Treating cancer when pRb and p53 cannot be reactivated.

Activation of oncoproteins and inactivation of tumor suppressors induces tumorigenesis. When these events happen upstream of pRb and p53, cancer therapies may initially succeed and then fail when pRb and p53 are activated and then re-inactivated. Therapies might succeed if they remain effective when pRb and p53 are genetically inactivated.

p27T187A knockin identifies Skp2/Cks1 pocket inhibitors for advanced prostate cancer.

SCF ubiquitinates Thr187-phosphorylated p27 for degradation. Overexpression of Skp2 coupled with underexpression of p27 are frequent characteristics of cancer cells. When the role of SCF-mediated p27 ubiquitination in cancer was specifically tested by p27 Thr187-to-Ala knockin (p27T187A KI), it was found dispensable for Kras-induced lung tumorigenesis but essential for Rb1-deficient pituitary tumorigenesis.

Deletions of Retinoblastoma 1 (Rb1) and Its Repressing Target S Phase Kinase-associated protein 2 (Skp2) Are Synthetic Lethal in Mouse Embryogenesis.

Tumor suppressor pRb represses Skp2, a substrate-recruiting subunit of the SCF(Skp2) ubiquitin ligase. Rb1(+/-) mice incur "two-hit" pituitary tumorigenesis; Skp2(-/-);Rb1(+/-) mice do not. Rb1(-/-) embryos die on embryonic day (E) 14.

Substituting threonine 187 with alanine in p27Kip1 prevents pituitary tumorigenesis by two-hit loss of Rb1 and enhances humoral immunity in old age.

p27Kip1 (p27) is an inhibitor of cyclin-dependent kinases. Inhibiting p27 protein degradation is an actively developing cancer therapy strategy. One focus has been to identify small molecule inhibitors to block recruitment of Thr-187-phosphorylated p27 (p27T187p) to SCF(Skp2/Cks1) ubiquitin ligase.

Antitumor mechanisms when pRb and p53 are genetically inactivated.

pRb and p53 are the two major tumor suppressors. Their inactivation is frequent when cancers develop and their reactivation is rationale of most cancer therapeutics. When pRb and p53 are genetically inactivated, cells irreparably lose the antitumor mechanisms afforded by them.

Skp2 suppresses apoptosis in Rb1-deficient tumours by limiting E2F1 activity.

One mechanism of tumour suppression by pRb is repressing E2F1. Hence, E2f1 deletion diminishes tumorigenesis following Rb1 loss. However, E2F1 promotes both proliferation and apoptosis.

Skp2 is required for survival of aberrantly proliferating Rb1-deficient cells and for tumorigenesis in Rb1+/- mice.

Heterozygosity of the retinoblastoma gene Rb1 elicits tumorigenesis in susceptible tissues following spontaneous loss of the remaining functional allele. Inactivation of previously studied retinoblastoma protein (pRb) targets partially inhibited tumorigenesis in Rb1(+/-) mice. Here we report that inactivation of pRb target Skp2 (refs.

An AR-Skp2 pathway for proliferation of androgen-dependent prostate-cancer cells.

Androgen-androgen-receptor (androgen-AR) signaling in normal prostate epithelium promotes terminal luminal epithelial cell differentiation. In androgen-dependent prostate-cancer cells, androgen-AR signaling gains the ability to promote both differentiation and proliferation. How this signaling promotes proliferation of androgen-dependent prostate-cancer cells and its relationship with the differentiation-promoting functions of the AR are important issues regarding the biology of androgen-dependent prostate-cancer cells.

Peripheral blood mononuclear and tumor cell pharmacodynamics of the novel epothilone B analogue, ixabepilone.

Background: We previously demonstrated that peak microtubule bundle formation (MBF) in peripheral blood mononuclear cells (PBMCs) occurs at the end of drug infusion and correlates with drug pharmacokinetics (PK). In the current study, a new expanded evaluation of drug target effect was undertaken.

Patients And Methods: Patients with advanced solid malignancies were treated with ixabepilone 40 mg/m2 administered as a 1-h i.

Skp2 contains a novel cyclin A binding domain that directly protects cyclin A from inhibition by p27Kip1.

Skp2 is well known as the F-box protein of the SCF(Skp2) x Roc1 complex targeting p27 for ubiquitylation. Skp2 also forms complexes with cyclin A, which is particularly abundant in cancer cells due to frequent Skp2 overexpression, but the mechanism and significance of this interaction remain unknown. Here, we report that Skp2-cyclin A interaction is mediated by novel interaction sequences on both Skp2 and cyclin A, distinguishing it from the well known RXL-hydrophobic patch interaction between cyclins and cyclin-binding proteins.

Tumour suppressor retinoblastoma protein Rb: a transcriptional regulator.

Rb was the first tumour suppressor identified through human genetic studies. The most significant achievement after almost twenty years since its cloning is the revelation that Rb possesses functions of a transcription regulator. Rb serves as a transducer between the cell cycle machinery and promoter-specific transcription factors.

Using kinetic studies to uncover new Rb functions in inhibiting cell cycle progression.

A well-established biological activity of the tumor suppressor Rb is blocking G1/S cell cycle progression when reintroduced into cultured Rb-deficient tumor cells. The best understood molecular mechanism underlying this function is that Rb binds the transcription factor E2F to repress expression of S phase genes such as cyclins E and A. A recent kinetic study of this model further revealed that Rb represses Skp2 to stabilize p27, which inhibits the kinase activity associated with cyclins E and A before the decline in their protein levels, to arrest the cell cycle.

An Rb-Skp2-p27 pathway mediates acute cell cycle inhibition by Rb and is retained in a partial-penetrance Rb mutant.

It is believed that Rb blocks G1-S transition by inhibiting expression of E2F regulated genes. Here, we report that the effects of E2F repression lag behind the onset of G1 cell cycle arrest in timed Rb reexpression experiments. In comparison, kinase inhibitor p27Kip1 protein accumulates with a faster kinetics.

Induction of androgen receptor-dependent apoptosis in prostate cancer cells by the retinoblastoma protein.

Re-expression of a tumor suppressor in tumor cells that lack it is an effective way to study its functional activities. However, because tumor cells contain multiple mutations, tumor suppressor functions that are dependent on (an)other regulators are unlikely to be identified by its re-expression alone if the other regulators are also mutated. In this study, we show that re-expression of retinoblastoma (RB) together with the androgen receptor (AR) in RB- and AR-deficient prostate cancer DU-145 cells resulted in an apoptotic activity, acting through the mitochondria damage-initiated caspase activation pathway, which was not present when RB, or the AR, was re-expressed alone.

Phase II clinical trial of parenteral hydroxyurea in combination with fluorouracil, interferon and filgrastim in the treatment of advanced pancreatic, gastric and neuroendocrine tumors.

Purpose: Combined inhibition of ribonucleotide reductase (RR) and thymidylate synthase (TS), the enzymes responsible for a balanced supply of nucleotides for DNA synthesis, has been shown to induce synergistic antiproliferative effects in vitro. In the clinic, prolonged infusion of the RR inhibitor, hydroxyurea (HU), may be more effective than bolus or oral administration of drug. The purpose of the current study was to determine whether dose intensification of parenteral hydroxyurea in combination with fluorouracil could enhance the response rates of the combination against refractory upper gastrointestinal malignancies.

Cyclin D1 governs adhesion and motility of macrophages.

The cyclin D1 gene encodes the regulatory subunit of a holoenzyme that phosphorylates and inactivates the retinoblastoma protein, thereby promoting cell-cycle progression. Cyclin D1 is overexpressed in hematopoetic and epithelial malignancies correlating with poor prognosis and metastasis in several cancer types. Because tumor-associated macrophages have been shown to enhance malignant progression and metastasis, and cyclin D1-deficient mice are resistant to oncogene-induced malignancies, we investigated the function of cyclin D1-/- bone marrow-derived macrophages.

Randomized phase II trial of embolization therapy versus chemoembolization therapy in previously treated patients with colorectal carcinoma metastatic to the liver.

Locoregional therapies are useful in treating patients with colorectal cancer metastatic to the liver. A prospective randomized phase II trial of hepatic artery embolization versus hepatic artery chemoembolization was conducted to evaluate the response rates and toxicities of these therapies in the second-line setting. Patients were required to have biopsy-proven adenocarcinoma of the colon or rectum metastatic to the liver, with the liver as the sole or predominant site of metastatic disease.

ErbB-2-induced mammary tumor growth: the role of cyclin D1 and p27Kip1.

The neu (c-erbB-2, HER2) proto-oncogene encodes a receptor tyrosine kinase that is a member of an important growth factor receptor family which includes the epidermal growth factor receptor (EGFR, ErbB1), ErbB3 and ErbB4. The neu is found over-expressed in 20-30% of human breast tumors. The c-erbB-2 is sufficient for the induction of mammary tumorigenesis in transgenic mice and the pathology of these mammary tumors strongly resembles human breast cancer.

Microvascular hyperpermeability in caveolin-1 (-/-) knock-out mice. Treatment with a specific nitric-oxide synthase inhibitor, L-NAME, restores normal microvascular permeability in Cav-1 null mice.

Microvascular permeability is mediated by (i) the caveolar transcytosis of molecules across endothelial cells and (ii) the paracellular movement of ions and nutrients. Recently, we derived Cav-1 (-/-) knock-out mice using standard homologous recombination techniques. These mice are viable but show a loss of endothelial cell caveolae and striking defects in caveolae-mediated endocytosis.

Androgen receptor acetylation governs trans activation and MEKK1-induced apoptosis without affecting in vitro sumoylation and trans-repression function.

The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both trans repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation.

Prolactin negatively regulates caveolin-1 gene expression in the mammary gland during lactation, via a Ras-dependent mechanism.

Caveolin-1 is a 22-kDa integral membrane protein that has been suggested to function as a negative regulator of mitogen-stimulated proliferation in a variety of cell types, including mammary epithelial cells. Because much of our insight into caveolin-1 function has come from the study of human breast tumor-derived cell lines in culture, the normal physiological regulators of caveolin-1 expression in the mammary gland remain unknown. Here, we examine caveolin-1 expression in mice at different stages of mammary gland development.

Inducible transgenics. New lessons on events governing the induction and commitment in mammary tumorigenesis.

Breast cancer arises from multiple genetic events that together contribute to the established, irreversible malignant phenotype. The development of inducible tissue-specific transgenics has allowed a careful dissection of the events required for induction and subsequent maintenance of tumorigenesis. Mammary gland targeted expression of oncogenic Ras or c-Myc is sufficient for the induction of mammary gland tumorigenesis in the rodent, and when overexpressed together the rate of tumor onset is substantially enhanced.

Cyclin D1 binds the androgen receptor and regulates hormone-dependent signaling in a p300/CBP-associated factor (P/CAF)-dependent manner.

The androgen receptor (AR) is a ligand-regulated member of the nuclear receptor superfamily. The cyclin D1 gene product, which encodes the regulatory subunit of holoenzymes that phosphorylate the retinoblastoma protein (pRB), promotes cellular proliferation and inhibits cellular differentiation in several different cell types. Herein the cyclin D1 gene product inhibited ligand-induced AR- enhancer function through a pRB-independent mechanism requiring the cyclin D1 carboxyl terminus.