In vivo and in vitro effects of a HIF‐1α inhibitor, RX‐0047

Abstract

HIF-1α plays a major role in activating gene transcription and is important for maintaining homeostasis under hypoxic conditions. Since tumors are often in a hypoxic state, HIF-1α is a potential target for the development of novel cancer therapeutics. This study was performed to determine the antitumoral efficacy of an antisense HIF-1α inhibitor, RX-0047 on different human cancer cell lines (MDA-MB 231, HME50-T, PC-3, Panc-1 and A549) in vitro. A549 lung cancer and PC-3 prostate cancer cells containing a luciferase gene reporter were used for in vivo xenograft animal models. Progressive tumor development was quantified using live animal BLI (bioluminescence imaging) in addition to ex vivo imaging and histology. All cell lines tested were sensitive to inhibition of cell growth with 10 nM and higher ranges of RX-0047, additionally RX-0047 sensitizes cells to ionizing radiation treatments. Finally, RX-0047 (30 mg/kg) inhibited the formation of human lung metastasis in xenograft mouse models and reduced tumor size in flank models. Keywordshypoxia; HIF-1α; RX-0047; cancer; bioluminescence imagingThe hypoxia-inducible factors (HIFs) are key transcriptional regulators of the hypoxic response in both adult and embryonic organisms. HIF-1 regulates cellular adaption to oxygen deficiency by regulating genes involved in erythropoiesis, iron metabolism, glycolysis, angiogenesis, inhibition of apoptosis, tissue matrix metabolism, cell proliferation and survival, which are key factors for tumor growth [Semenza, 2002;Bracken et al., 2003;Goda et al., 2003]. Loss of HIF-1α activity dramatically decreases tumor growth, vascularization, and energy metabolism, whereas overexpression of HIF-1α increases HIF-1 transcription factor activity and promotes tumor growth [Powis and Kirkpatrick, 2004].HIF-1 is a heterodimer consisting of the oxygen sensitive HIF-1α (120 kDa) and the constitutively expressed HIF-1β subunit (80 kDa) which is also known as aryl hydro-carbon receptor nuclear translocator, ARNT . Both subunits contain a basic-helix-loop-helix motif and a Per Arnt Sim (PAS) protein-protein interaction domain . Recent studies demonstrated that hydroxylation of HIF-1α at two NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript specific proline residues (Pro-402 and Pro-564) is catalyzed by prolyl-4-hydroxylase (PHD) using molecular oxygen, Fe +2 and oxoglutarate as substrates [Ivan et al., 2001]. Under normoxic conditions, the tumor suppressor von Hippel-Lindau (VHL) protein specifically interacts with hydroxylated HIF-1α and mediates the assembly of a complex that activates an ubiquitin-dependent proteasome system, therefore HIF-1α subunit is kept low due to massive ubiquitination and subsequent proteosomal degradation [Salceda and Caro, 1997;Maxwell et al., 1999]. However, during hypoxia, proline is not hydroxylated, and ubiquitination is inhibited, causing accumulation of the HIF-1α protein [Salceda and Caro, 1997;Maxwell et al., 1999;Bruick, 2003]. Stabilized HIF-1α protein translocates into the nu...

Diverging prognostic impacts of hypoxic markers according to NSCLC histology

“…In this context, our data further highlight the importance of histological NSCLC subtypes. As inhibitors of HIF-1 and CAIX have been developed and are under examination [54][55][56], we have shown that targeting the HIFs or their associated markers may yield diverging effects according to histological subtypes as a result of the different prognostic impacts of these markers. Increased knowledge about these hypoxia and metabolic markers in NSCLC tumor and stromal compartments will be vital in identifying different cancer cell phenotypes as candidates for specific molecular targeting.…”

Identification of a Survival-independent Metastasis-enhancing Role of Hypoxia-inducible Factor-1α with a Hypoxia-tolerant Tumor Cell Line

“…Liao et al (12) showed that lack of HIF-1␣ resulted in delayed tumor onset, reduced tumor growth, and a diminished number of blood vessels, leading to a later onset of lung metastasis of the spontaneous PymT-model. Other studies elucidated that HIF-1␣ depletion reduced the size of experimental bone metastases of a human breast carcinoma cell line in nude mice (13) and that inhibition of HIF-1␣ by a synthetic inhibitor blocked outgrowth of pulmonary metastases of a human lung carcinoma in an experimental xenograft metastasis assay (41). However, these studies could not resolve whether the antitumorigenic and antimetastatic effect of HIF-1␣ deficiency can only be attributed to decreased survival of tumor cells within unfavorable microenvironments or whether HIF-1␣ directly impacts on tumor cell invasiveness in vivo, e.g.…”

Construction of Mutant TKGFP for Real-Time Imaging of Temporal Dynamics of HIF-1 Signal Transduction Activity Mediated by Hypoxia and Reoxygenation in Tumors in Living Mice

“…Hypoxia-responsive elements linked with luciferase reporter genes combined with optical imaging systems have been used for real-time observation of HIF-1 signal transduction activity in living subjects (36)(37)(38). However, these approaches are limited by depth penetration and the nature of tomography and cannot monitor the spatial heterogeneity of HIF-1 signal transduction activity.…”