Forchlorfenuron Disrupts SEPT9_i1 Filaments and Inhibits HIF-1

Abstract

Forchlorfenuron (FCF) is a synthetic plant cytokinin that has been shown to alter yeast and mammalian septin organization. Septins are a highly conserved family of GTP-binding cytoskeletal proteins. Mammalian septins are involved in diverse cellular processes including tumorigenesis. We have been studying the interaction between septin 9 isoform 1 (SEPT9_i1) and hypoxia inducible factor-1α (HIF-1α), the oxygen regulated subunit of HIF-1. HIF-1 is a key transcription factor in the hypoxic responses pathway, and its activation has been observed in carcinogenesis and numerous cancers. SEPT9_i1/HIF-1α interaction plays an important role in upregulation of HIF-1 transcriptional activity by preventing HIF-1α’s ubiquitination and degradation leading to increased tumor growth and angiogenesis. We tested the hypothesis whether FCF affects SEPT9_i1 filamentous structures and consequently HIF-1 pathway in cancer cells. We showed that FCF suppresses tumorigenic properties, including proliferation, migration and transformation, in prostate cancer cells. FCF did not alter SEPT9_i1 steady state protein expression levels but it affected its filamentous structures and subcellular localization. FCF induced degradation of HIF-1α protein in a dose- and time-dependent manner. This inhibition was also shown in other common cancer types tested. Rapid degradation of HIF-1α protein levels was accompanied by respective inhibition in HIF-1α transcriptional activity. Moreover, HIF-1α protein half-life was markedly decreased in the presence of FCF compared with that in the absence of FCF. The FCF-induced degradation of HIF-1α was mediated in a significant part via the proteasome. To the best of our knowledge, this is the first demonstration of specific manipulation of septin filaments by pharmacological means having downstream inhibitory effects on the HIF-1 pathway.

In Silico Docking of Forchlorfenuron (FCF) to Septins Suggests that FCF Interferes with GTP Binding

“…FCF treatments have been reported to phenocopy the effects of septin-depletion affecting biological processes such as cell division and cell motility [19], glucose uptake [28], store operated calcium influx [29], ciliogenesis [30] and embryonic gastrulation [31]. Recent work also showed that FCF enhances septin-mediated degradation of the hypoxia inducible factor-1a and decreases the proliferation of prostate cancer cells [32]. While in vitro biochemical studies indicate that FCF affects directly the organization of purified recombinant septins [19], FCF has been shown to bind a cytokinin-binding protein from mung bean seedlings [33].…”

Off-Target Effects of the Septin Drug Forchlorfenuron on Nonplant Eukaryotes

“…In these cell types, FCF may be a useful tool to analyze higher-order septin dynamics, but conclusions about septin functions in various cellular processes based on FCF-induced effects should be made with the utmost caution. In systems in which septin genes are not easily mutated, numerous studies have attempted to address this issue using RNA interference-mediated depletion of septins, and in most cases this mimics the FCF effect of interest (30)(31)(32)52). However, it has been well established that experimental activation of the RNA interference system via the introduction of synthetic small RNA molecules is associated with cytotoxicity and various indicators of general cellular stress (53)(54)(55)(56)(57), not unlike what we have documented for FCF in multiple cell types.…”

“…Septins have been found to be involved in the mammalian hypoxic response (30), calcium signaling (31), and glucose uptake (32), as well as algal cell division (3) and muscle contraction in blood flukes (33), partly on the basis of experiments using FCF as an inhibitor of septin function. Two major untested assumptions in these studies are that septins from different species respond the same way to FCF, and that off-target effects of the drug are negligible at working concentrations.…”

Septins: Regulators of Protein Stability

“…In the presence of HSP90 inhibitors, the HIF-1α-HSP90 association is disrupted, leading to a competition between RACK1, which promotes HIF-1α degradation, and septin 9, which confers HIF-1α stabilization (Amir et al, 2009). In support of this HIF-1α-stabilizing role of septin 9, disruption of normal assembly-disassembly of septin oligomers with FCF induces degradation of HIF-1α, and inhibition in HIF-1α transcriptional activity in various cancer cell types (Vardi-Oknin et al, 2013). …”