ABSTRACT
Histone deacetylase inhibitors (HDACi) can elicit a range of biological responses that affect tumor growth and survival, including inhibition of cell cycle progression, induction of tumor cell-selective apoptosis, suppression of angiogenesis, and modulation of immune responses, and show promising activity against hematological malignancies in clinical trials. Using the Emu-myc model of B cell lymphoma, we screened tumors with defined genetic alterations in apoptotic pathways for therapeutic responsiveness to the HDACi vorinostat. We demonstrated a direct correlation between induction of tumor cell apoptosis in vivo and therapeutic efficacy. Vorinostat did not require p53 activity or a functional death receptor pathway to kill Emu-myc lymphomas and mediate a therapeutic response but depended on activation of the intrinsic apoptotic pathway with the proapoptotic BH3-only proteins Bid and Bim playing an important role. Our studies provide important information regarding the mechanisms of action of HDACi that have broad implications regarding stratification of patients receiving HDACi therapy alone or in combination with other anticancer agents.
Subject(s)
Apoptosis/drug effects , Enzyme Inhibitors/therapeutic use , Histone Deacetylase Inhibitors , Hydroxamic Acids/therapeutic use , Lymphoma, B-Cell/drug therapy , Animals , Apoptosis Regulatory Proteins/physiology , BH3 Interacting Domain Death Agonist Protein/physiology , Bcl-2-Like Protein 11 , Genes, myc , Membrane Proteins/physiology , Mice , Mice, Inbred C57BL , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-bcl-2/physiology , TNF-Related Apoptosis-Inducing Ligand/physiology , Tumor Suppressor Protein p53/physiology , Vorinostat , bcl-X Protein/physiologyABSTRACT
Ets proteins are transcription factors, which share a unique DNA binding domain, the Ets domain. Some members of the Ets family are implicated in tumorigenesis. Ets1, the founder of the Ets family, is predominantly expressed in invasive tumors and able to activate certain genes encoding ECM-degrading proteases. We used RNA-interference in combination with DNA chip analysis to identify Ets1-regulated genes in MDA-MB-231 breast cancer cells. Of the Ets1-responsive proteases, matrix metalloproteases MMP1 and MMP9, but not MMP3 or uPA, showed reduced RNA levels when endogenous Ets1 expression was suppressed. These data suggest that Ets1 regulates only a certain subset of ECM-degrading proteases. How Ets1 is regulated in invasive breast cancer cells is unknown. The observations that protein kinase C inhibitors abrogated Ets1 expression and that protein kinase C was able to increase Ets1-dependent transcription imply that protein kinase C is a potential regulator of Ets1 activity in breast cancer cells.
Subject(s)
Breast Neoplasms/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogenes , Transcription Factors/genetics , Breast Neoplasms/enzymology , Breast Neoplasms/pathology , Disease Progression , Female , Genetic Markers , Humans , Protein Kinase C/metabolism , Proto-Oncogene Mas , Proto-Oncogene Protein c-ets-1 , Proto-Oncogene Proteins c-etsABSTRACT
The majority of breast cancers metastasizing to bone secrete parathyroid hormone-related protein (PTHrP). PTHrP induces local osteolysis that leads to activation of bone matrix-borne transforming growth factor beta (TGF beta). In turn, TGF beta stimulates PTHrP expression and, thereby, accelerates bone destruction. We studied the mechanism by which TGF beta activates PTHrP in invasive MDA-MB-231 breast cancer cells. We demonstrate that TGF beta 1 up-regulates specifically the level of PTHrP P3 promoter-derived RNA in an actinomycin D-sensitive fashion. Transient transfection studies revealed that TGF beta 1 and its effector Smad3 are able to activate the P3 promoter. This effect depended upon an AGAC box and a previously described Ets binding site. Addition of Ets1 greatly enhanced the Smad3/TGF beta-mediated activation. Ets2 had also some effect, whereas other Ets proteins, Elf-1, Ese-1, and Erf-1, failed to cooperate with Smad3. In comparison, Ets1 did not increase Smad3/TGF beta-induced stimulation of the TGF beta-responsive plasminogen activator inhibitor 1 (PAI-1) promoter. Smad3 and Smad4 were able to specifically interact with the PTHrP P3-AGAC box and to bind to the P3 promoter together with Ets1. Inhibition of endogenous Ets1 expression by calphostin C abrogated TGF beta-induced up-regulation of the P3 transcript, whereas it did not affect the TGF beta effect on PAI expression. In TGF beta receptor II- and Ets1-deficient, noninvasive MCF-7 breast cancer cells, TGF beta 1 neither influenced endogenous PTHrP expression nor stimulated the PTHrP P3 promoter. These data suggest that TGF beta activates PTHrP expression by specifically up-regulating transcription from the PTHrP P3 promoter through a novel Smad3/Ets1 synergism.
