ABSTRACT
BACKGROUND: The mitogen-activated protein kinases (MAPKs) regulate cell growth, differentiation, and stress responses, and many critical signaling pathways are subject to cross-regulation by MAPK signaling. Previous studies have yielded evidence of cross-talk between the MAPK pathways and androgen receptor (AR) signaling, which plays a critical role in growth control of both normal prostate and prostate cancer (PCa). Objective of this study was to evaluate the expression of MAPK-like protein nemo-like kinase (NLK) in PCa and its effects on AR-mediated transcription. METHODS: Real-time PCR and IHC were used to evaluate levels of NLK in prostatic samples. Effects of over-expression of NLK on apoptosis and proliferation were determined using Western blot and flow cytometry. Effects on AR signaling were evaluated using over-expression and knockdown of NLK in PCa cells in combination with PCR, Western blotting and reporter assays. RESULTS: Our results show that the expression of NLK is decreased in PCa metastases in comparison to normal prostate epithelium and primary PCa. Our results also show that over-expression of NLK resulted in induction of apoptosis, which was more pronounced in AR-expressing LNCaP versus AR-negative PC-3 cells. Higher levels of NLK decreased levels of AR mRNA and protein as well as inhibited AR-mediated transcription. CONCLUSIONS: NLK expression is altered during PCa progression and it is involved in regulation of AR signaling in these cells. A deeper understanding of the roles of NLK in regulation of AR-mediated transcription and control of PCa progression may point the way to new modes of therapeutic intervention in this disease.
Subject(s)
Apoptosis/physiology , Intracellular Signaling Peptides and Proteins/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Protein Serine-Threonine Kinases/metabolism , Receptors, Androgen/metabolism , Animals , CHO Cells , Cell Division/physiology , Cricetinae , Cricetulus , Gene Expression Regulation, Enzymologic , Gene Expression Regulation, Neoplastic , Humans , Intracellular Signaling Peptides and Proteins/genetics , MAP Kinase Signaling System/physiology , Male , Protein Serine-Threonine Kinases/genetics , RNA, Messenger/metabolism , Receptors, Androgen/genetics , Transcription, Genetic/physiology , TransfectionABSTRACT
Morbidity and mortality of advanced prostate cancer (CaP) are associated with bone metastases. Bone metastases of prostate cancer stimulate new bone formation, resulting in osteoblastic metastases. Very little is known about how migrating CaP cells settle in the bone tissues and induce bone lesions, but recent studies have suggested that factors known as Wnts, which are expressed by CaP, can promote establishment of CaP cells in the bone microenvironment and stimulate bone formation. Signaling via the Wnt pathway is important in embryogenesis and development, and has also been shown to be important in cancer development and progression. CaP cells exhibit increased Wnt signaling vs. normal prostate epithelium, and Wnt has recently been shown to play a central role in bone development, regulating factors critical in control of osteoblast and osteoclast differentiation. In this review we have focused on the roles of Wnt signaling in CaP, bone, and CaP bone metastases.
Subject(s)
Bone Neoplasms/secondary , Bone and Bones/metabolism , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Wnt Proteins/physiology , Humans , Male , Signal Transduction , Wnt Proteins/geneticsABSTRACT
Molecular modeling studies led to the identification of LFM-A13 (alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide) as a potent inhibitor of Polo-like kinase (Plk). LFM-A13 inhibited recombinant purified Plx1, the Xenopus homolog of Plk, in a concentration-dependent fashion, as measured by autophosphorylation and phosphorylation of a substrate Cdc25 peptide. LFM-A13 was a selective Plk inhibitor. While the human PLK3 kinase was also inhibited by LFM-A13 with an IC(50) value of 61 microM, none of the 7 other serine/threonine kinases, including CDK1, CDK2, CDK3, CHK1, IKK, MAPK1 or SAPK2a, none of the 10 tyrosine kinases, including ABL, BRK, BMX, c-KIT, FYN, IGF1R, PDGFR, JAK2, MET, or YES, or the lipid kinase PI3Kgamma were inhibited (IC(50) values >200-500 microM). The mode of Plk3 inhibition by LFM-A13 was competitive with respect to ATP with a K(i) value of 7.2 microM from Dixon plots. LFM-A13 blocked the cell division in a zebrafish (ZF) embryo model at the 16-cell stage of the embryonic development followed by total cell fusion and lysis. LFM-A13 prevented bipolar mitotic spindle assembly in human breast cancer cells and glioblastoma cells and when microinjected into living epithelial cells at the prometaphase stage of cell division, it caused a total mitotic arrest. Notably, LFM-A13-delayed tumor progression in the MMTV/neu transgenic mouse model of HER2 positive breast cancer at least as effectively as paclitaxel and gemcitabine. LFM-A13 showed a favorable toxicity profile in mice and rats. In particular there was no evidence of hematologic toxicity as documented by peripheral blood counts and bone marrow examinations. These results establish LFM-A13 as a small molecule inhibitor of Plk with in vitro and in vivo anti-proliferative activity against human breast cancer.
Subject(s)
Amides/chemical synthesis , Amides/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacology , Breast Neoplasms/drug therapy , Cell Cycle Proteins/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Nitriles/chemical synthesis , Nitriles/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins/antagonists & inhibitors , Amides/toxicity , Animals , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , DNA, Recombinant/chemical synthesis , DNA, Recombinant/pharmacology , Disease Progression , Drug Screening Assays, Antitumor , Female , Fluorescence , Humans , Mice , Mice, Inbred BALB C , Mice, Transgenic , Microinjections , Microscopy, Confocal , Models, Molecular , Nitriles/toxicity , Rats , Zebrafish , Polo-Like Kinase 1ABSTRACT
Inherited and somatic mutations in the adenomatous polyposis coli occur in most colon cancers, leading to activation of beta-catenin-responsive genes. To identify small molecule antagonists of this pathway, we challenged transformed colorectal cells with a secondary structure-templated chemical library, looking for compounds that inhibit a beta-catenin-responsive reporter. We identified ICG-001, a small molecule that down-regulates beta-catenin/T cell factor signaling by specifically binding to cyclic AMP response element-binding protein. ICG-001 selectively induces apoptosis in transformed cells but not in normal colon cells, reduces in vitro growth of colon carcinoma cells, and is efficacious in the Min mouse and nude mouse xenograft models of colon cancer.