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Purpose: Clear cell renal cell carcinoma (ccRCC) is frequently associated with inactivation of the von Hippel-Lindau tumor suppressor, resulting in activation of HIF-1a and HIF-2a. The current paradigm, established using mechanistic cell-based studies, supports a tumor promoting role for HIF-2a, and a tumor suppressor role for HIF-1a. However, few studies have comprehensively examined the clinical relevance of this paradigm. Furthermore, the hypoxia-associated factor (HAF), which regulates the HIFs, has not been comprehensively evaluated in ccRCC.Experimental Design: To assess the involvement of HAF/HIFs in ccRCC, we analyzed their relationship to tumor grade/stage/ outcome using tissue from 380 patients, and validated these associations using tissue from 72 additional patients and a further 57 patients treated with antiangiogenic therapy for associations with response. Further characterization was performed using single-cell mRNA sequencing (scRNA-seq), RNA-in situ hybridization (RNA-ISH), and IHC.Results: HIF-1a was primarily expressed in tumor-associated macrophages (TAMs), whereas HIF-2a and HAF were expressed primarily in tumor cells. TAM-associated HIF-1a was significantly associated with high tumor grade and increased metastasis and was independently associated with decreased overall survival. Furthermore, elevated TAM HIF-1a was significantly associated with resistance to antiangiogenic therapy. In contrast, high HAF or HIF-2a were associated with low grade, decreased metastasis, and increased overall survival. scRNA-seq, RNA-ISH, and Western blotting confirmed the expression of HIF-1a in M2-polarized CD163-expressing TAMs.Conclusions: These findings highlight a potential role of TAM HIF-1a in ccRCC progression and support the reevaluation of HIF-1a as a therapeutic target and marker of disease progression.

Background Solid tumours are less oxygenated than normal tissues. This is called tumour hypoxia and leads to resistance to radiotherapy and chemotherapy. The molecular mechanisms underlying such resistance have been investigated in a range of tumour types, including the adult brain tumours glioblastoma, yet little is known for paediatric brain tumours. Medulloblastoma (MB) is the most common malignant brain tumour in children. We aimed to elucidate the impact of hypoxia on the sensitivity of MB cells to chemo- and radiotherapy. Methods We used two MB cell line (D283-MED and MEB-Med8A) and a widely used glioblastoma cell line (U87MG) for comparison. We applied a range of molecular and cellular techniques to measure cell survival, cell cycle progression, protein expression and DNA damage combined with a transcriptomic micro-array approach in D283-MED cells, for global gene expression analysis in acute and chronic hypoxic conditions. Results In D283-MED and U87MG, chronic hypoxia (5 days), but not acute hypoxia (24 h) induced resistance to chemotherapy and X-ray irradiation. This acquired resistance upon chronic hypoxia was present but less pronounced in MEB-Med8A cells. Using transcriptomic analysis in D283-MED cells, we found a large transcriptional remodelling upon long term hypoxia, in particular the expression of a number of genes involved in detection and repair of double strand breaks (DSB) was altered. The levels of Nibrin (NBN) and MRE11, members of the MRN complex (MRE11/Rad50/NBN) responsible for DSB recognition, were significantly down-regulated. This was associated with a reduction of Ataxia Telangiectasia Mutated ( ATM) activation by etoposide, indicating a profound dampening of the DNA damage signalling in hypoxic conditions. As a consequence, p53 activation by etoposide was reduced, and cell survival enhanced. Whilst U87MG shared the same dampened p53 activity, upon chemotherapeutic drug treatment in chronic hypoxic conditions, these cells used a different mechanism, independent of the DNA damage pathway. Conclusion Together our results demonstrate a new mechanism explaining hypoxia-induced resistance involving the alteration of the response to DSB in D283-MED cells, but also highlight the cell type to cell type diversity and the necessity to take into account the differing tumour genetic make-up when considering re-sensitisation therapeutic protocols. Electronic supplementary material The online version of this article (10.1186/s12885-019-5476-9) contains supplementary material, which is available to authorized users.

Clear cell renal cell carcinoma (ccRCC), the most common form of kidney cancer, is typically initiated by inactivation of the von Hippel Lindau (VHL) gene, which results in the constitutive activation of the hypoxia inducible factors, HIF-1α and HIF-2α. Using a high throughput screen, we identify novel compounds that decrease HIF-1/2α levels and induce ferroptosis by targeting Iron Sulfur Cluster Assembly 2 (ISCA2), a component of the late mitochondrial Iron Sulfur Cluster (L-ISC) assembly complex. ISCA2 inhibition either pharmacologically or using siRNA decreases HIF-2α protein levels by blocking iron-responsive element (IRE)-dependent translation, and at higher concentrations, also decreases HIF-1α translation through unknown mechanisms. Additionally, ISCA2 inhibition triggers the iron starvation response, resulting in iron/metals overload and death via ferroptosis. ISCA2 levels are decreased in ccRCC compared to normal kidney, and decreased ISCA2 levels are associated with pVHL loss and with sensitivity to ferroptosis induced by ISCA2 inhibition. Strikingly, pharmacological inhibition of ISCA2 using an orally available ISCA2 inhibitor significantly reduced ccRCC xenograft growth in vivo, decreased HIF-α levels and increased lipid peroxidation, suggesting increased ferroptosis in vivo. Thus, the targeting of ISCA2 may be a promising therapeutic strategy to inhibit HIF-1/2α and to induce ferroptosis in pVHL deficient cells.

Glioblastoma, a grade IV astrocytoma, has a poor survival rate in part due to ineffective treatment options available. These tumours are heterogeneous with areas of low oxygen levels, termed hypoxic regions. Many intra-cellular signalling pathways, including DNA repair, can be altered by hypoxia. Since DNA damage induction and subsequent activation of DNA repair mechanisms is the cornerstone of glioblastoma treatment, alterations to DNA repair mechanisms could have a direct influence on treatment success. Our aim was to elucidate the impact of chronic hypoxia on DNA repair gene expression in a range of glioblastoma cell lines. We adopted a NanoString transcriptomic approach to examine the expression of 180 DNA repair related genes in four classical glioblastoma cell lines (U87-MG, U251-MG, D566-MG, T98G) exposed to 5 days of normoxia (21% O 2 ), moderate (1% O 2 ) or severe (0.1% O 2 ) hypoxia. We observed altered gene expression in several DNA repair pathways including homologous recombination repair, non-homologous end-joining and mismatch repair, with hypoxia primarily resulting in downregulation of gene expression. The extend of gene expression changes was function of the hypoxic severity. Some, but not all, of these downregulations were directly under the control of HIF activity. For example, the downregulation of LIG4, a key component of non-homologous end-joining, was reversed upon inhibition of the hypoxia inducible factor (HIF). In contrast, the downregulation of the mismatch repair gene, PMS2, was not affected by HIF inhibition. This suggests that numerous molecular mechanisms lead to hypoxia-induced reprogramming of the transcriptional landscape of DNA repair. Whilst the global impact of hypoxia on DNA repair gene expression is likely to lead to genomic instability, tumorigenesis and reduced sensitivity to anti-cancer treatment, treatment re-sensitizing might require additional approaches to a simple HIF inhibition.