changes were observed in Cx43 mRNA or protein expression when comparing the two lines. Immunofluorescence analysis demonstrated that a decrease in Cad11 may have affected its co-localization with Cx43. This, in turn, may have affected the proper formation of intercellular channels and thereby communication. These observations suggest that loss of Rb1 affects adhesion and communication in osteoblasts resulting in the expression of an adipocyte phenotype. Citation Format: Elisha Pendleton, Anthony Ketner, Thomas Bodenstine, Nalini Chandar. Role of retinoblastoma gene in maintenance of osteoblast function and communication [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2476.