Hypoxia & tumor-associated angiogenesis

Oxygen pressure within solid tumors is heterogenous, ranging from about 5% O2 in well vascularized regions to anoxia near necrotic areas, but is on average in the hypoxic range (about 1% O2). Hypoxia arises from a less ordered vasculature in malignant tumors, often described as chaotic as compared to normal tissues. In order to survive in this stressful environment, tumor cells have developed a coordinated set of responses, orchestrating their adaptation to hypoxia. The outcomes of the cellular responses to hypoxia are aggressive disease, resistance to therapy, and decreased patient survival. Adaptation to hypoxia is primarily mediated by hypoxia inducible transcription factor (HIF) complexes, which become stabilized and activated at low oxygen levels. HIF is composed of an α subunit (HIF-1α, HIF-2α and HIF-3α subunits) and a ß subunit (HIF-1ß/ARNT). Whereas HIF-1ß is constitutively present, the HIF-α members are highly unstable.

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Normal vasculature

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Tumor vasculature

Tumors contain regions of hypoxia and necrosis because their vasculature can not supply oxygen and other vital nutrients to all the cells. Whereas normal vasculature is hierarchically organized, with vessels that are sufficiently close to ensure adequate nutrient and oxygen supply to all cells, tumor vessels are chaotic, dilated, tortuous and are often far apart and have sluggish blood flow. As a consequence, areas of hypoxia and necrosis often develop distant from blood vessels. In addition to these regions of chronic (or diffusion-limited) hypoxia, areas of acute (or perfusion-limited) hypoxia can develop in tumors as a result of the temporary closure or reduced flow in certain vessels. AV, arteriovenous.
from J. Martin Brown & William R. Wilson Nature Reviews Cancer 4, 437-447

In normoxia, HIF-α are subject to a regulatory process involving the enzymatic hydroxylation of conserved prolyl and asparaginyl residues, thus leading to rapid von Hippel-Lindau protein (pVHL)-mediated ubiquitination and proteasomal degradation. As a result, intratumoral hypoxia, or genetic mutations that disrupt the function of pVHL, induce the activation of this pathway. Increased HIF-α activity leads to the upregulation of genes and induces expression of proteins that promote neoangiogenesis, anaerobic metabolism, and many other survival pathways. Overwhelming evidence based on immunohistochemical studies of human tumor sections indicates that both HIFs are overexpressed in the majority of human cancers. Moreover this overexpression in human tumors is associated with poor prognosis and poor outcome to radiation and chemotherapy. Studies demonstrating the importance of HIFs expression for tumor survival have made HIFs as attractive targets for cancer therapy.

SphK1/S1P signaling regulates HIF-1 level and activity in multiple cancer cell models

In 2008, we have shown for the first time that SphK1 could regulate HIF-1 accumulation under hypoxia. Our studies conducted in glioblastoma, prostate, breast, lung, kidney cancer cell models suggest a canonical role for SphK1 in adaptation to hypoxia. A sharp stimulation of SphK1 activity occurred rapidly (within 1-2 hrs) yet transiently under hypoxia indicating a likely post-traductionnal effect, with SphK1 activation invariably preceding HIF-1α accumulation. SphK1 stimulation appeared to depend on reactive oxygen species (ROS) production as the ROS scavenger N-Acetyl Cysteine could prevent both SphK1 stimulation and HIF-1α accumulation. How does generation of ROS result in SphK1 stimulation is currently unknown. A large body of evidence suggests that ROS can modulate HIF-1α level through direct inhibition of prolyl-hydroxylases but also indirectly via activation of the Akt/GSK3ß signaling. We found that the SphK1-mediated accumulaton of HIF-1α levels under hypoxia relied on the Akt/GSK3ß pathway (see scheme below). How does the biolipid S1P produced upon SphK1 stimulation activate the Akt/GSK3ß signaling (intracellular versus autocrine effect) is currently under investigation. Demonstrating the instrumental role of SphK1, both pharmacological and RNA silencing inhibition of SphK1 activity could prevent activation of the Akt/GSK3ß signaling, accumulation of HIF-1α and its transcriptional activity in all human cancer cell lineages. The major regulatory mechanism of HIF-1 accumulation under hypoxia is its pVHL-mediated proteasomal degradation, and we established that HIF-1α degradation triggered by SphK1 inhibition was controlled by the proteasome via a pVHL-dependent mechanism as demonstrated by inhibition of the proteasome by the MG132 compound or using pVHL-deficient and reconstituted pVHL cell models.

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Ader et al, Cancer Res, 2008

Neutralizing S1P inhibits intratumoral hypoxia and chemosensitizes in prostate cancer

Taking advantage of a monoclonal antibody that neutralizes extracellular S1P (sphingomab), we have shown that inhibition of the S1P extracellular signaling blocks HIF-1 accumulation and its transcriptional activity in several cancer cell models exposed to hypoxia. Using an orthotopic xenograft model of prostate cancer, we showed that sphingomab reduces hypoxia and modifies vessel architecture within 5 days of treatment, leading to increased intratumoral blood perfusion. Supporting the notion that a transient vascular normalization of tumor vessels is the mechanism by which sphingomab exerts its effects, we demonstrated that administration of the antibody for 5 days before chemotherapy was more effective at local tumor control and metastatic dissemination than any other treatment scheduling. These findings validate sphingomab as a potential new normalization agent that could contribute to successful sensitization of hypoxic tumors to chemotherapy.

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HIF-1α staining of representative regions of tumor sections from animals treated for 5 days with 50 mg/kg anti-S1P mAb or IgG control shows a clear reduction in HIF-1α expression in animals treated with anti-S1P mAb. Scale bar, 100 µM.

Representative images of immunofluorescence double staining for endothelial cells (CD34) and pericytes (αSMA) in paraffin sections obtained from tumors of animals treated with 50 mg/kg anti-S1P mAb or IgG control, showing a vascular remodeling with improved pericyte coverage.Red, CD34 staining; green, αSMA staining. Counterstaining was done with DAPI .

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Ader et al, Oncotarget, 2015

SphK1/S1P signaling is essential in regulating HIF-2 activity in various cancer cell models including ccRCC

More recently, we established that the SphK1/S1P signaling also regulates the transcription factor hypoxia inducible HIF-2α in diverse cancer cell lineages notably cell renal carcinoma (ccRCC), where HIF-2α has been established as a driver of a more aggressive disease. In ccRCC lacking a functional VHL gene and overexpressing HIF-2α, SphK1 controls HIF-2α expression and transcriptional activity through a ROS-dependent phospholipase D (PLD) driven mechanism. SphK1 silencing promotes a VHL-independent HIF-2α loss of expression and activity and reduces cell proliferation in ccRCC. Importantly, down-regulation of SphK1 is associated with impaired Akt and mTOR signaling in ccRCC. Using an anti-S1P mAb, we show that inhibition of S1P extracellular signaling blocks HIF-2α accumulation in ccRCC cell lines, an effect mimicked when the S1P transporter Spns2 is silenced. These findings demonstrate that SphK1/S1P signaling may act as a canonical regulator of HIF-2α expression in ccRCC, giving support to its inhibition as a therapeutic strategy that could contribute to reduce HIF-2 activity in ccRCC.

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Bouquerel et al, Oncogenesis, 2016

FTY720 (Fingolimod) induces vascular remodeling and chemosensitizes in ccRCC models

Here, we report that FTY720 – an inhibitor of the S1P signaling pathway – inhibits both HIF-1α and HIF-2α accumulation in several human cancer cell lines. In a ccRCC heterotopic xenograft model, we show that FTY720 transiently decreases HIF-1α and HIF-2α intratumoral level and modifies tumor vessel architecture within 5 days of treatment, suggesting a vascular normalization. In mice bearing subcutaneous ccRCC tumor, FTY720 and a gemcitabine-based chemotherapy alone display a limited effect whereas in combination there is a synergy of effect on tumor size without toxicity. Noteworthy, administration of FTY720 for 5 days before chemotherapy is not associated with a more effective tumor control, suggesting a mode of action mainly independent of the vascular remodeling. In conclusion, these findings demonstrate that FTY720 could successfully sensitize ccRCC to chemotherapy and establish this molecule as a potent therapeutic agent for ccRCC treatment, independently of drug scheduling.

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Gstalder et al, Mol Cancer Ther., 2016