Interaction of HIF and USF Signaling Pathways in Human Genes Flanked by Hypoxia-Response Elements and E-box Palindromes

Abstract

Rampant activity of the hypoxia-inducible factor (HIF)-1 in cancer is frequently associated with the malignant progression into a harder-to-treat, increasingly aggressive phenotype. Clearly, anti-HIF strategies in cancer cells are of considerable clinical interest. One way to fine-tune, or inhibit, HIF's transcriptional outflow independently of hydroxylase activities could be through competing transcription factors. A CACGTG-binding activity in human hepatoma cells was previously found to restrict HIF's access to hypoxia response cis-elements (HRE) in a Daphnia globin gene promoter construct (phb2). The CACGTG factor, and its impact on hypoxia-responsive human genes, was analyzed in this study by genome-wide computational scans as well as gene-specific quantitative PCR, reporter and DNA-binding assays in hepatoma (Hep3B), cervical carcinoma (HeLa), and breast carcinoma (MCF7) cells. Among six basic helix-loop-helix transcription factors known to target CACGTG palindromes, we identified upstream stimulatory factor (USF)-1/2 as predominant phb2 CACGTG constituents in Hep3B, HeLa, and MCF7 cells. Human genes with adjacent or overlapping HRE and CACGTG motifs included with lactate dehydrogenase A (LDHA) and Bcl-2/E1B 19 kDa interacting protein 3 (BNIP3) hypoxia-induced HIF-1 targets. Parallel recruitment of HIF-1a and USF1/2a to the respective promoter chromatin was verified for all cell lines investigated. Mutual complementing (LDHA) or moderating (BNIP3) cross-talk was seen upon overexpression or silencing of HIF-1a and USF1/2a. Distinct (LDHA) or overlapping (BNIP3) promoter-binding sites for HIF-1 and USFs were subsequently characterized. We propose that, depending on abundance or activity of its protein constituents, O 2 -independent USF signaling can function to fine-tune or interfere with HIF-mediated transcription in cancer cells. Mol Cancer Res; 9(11); 1520-36. Ó2011 AACR.

Upstream Stimulatory Factor 2 and Hypoxia-Inducible Factor 2α (HIF2α) Cooperatively Activate HIF2 Target Genes during Hypoxia

“…Conversely, reduction of USF2 expression by siRNA increased hypoxic activation of some HIF1 target genes, such as KCTD11, BNIP3L, and GRP111, in hypoxic Hep3B cells (Table 1). This inhibitory role of USF2 for some HIF1 target genes is consistent with previous reports indicating that HREs of some HIF1 target genes, such as LDHA, BNIP3, and LPK, are bound by USF1/USF2 under normoxia or in response to glucose activation but by HIF1 in hypoxic cells (49,62). However, while the previous reports indicated that USF1/USF2 and HIF1 compete for the same binding site on some HIF1 target promoters during hypoxia, we show that USF2 and HIF2 bind to different sites of HIF2 target genes in an uncompetitive manner in vitro (Fig.…”

STAT3 or USF2 Contributes to HIF Target Gene Specificity

“…1D, P < 0.05 vs HIF1α only). This inhibitory role of USF2 for some HIF1 target genes is consistent with previous reports indicating that HBSs of some HIF1 target genes, such as LDHA , BNIP3 , and LPK , are bound by USF1/USF2 under normoxia but by HIF1 in hypoxic cells [34] as we also observed previously [29]. The weak activation of HIF1 target gene reporters by HIF2 was not changed by co-transfection of STAT3 or USF2 (Fig.…”

“…9A). This could not be explained by USF2’s role in binding to HBS and activating HIF1 target genes under normoxia [43] as RCC4 cells exhibit constitutive HIF activity.…”

Targeted CDX2 expression inhibits aggressive phenotypes of colon cancer cells in vitro and in vivo

“…Hypoxia is a major feature of solid tumors and induces hypoxia-inducible factor-1α (HIF-1α) which binds to the hypoxia-response elements (HREs) of various target genes and activates their transcription to regulate glucose transport and angiogenesis, and potentially enhance the survival of tumor cells (18–22). Our previous studies have shown that hypoxia modulates the downregulation of CDX2 in colorectal cancer (23).…”