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Supplementary Data from Metallothionein Induction by Hypoxia Involves Cooperative Interactions between Metal-Responsive Transcription Factor-1 and Hypoxia-Inducible Transcription Factor-1α

Mammalian metallothionein (MT) genes are transcriptionally activated by the essential metal zinc as well as by environmental stresses, including toxic metal overload and redox fluctuations. In addition to playing a key role in zinc homeostasis, MT proteins can protect against metal- and oxidant-induced cellular damage, and may participate in other fundamental physiologic and pathologic processes such as cell survival, proliferation, and neoplasia. Previously, our group reported a requirement for metal-responsive transcription factor-1 (MTF-1) in hypoxia-induced transcription of mouse MT-I and human MT-IIA genes. Here, we provide evidence that the protumorigenic hypoxia-inducible transcription factor-1α (HIF-1α) is essential for induction of MT-1 by hypoxia, but not zinc. Chromatin immunoprecipitation assays revealed that MTF-1 and HIF-1α are both recruited to the mouse MT-I promoter in response to hypoxia, but not zinc. In the absence of HIF-1α, MTF-1 is recruited to the MT-I promoter but fails to activate MT-I gene expression in response to hypoxia. Thus, HIF-1α seems to function as a coactivator of MT-I gene transcription by interacting with MTF-1 during hypoxia. Coimmunoprecipitation studies suggest interaction between MTF-1 and HIF-1α, either directly or as mediated by other factors. It is proposed that association of these important transcription factors in a multiprotein complex represents a common strategy to control unique sets of hypoxia-inducible genes in both normal and diseased tissue. (Mol Cancer Res 2008;6(3):483–90)

The orphan nuclear receptor estrogen-related receptor α (ERRα) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRα, bound to a co-activator protein (e.g., peroxisome proliferator receptor γ co-activator-1α, PGC-1α), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRα is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRα, but not to ERRβ or ERRγ, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRα's transcriptional activity in reporter gene assays, and prevents endogenous PGC-1α and ERRα from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30 mg/kg given once daily and 100 mg/kg given once weekly. In a combination study, SR16388 (10 mg/kg, once daily) and paclitaxel (7.5 mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRα biology.

The peroxisome proliferator-activated receptor delta (PPARdelta) is a ligand-activated, nuclear receptor transcription factor that has a documented role in glucose and lipid homeostasis. Recent studies have implicated this nuclear receptor in numerous aspects of oncogenesis. We report herein the characterization of a novel small-molecule (SR13904) that inhibits PPARdelta agonist-induced transactivation and functions as a PPARdelta antagonist. SR13904 also antagonizes PPARgamma transactivation, albeit with much weaker potency. SR13904 displays inhibitory effects on cellular proliferation and survival in several human carcinoma lines, including lung, breast and liver. These inhibitory effects of SR13904 on tumor cells were linked to a G(1)/S cell cycle block and increased apoptosis. Molecular studies show that SR13904 treatment of a lung cancer cell line, A549, results in markedly reduced levels of a number of cell cycle proteins including cyclin A and D, and cyclin dependent kinase (CDK) 2 and 4. The inhibitory effects on CDK2 appear to be transcriptional. Several of these cell cycle-related genes are known to be upregulated by PPARdelta. The antitumor activities of SR13904 suggest that antagonism of PPARdelta-mediated transactivation may inhibit tumorigenesis and that pharmacological inhibition of PPARdelta may be a potential strategy for treatment or prevention of cancer.

Hypoxia-inducible factor-1 (HIF-1), the major transcriptional regulator of the mammalian cellular response to low oxygen (hypoxia), is embedded within a complex network of signaling pathways. We have been investigating the importance of another stress-responsive transcription factor, MTF-1, for the adaptation of cells to hypoxia. This article reports that MTF-1 plays a central role in hypoxic cells by contributing to HIF-1 activity. Loss of MTF-1 in transformed Mtf1 null mouse embryonic fibroblasts (MEFs) results in an attenuation of nuclear HIF-1α protein accumulation, HIF-1 transcriptional activity, and expression of an established HIF-1 target gene, glucose transporter-1 ( Glut1 ). Mtf1 null ( Mtf1 KO) MEFs also have constitutively higher levels of both glutathione (GSH) and the rate-limiting enzyme involved in GSH synthesis—glutamate cysteine ligase catalytic subunit—than wild type cells. The altered cellular redox state arising from increased GSH may perturb oxygen-sensing mechanisms in hypoxic Mtf1 KO cells and decrease the accumulation of HIF-1α protein. Together, these novel findings define a role for MTF-1 in the regulation of HIF-1 activity.

The orphan nuclear receptor estrogen-related receptor alpha (ERRalpha) has been implicated in the development of various human malignancies, including breast, prostate, ovary, and colon cancer. ERRalpha, bound to a co-activator protein (e.g., peroxisome proliferator receptor gamma co-activator-1alpha, PGC-1alpha), regulates cellular energy metabolism by activating transcription of genes involved in various metabolic processes, such as mitochondrial genesis, oxidative phosphorylation, and fatty acid oxidation. Accumulating evidence suggests that ERRalpha is a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor microenvironments. This report describes a novel steroidal antiestrogen (SR16388) that binds selectively to ERRalpha, but not to ERRbeta or ERRgamma, as determined using a time-resolved fluorescence resonance energy transfer assay. SR16388 potently inhibits ERRalpha's transcriptional activity in reporter gene assays, and prevents endogenous PGC-1alpha and ERRalpha from being recruited to the promoters or enhancers of target genes. Representative in vivo results show that SR16388 inhibited the growth of human prostate tumor xenografts in nude mice as a single agent at 30mg/kg given once daily and 100mg/kg given once weekly. In a combination study, SR16388 (10mg/kg, once daily) and paclitaxel (7.5mg/kg, twice weekly) inhibited the growth of prostate tumor xenografts in nude mice by 61% compared to untreated xenograft tumors. SR16388 also inhibited the proliferation of diverse human tumor cell lines after a 24-h exposure to the compound. SR16388 thus has utility both as an experimental antitumor agent and as a chemical probe of ERRalpha biology.

We previously identified metal-responsive transcription factor-1 (MTF-1) as a positive contributor to mouse fibrosarcoma growth through effects on cell survival, proliferation, tumor angiogenesis and extracellular matrix remodeling. In the present study, we investigated MTF-1 protein expression in human tissues by specific immunostaining of both normal and tumor tissue samples. Immunohistochemical (IHC) staining of a human tissue microarray (TMA), using a unique anti-human MTF-1 antibody, indicated constitutive MTF-1 expression in most normal tissues, with liver and testis displaying comparatively high levels of expression. Nevertheless, MTF-1 protein levels were found to be significantly elevated in diverse human tumor types, including breast, lung and cervical carcinomas. IHC analysis of a separate panel of full-size tissue sections of human breast cancers, including tumor and normal adjacent, surrounding tissue, confirmed and extended the results of the TMA analysis. Taken with our previous findings, this new study suggests a role for MTF-1 in human tumor development, growth or spread. Moreover, the study suggests that MTF-1 could be a novel therapeutic target that offers the opportunity to manipulate metal or redox homeostasis in tumor cells.

Mammalian metallothionein (MT) genes are transcriptionally activated by the essential metal zinc as well as by environmental stresses, including toxic metal overload and redox fluctuations. In addition to playing a key role in zinc homeostasis, MT proteins can protect against metal- and oxidant-induced cellular damage, and may participate in other fundamental physiologic and pathologic processes such as cell survival, proliferation, and neoplasia. Previously, our group reported a requirement for metal-responsive transcription factor-1 (MTF-1) in hypoxia-induced transcription of mouse MT-I and human MT-IIA genes. Here, we provide evidence that the protumorigenic hypoxia-inducible transcription factor-1α (HIF-1α) is essential for induction of MT-1 by hypoxia, but not zinc. Chromatin immunoprecipitation assays revealed that MTF-1 and HIF-1α are both recruited to the mouse MT-I promoter in response to hypoxia, but not zinc. In the absence of HIF-1α, MTF-1 is recruited to the MT-I promoter but fails to activate MT-I gene expression in response to hypoxia. Thus, HIF-1α seems to function as a coactivator of MT-I gene transcription by interacting with MTF-1 during hypoxia. Coimmunoprecipitation studies suggest interaction between MTF-1 and HIF-1α, either directly or as mediated by other factors. It is proposed that association of these important transcription factors in a multiprotein complex represents a common strategy to control unique sets of hypoxia-inducible genes in both normal and diseased tissue. (Mol Cancer Res 2008;6(3):483–90)