Elizabeth R. Rayburn

Wnt / β-Catenin Signaling Pathway as Novel Cancer Drug Targets

Wnt proteins are a large family of secreted glycoproteins. Wnt proteins bind to the Frizzled receptors and LRP5/6 co-receptors, and through stabilizing the critical mediator beta-catenin, initiate a complex signaling cascade that plays an important role in regulating cell proliferation and differentiation. Deregulation of the canonical Wnt/beta-catenin signaling pathway, mostly by inactivating mutations of the APC tumor suppressor, or oncogenic mutations of beta-catenin, has been implicated in colorectal tumorigenesis. Although oncogenic mutations of beta-catenin have only been discovered in a small fraction of non-colon cancers, elevated levels of beta-catenin protein, a hallmark of activated canonical Wnt pathway, have been observed in most common forms of human malignancies, indicating that activation of this pathway may play an important role in tumor development. Over the past 15 years, our understanding of this signaling pathway has significantly improved with the identification of key regulatory proteins and the important downstream targets of beta-catenin/Tcf transactivation complex. Given the fact that Wnt/beta-catenin signaling is tightly regulated at multiple cellular levels, the pathway itself offers ample targeting nodal points for cancer drug development. In this review, we discuss some of the strategies that are being used or can be explored to target key components of the Wnt/beta-catenin signaling pathway in rational cancer drug discovery.

Ribosomal protein S7 as a novel modulator of p53–MDM2 interaction: binding to MDM2, stabilization of p53 protein, and activation of p53 function

As a major negative regulator of p53, the MDM2 oncogene plays an important role in carcinogenesis and tumor progression. MDM2 promotes p53 proteasomal degradation and negatively regulates p53 function. The mechanisms by which the MDM2–p53 interaction is regulated are not fully understood, although several MDM2-interacting molecules have recently been identified. To search for novel MDM2-binding partners, we screened a human prostate cDNA library by the yeast two-hybrid assay using full-length MDM2 protein as the bait. Among the candidate proteins, ribosomal protein S7 was identified and confirmed as a novel MDM2–interacting protein. Herein, we demonstrate that S7 binds to MDM2, in vitro and in vivo, and that the interaction between MDM2 and S7 leads to modulation of MDM2-p53 binding by forming a ternary complex among MDM2, p53 and S7. This results in the stabilization of p53 protein through abrogation of MDM2-mediated p53 ubiquitination. Consequently, S7 overexpression increases p53 transactivational activities, induces apoptosis, and inhibits cell proliferation. The identification of S7 as a novel MDM2-interacting partner contributes to elucidation of the complex regulation of the MDM2–p53 interaction and has implications in cancer prevention and therapy.

MDM2 and Human Malignancies: Expression, Clinical Pathology, Prognostic Markers, and Implications for Chemotherapy

The human homologue of the mouse double minute 2 (MDM2) oncogene is overexpressed in more than forty different types of malignancies, including solid tumors, sarcomas and leukemias. Because of its prevalent expression and its interactions with p53 and other signaling molecules, MDM2 plays a central role in cancer development and progression. The expression of this oncoprotein is being studied by researchers world-wide, and the amount of data published about it is increasing exponentially. Although there are some conflicting data about the effects of MDM2 expression in individual cancers, the overall evidence is convincing, indicating that increased MDM2 expression is related to a worse clinical prognosis. There is an increased likelihood of distant metastases, as well as a decreased response to therapeutic intervention in MDM2-positive cancers. MDM2 may also serve as a diagnostic marker, not only for cancer stage, but to differentiate between similar cancers. MDM2 may also be associated with drug resistance in cancer chemotherapy. These findings make studying the oncoprotein necessary to aid in our understanding of cancer development, to identify novel cancer drug targets, and to increase the efficacy of cancer therapy.

Antisense, RNAi, and Gene Silencing Strategies for Therapy: Mission Possible or Impossible?

Antisense oligonucleotides can regulate gene expression in living cells. As such, they regulate cell function and division, and can modulate cellular responses to internal and external stresses and stimuli. Although encouraging results from preclinical and clinical studies have been obtained and significant progress has been made in developing these agents as drugs, they are not yet recognized as effective therapeutics. Several major hurdles remain to be overcome, including problems with efficacy, off-target effects, delivery and side effects. The lessons learned from antisense drug development can help in the development of other oligonucleotide-based therapeutics such as CpG oligonucleotides, RNAi and miRNA.

Stabilization of E2F1 protein by MDM2 through the E2F1 ubiquitination pathway

Although previous studies suggested that the tumorigenicity of mouse double minute 2 (MDM2) was due to its negative regulation of p53, the p53-independent interactions may be equally as important. During recent studies utilizing MDM2 inhibitors, we noted that E2F transcription factor 1 (E2F1) was downregulated upon inhibition of MDM2, regardless of the p53 status of the cancer. The present study investigated the mechanisms responsible for the MDM2-mediated increase in E2F1 expression. MDM2 prolongs the half-life of the E2F1 protein by inhibiting its ubiquitination. MDM2 displaces SCFSKP2, the E2F1 E3 ligase. Direct binding between MDM2 and E2F1 is necessary for the negative effects of MDM2 on E2F1 ubiquitination, and deletion of the MDM2 nuclear localization signal does not result in loss of the ability to increase the E2F1 protein level. The downregulation of E2F1 upon MDM2 inhibition was not due to either pRB or p14Arf. In addition, E2F1 was responsible for at least part of the inhibition of cell proliferation induced by MDM2 knockdown. In conclusion, the present study provides evidence that stabilization of the E2F1 protein is likely another p53-independent component of MDM2-mediated tumorigenesis. More knowledge about the MDM2–E2F1 interaction may be helpful in developing novel anticancer therapies.

Experimental therapy of prostate cancer with novel natural product anti-cancer ginsenosides.

Ginseng and its components exert various biological effects, including antioxidant, anti‐carcinogenic, anti‐mutagenic, and anti‐tumor activity, and recent research has focused on their value in human cancer prevention and treatment. We recently isolated 25‐hydroxyprotopanaxadiol (25‐OH‐PPD) and 25‐hydroxyprotopanaxatriol (25‐OH‐PPT) from Panax ginseng and evaluated their anti‐cancer activity in vitro.

Novel ginsenosides 25-OH-PPD and 25-OCH3-PPD as experimental therapy for pancreatic cancer: anticancer activity and mechanisms of action

We recently isolated and characterized two novel ginsenosides, 25-OH-PPD and 25-OCH(3)-PPD. We investigated whether these ginsenosides could represent safe and effective therapeutic agents for pancreatic cancer. In vitro and in vivo studies demonstrated that both compounds inhibited proliferation, caused cell cycle arrest, and induced apoptosis. They also both inhibited the growth of xenograft tumors without any host toxicity. Preliminary investigations into the mechanisms of action of the compounds suggest that their effects may be partially mediated by their inhibition of the MDM2 oncogene and related pathways. The data presented here support further evaluation of the ginsenosides for pancreatic cancer therapy.

RYBP stabilizes p53 by modulating MDM2.

The mouse double minute 2 (MDM2)–p53 interaction regulates the activity of p53 and is a potential target for human cancer therapy. Here, we report that RYBP (RING1‐ and YY1‐binding protein), a member of the polycomb group (PcG), interacts with MDM2 and decreases MDM2‐mediated p53 ubiquitination, leading to stabilization of p53 and an increase in p53 activity. RYBP induces cell‐cycle arrest and is involved in the p53 response to DNA damage. Expression of RYBP is decreased in human cancer tissues compared with adjacent normal tissues. These results show that RYBP is a new regulator of the MDM2–p53 loop and that it has tumour suppressor activity.

Anti-lung cancer effects of novel ginsenoside 25-OCH3-PPD

20(S)-25-methoxyl-dammarane-3beta, 12beta, 20-triol (25-OCH(3)-PPD), a newly identified natural product from Panax notoginseng, exhibits activity against a variety of cancer cells. Herein, we report the effects of this compound on human A549, H358, and H838 lung cancer cells, and compare these effects with a control lung epithelial cell line, BEAS-2B. 25-OCH(3)-PPD decreased survival, inhibited proliferation, and induced apoptosis and G1 cell cycle arrest in the lung cancer cell lines. The P. notoginseng compound also decreased the levels of proteins associated with cell proliferation and cell survival. Moreover, 25-OCH(3)-PPD inhibited the growth of A549 lung cancer xenograft tumors. 25-OCH(3)-PPD demonstrated low toxicity to non-cancer cells, and no observable toxicity was seen when the compound was administered to animals. In conclusion, our preclinical data indicate that 25-OCH(3)-PPD is a potential therapeutic agent in vitro and in vivo, and further preclinical and clinical development of this agent for lung cancer is warranted.

Chemotherapy and chemosensitization of non-small cell lung cancer with a novel immunomodulatory oligonucleotide targeting Toll-like receptor 9.

Lung cancer is a leading cause of death world-wide and the long-term survival rate for lung cancer patients is one of the lowest for any cancer. New therapies are urgently needed. The present study was designed to evaluate an immunomodulatory oligonucleotide as a novel type of therapy for lung cancer. The in vivo effects of the immunomodulatory oligonucleotides were determined in four tumor models derived from human non–small cell lung cancer (NSCLC) cell lines (A549, H1299, H358, and H520), administered alone or in combination with conventional chemotherapeutic agents used to treat lung cancer. The in vitro effects of the immunomodulatory oligonucleotide on the growth, apoptosis, and proliferation of NSCLC cells were also determined. We also examined NSCLC cells for expression of Toll-like receptor 9 (TLR9), the receptor for the immunomodulatory oligonucleotide. We showed several important findings: (a) treatment with the immunomodulatory oligonucleotide led to potent antitumor effects, inhibiting tumor growth by at least 60% in all four in vivo models; (b) combination with the immunomodulatory oligonucleotide led to enhanced effects following treatment with gemcitabine or Alimta; (c) the immunomodulatory oligonucleotide increased apoptosis, decreased proliferation, and decreased survival in A549 cells in vitro; and (d) both TLR9 mRNA and protein were expressed in NSCLC cells. The immunomodulatory oligonucleotide has potent antitumor effects as monotherapy and in combination with conventional chemotherapeutic agents, and may act directly on NSCLC cells via TLR9. The present study provides a rationale for developing the immunomodulatory oligonucleotide for lung cancer therapy. [Mol Cancer Ther 2006;5(6):1585–92]