While the functions of hypoxia-inducible factor 1␣ (HIF1␣)/aryl hydrocarbon receptor nuclear translocator (ARNT) and HIF2␣/ARNT (HIF2) proteins in activating hypoxia-inducible genes are well established, the role of other transcription factors in the hypoxic transcriptional response is less clear. We report here for the first time that the basic helix-loop-helix-leucine-zip transcription factor upstream stimulatory factor 2 (USF2) is required for the hypoxic transcriptional response, specifically, for hypoxic activation of HIF2 target genes. We show that inhibiting USF2 activity greatly reduces hypoxic induction of HIF2 target genes in cell lines that have USF2 activity, while inducing USF2 activity in cells lacking USF2 activity restores hypoxic induction of HIF2 target genes. Mechanistically, USF2 activates HIF2 target genes by binding to HIF2 target gene promoters, interacting with HIF2␣ protein, and recruiting coactivators CBP and p300 to form enhanceosome complexes that contain HIF2␣, USF2, CBP, p300, and RNA polymerase II on HIF2 target gene promoters. Functionally, the effect of USF2 knockdown on proliferation, motility, and clonogenic survival of HIF2-dependent tumor cells in vitro is phenocopied by HIF2␣ knockdown, indicating that USF2 works with HIF2 to activate HIF2 target genes and to drive HIF2-depedent tumorigenesis.A hypoxic microenvironment is frequently found in solid tumors. The transcriptional response mediated by hypoxia-inducible factor 1␣ (HIF1␣)/aryl hydrocarbon receptor nuclear translocator (ARNT) (HIF1) and HIF2␣/ARNT (HIF2) plays a critical role in malignant progression by increasing expression of genes involved in angiogenesis, anaerobic metabolism, and other processes that enable tumor cells to survive and/or escape their O 2 -deficient microenvironment (25,53,56,93).It is well established that multiple transcription factors (TFs) are required to achieve maximal activation of target genes in response to a specific stimulus. This multifactorial transcription complex has been termed the "enhanceosome" (100). Individual factors in the enhanceosome complex may promote transcription initiation by recruiting RNA polymerase II (Pol II)/general transcription factors and/or recruiting chromatin-modifying enzymes, such as histone acetylases and chromatin remodeling complexes. In addition, TFs such as Myc increase gene expression by recruiting elongation factors to regulate Pol II pause release (77). Thus, reduced levels of transcription could occur in the absence of factors that have redundant functions within the enhanceosome, while other transcription factors having unique functions are absolutely required for gene activation.The role of HIF1 and HIF2 in activating hypoxia-inducible genes is well established (21,37,48,79,103). However, the other transcription factors required for hypoxic activation of HIF target genes have been much less studied. Based on the enhanceosome concept, we hypothesized that another transcription factor(s) is required to activate HIF target genes during hypoxia. We ...

160 Background: Radium-223 is a targeted alpha therapy that improves survival in men with mCRPC. The biologic basis for radium-223 efficacy is not completely understood. We hypothesized that PC osteomimicry, a form of epithelial plasticity leading to an osteoblastic phenotype, may contribute to the intralesional deposition of radium-223 and subsequent irradiation of the tumor microenvironment. Methods: We conducted a pharmacodynamic study of radium-223 in men with bone predominant mCRPC to investigate genomic and phenotypic alterations in circulating tumor cells (CTCs), ctDNA, and metastases. Prior to radium and 3 and 6 months after radium, liquid and metastatic biopsies were collected, including CTCs for phenotypic characterization and CTC/ctDNA genomic analysis. The primary objective was to describe the prevalence of CTC bone alkaline phosphatase (BAP) over time. We measured radium-223 decay products in tumor and surrounding normal bone during treatment. Results: We enrolled 20 men with heavily pre-treated symptomatic bone predominant mCRPC and treated with radium-223 over a median of 6 doses. 55% had elevated serum BAP. PFS was 5.5 mo; OS was 13.3 mo; 10% had unfavorable (≥5) to favorable ( < 5) Cellsearch CTC conversion; 5% had ≥30% PSA decline. We found evidence of persistent CTC BAP expression in both EpCAM+ and EpCAM- CTCs in the majority of men over time during radium treatment. We identified genomic gain of key osteomimicry regions in CTC DNA, including loci for BAP, osteopontin, and OB-cadherin. Radium-223 uptake was observed in tumor to a greater degree than surrounding normal bone. CTC DNA and matched ctDNA and CTC cultures suggested persistence of CTCs with aggressive genomic alterations such as AR, FOXA1, and MYC gain and PTEN and RB1 loss. We established multiple CTC cultures and one CTC PDX; cells exhibited evidence of epithelial plasticity with BAP expression. Conclusions: Osteomimicry may contribute to the uptake of Radium-223 within bone metastases and may thereby enhance the therapeutic benefit of radium-223. We found genomic and phenotypic evidence of osteomimicry in CTCs and CTC cultures from men with mCRPC. Clinical trial information: NCT02204943.

Despite initial and often dramatic responses of specific NSCLC tumors to EGFR TKIs such as gefitinib and erlotinib, nearly all develop resistance and eventually relapse. We have investigated both rapid and chronic mechanisms of resistance to EGFR-specific TKIs in lung cancer cell lines. Following 3 to 4 day treatments with gefitinib, FGFR2 and FGFR3 mRNA and protein are selectively and rapidly (24 to 48 hrs) increased in NSCLC cell lines in which EGFR is the dominant oncogenic driver via mutation or amplification. This induction is mediated, in part, by transcriptional mechanisms and indicates that EGFR signaling functions to repress expression of FGFR2 and FGFR3. Moreover, EGFR TKI-induced FGFR2 and FGFR3 are capable of mediating FGF-stimulated ERK activation and transformed growth in the continued presence of EGFR TKIs. These studies highlight FGFR2 and FGFR3 induction as a rapid molecular response that may modulate initial sensitivity of EGFR-driven lung tumors to EGFR-specific TKIs. We have also employed NSCLC cell lines bearing activating mutations in EGFR and rendered them resistant to EGFR-specific TKIs, gefitinib (reversible) and BIBW2992 (irreversible), through chronic (2–3 months) adaptation in tissue culture. To date, three (HCC4006, H1650, HCC2279) of the eight chronically-adapted NSCLC cell lines exhibit a marked induction of FGF2 and FGFR1 upon acquisition of gefitinib resistance (Table 1). Additionally, two of eight undergo marked loss of epithelial differentiation as measured by decreased E-cadherin. The induction of FGFR1 was not mediated by gene amplification as assessed by FISH analysis. Importantly, these chronically-adapted cell lines were highly sensitive to both FGFR-specific TKIs and an FGF ligand trap, FP-1039, as measured by ERK signaling and growth assays. Thus, induction of FGF2 and FGFR1 following chronic adaptation to EGFR-specific TKIs may provide a novel autocrine receptor tyrosine kinase-driven growth pathway in a subset of lung tumors that were initially sensitive to EGFR-specific TKIs. We are presently exploring the in vivo relevance of this mechanism using primary tumor specimens obtained pretreatment and following tumor progression on EGFR-specific TKIs. Combined, these studies indicate that FGFR-specific TKIs may be valuable additions to existing targeted therapeutic strategies with EGFR-specific TKIs to reduce acquired resistance. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B70.

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