No abstract

SummaryAMER1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane

RAP46 is a eukaryotic cochaperone that associates with several proteins, including the heat shock protein hsp70/hsc70 and the glucocorticoid receptor (GR). Here we show a downregulation of GR-mediated transactivation by RAP46 via a mechanism independent of a cytoplasmic action of this cochaperone. We demonstrate a specific cytoplasmic–nuclear recruitment of RAP46 by the liganded GR that results in inhibition of the transactivation function of the receptor. A repeated sequence motif [EEX4]8 at the NH2 terminus of RAP46 or BAG-1L, a larger isoform of RAP46, is responsible for this downregulation of GR activity. BAG-1, a shorter isoform with only a duplication of the [EEX4] sequence, does not inhibit GR activity. The [EEX4]8 motif, when linked to an otherwise unrelated protein, abrogated the inhibitory action of endogenous RAP46 on GR-mediated transactivation. The nuclear effects of RAP46 and BAG-1L are specific since GR-mediated inhibition of AP-1 activity was not affected. These studies identify the [EEX4]8 sequence as a signature motif for inhibition of GR-mediated transactivation and demonstrate a specific nuclear action of a eukaryotic cochaperone in the regulation of GR activity.

Matrix metalloproteinases belong to a family of structurally related enzymes that plays important role in tissue morphogenesis, differentiation, and wound healing. Their expression is negatively regulated by several members of the steroid hormone receptor family. This is thought to occur through interaction of the steroid receptors with the transcription factor AP-1 that is otherwise required for positive regulation. Here, we demonstrate that AP-1 is not always a target for downregulation of expression of matrix metalloproteinases by steroid receptors. Androgen receptor negatively regulates matrix metalloproteinase-1 expression not through AP-1 but through a family of Ets-related transcription factors that are also required for positive regulation. This negative regulation is specific for the androgen receptor. It does not require the DNA binding activity but needs amino-terminal sequences of the receptor. These results identify a novel regulatory pathway for negative regulation utilized by a member of the steroid hormone receptor family for down-regulating the expression of matrix metalloproteinases.Matrix metalloproteinases (MMPs) 1 are composed of a family of metal-ion requiring enzymes that degrades components of the extracellular matrix. They can be subdivided into several classes on the basis of their substrate specificity. These classes include the interstitial collagenases, which degrade type I, II, and III collagens, the type IV collagenases, which degrade basement membrane collagens type IV and V, the stromelysins, which degrade proteoglycans, fibronectin, and laminin, and matrilysin, which has a wide range of substrates including proteoglycans, fibronectin, gelatin, and elastin (for reviews see Refs. 1 and 2).Regulated expression of MMPs accompanies changes in tissue organization in normal growth processes and pathological conditions. These are most evident in organogenesis, neovascularization, tissue repair, inflammation, arthritis, tumor invasion, and metastasis (1). In these processes, positive and negative regulation of expression of MMP genes by a variety of growth factors, cytokines, oncogenes, tumor promoters, and steroid hormones are required (2, 3). The steroid hormones in particular, negatively regulate the expression of the MMPs through the action of their corresponding receptors (4, 5).Glucocorticoid receptor (GR) exerts its negative regulation by interacting with the transcription factor AP-1, an important regulator of expression of several MMP genes (4 -8). Progesterone acting through its receptor in stromal cells of the endometrium down-regulates the levels of MMP mRNAs of the stromelysin family through a mechanism that is not yet known (9, 10). In the epithelial cells of the endometrium, progesterone down-regulates the expression of matrilysin through the release of transforming growth factor ␤1 from the stromal cells (10). Although androgen receptor (AR) represses the expression of several genes (11-17), it is not clear how this is effected nor is it clear whether MMPs belong to the...

RAP46 was first identified by its ability to bind the glucocorticoid receptor. It has since been reported to bind several cellular proteins, including the anti-apoptotic protein Bcl-2, but the biological significance of these interactions is unknown. Here we show that RAP46 binds the hinge region of the glucocorticoid receptor and inhibits DNA binding and transactivation by the receptor. We further show that overexpression of RAP46 in mouse thymoma S49.1 cells inhibits glucocorticoid-induced apoptosis. Conversely, glucocorticoid-induced apoptosis and transactivation were enhanced after treating S49.1 cells with the immunosuppressant rapamycin, which down-regulates cellular levels of BAG-1, the mouse homolog of RAP46. The effect of rapamycin can, however, be overcome by overexpression of RAP46. These results together identify RAP46 as a protein that controls glucocorticoid-induced apoptosis through its negative regulatory action on the transactivation property of the glucocorticoid receptor.

Mutations in the p53 tumor suppressor gene are the most frequent genetic alteration in cancer and are often associated with progression from benign to invasive stages with metastatic potential. Mutations inactivate tumor suppression by p53, and some endow the protein with novel gain of function (GOF) properties that actively promote tumor progression and metastasis. By comparative gene expression profiling of p53-mutated and p53-depleted cancer cells, we identified ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) as a mutant p53 target gene, which functions as a uridine 5′-diphosphatase (UDPase) in the endoplasmic reticulum (ER) to promote the folding of N-glycosylated membrane proteins. A comprehensive pan-cancer analysis revealed a highly significant correlation between p53 GOF mutations and ENTPD5 expression. Mechanistically, mutp53 is recruited by Sp1 to the ENTPD5 core promoter to induce its expression. We show ENTPD5 to be a mediator of mutant p53 GOF activity in clonogenic growth, architectural tissue remodeling, migration, invasion, and lung colonization in an experimental metastasis mouse model. Our study reveals folding of N-glycosylated membrane proteins in the ER as a mechanism underlying the metastatic progression of tumors with mutp53 that could provide new possibilities for cancer treatment.M utations in the TP53 tumor suppressor gene are the most frequent genetic alterations in human cancer and commonly compromise the gene's tumor suppressor activity. p53-knockout mice succumb to tumors very early in life, arguing that the loss of function associated with p53 mutations is sufficient on its own to explain the high mutation frequency observed in patients with cancer (1). However, in striking contrast to mutations in other tumor suppressor genes, the vast majority of TP53 gene alterations in patients with cancer neither ablate p53 expression nor produce unstable or truncated proteins. Instead, p53 mutations are mostly missense mutations resulting in expression of mutant p53 (mutp53) proteins with only single-amino acid substitutions that accumulate to steady-state levels greatly exceeding those of wild-type p53 (wtp53) in normal tissues. Immunohistochemical positivity for p53 is therefore considered a diagnostic marker for the presence of a TP53 mutation (2). The high prevalence of missense mutations suggests a selective advantage during cancer progression, so it was hypothesized early on in p53 research that p53 mutations are neomorphic and endow the mutp53 protein with novel oncogenic functions that actively promote cancer progression and therapy resistance (2). These oncogenic properties are generally referred to as the mutp53 gain of function (GOF).Over the years, substantial experimental evidence for mutp53 GOF has accumulated (3-5). For example, mice expressing cancer-associated p53 hot spot mutations from the endogenous Trp53 gene locus are remarkably different from p53-deficient mice: tumorigenesis is accelerated, and the spectrum of tumors is shifted toward carcinomas and more meta...

Biallelic mutation of the ADENOMATOUS POLYPOSIS COLI (APC) gene is a hallmark of sporadic colorectal cancer and colorectal, duodenal and desmoid tumours that develop in familial adenomatous polyposis (FAP) patients. The mutations affecting both APC alleles are interdependent, the position of the first APC mutation determining where the second hit will occur. This results in a complex pattern of mutation distribution in the APC sequence that translates into the stabilization of beta-catenin that in turn feeds the affected cells with a permanent mitogenic signal. We describe here a new APC domain, the beta-catenin inhibitory domain (CID) of APC located between the second and third 20 amino acid repeats and therefore present in many truncated APC products found in human tumours. In truncated APC, the CID is absolutely necessary to down-regulate the transcriptional activity and the level of beta-catenin, even when an axin/conductin binding site is present. The activity of the CID is dramatically reduced in several colon cancer cell lines and can be inhibited by shorter truncated APC lacking the CID. The CID is a direct target of the selective pressure acting on APC during tumourigenesis. It explains the interdependence of both APC mutations, not only in colorectal but also in duodenal and desmoid tumours.

Cancer cells have a characteristic metabolism, mostly caused by alterations in signal transduction networks rather than mutations in metabolic enzymes. For metabolic drugs to be cancer-selective, signaling alterations need to be identified that confer a druggable vulnerability. Here, we demonstrate that many tumor cells with an acquired cancer drug resistance exhibit increased sensitivity to mechanistically distinct inhibitors of cancer metabolism. We demonstrate that this metabolic vulnerability is driven by mTORC1, which promotes resistance to chemotherapy and targeted cancer drugs, but simultaneously suppresses autophagy. We show that autophagy is essential for tumor cells to cope with therapeutic perturbation of metabolism and that mTORC1-mediated suppression of autophagy is required and sufficient for generating a metabolic vulnerability leading to energy crisis and apoptosis. Our study links mTOR-induced cancer drug resistance to autophagy defects as a cause of a metabolic liability and opens a therapeutic window for the treatment of otherwise therapy-refractory tumor patients.