Macrophage miR-210 induction and metabolic reprogramming in response to pathogen interaction boost life-threatening inflammation.

Unbalanced immune responses to pathogens can be life-threatening although the underlying regulatory mechanisms remain unknown. Here, we show a hypoxia-inducible factor 1α-dependent microRNA (miR)-210 up-regulation in monocytes and macrophages upon pathogen interaction. MiR-210 knockout in the hematopoietic lineage or in monocytes/macrophages mitigated the symptoms of endotoxemia, bacteremia, sepsis, and parasitosis, limiting the cytokine storm, organ damage/dysfunction, pathogen spreading, and lethality. Similarly, pharmacologic miR-210 inhibition improved the survival of septic mice. Mechanistically, miR-210 induction in activated macrophages supported a switch toward a proinflammatory state by lessening mitochondria respiration in favor of glycolysis, partly achieved by downmodulating the iron-sulfur cluster assembly enzyme ISCU. In humans, augmented miR-210 levels in circulating monocytes correlated with the incidence of sepsis, while serum levels of monocyte/macrophage-derived miR-210 were associated with sepsis mortality. Together, our data identify miR-210 as a fine-tuning regulator of macrophage metabolism and inflammatory responses, suggesting miR-210-based therapeutic and diagnostic strategies.

PHD3 Controls Lung Cancer Metastasis and Resistance to EGFR Inhibitors through TGFα.

Lung cancer is the leading cause of cancer-related death worldwide, in large part due to its high propensity to metastasize and to develop therapy resistance. Adaptive responses to hypoxia and epithelial-mesenchymal transition (EMT) are linked to tumor metastasis and drug resistance, but little is known about how oxygen sensing and EMT intersect to control these hallmarks of cancer. Here, we show that the oxygen sensor PHD3 links hypoxic signaling and EMT regulation in the lung tumor microenvironment. PHD3 was repressed by signals that induce EMT and acted as a negative regulator of EMT, metastasis, and therapeutic resistance. PHD3 depletion in tumors, which can be caused by the EMT inducer TGFß or by promoter methylation, enhanced EMT and spontaneous metastasis via HIF-dependent upregulation of the EGFR ligand TGFα. In turn, TGFα stimulated EGFR, which potentiated SMAD signaling, reinforcing EMT and metastasis. In clinical specimens of lung cancer, reduced PHD3 expression was linked to poor prognosis and to therapeutic resistance against EGFR inhibitors such as erlotinib. Reexpression of PHD3 in lung cancer cells suppressed EMT and metastasis and restored sensitivity to erlotinib. Taken together, our results establish a key function for PHD3 in metastasis and drug resistance and suggest opportunities to improve patient treatment by interfering with the feedforward signaling mechanisms activated by PHD3 silencing.Significance: This study links the oxygen sensor PHD3 to metastasis and drug resistance in cancer, with implications for therapeutic improvement by targeting this system. Cancer Res; 78(7); 1805-19. ©2018 AACR.

Tumor matrix stiffness promotes metastatic cancer cell interaction with the endothelium.

Tumor progression alters the composition and physical properties of the extracellular matrix. Particularly, increased matrix stiffness has profound effects on tumor growth and metastasis. While endothelial cells are key players in cancer progression, the influence of tumor stiffness on the endothelium and the impact on metastasis is unknown. Through quantitative mass spectrometry, we find that the matricellular protein CCN1/CYR61 is highly regulated by stiffness in endothelial cells. We show that stiffness-induced CCN1 activates ß-catenin nuclear translocation and signaling and that this contributes to upregulate N-cadherin levels on the surface of the endothelium, in vitro This facilitates N-cadherin-dependent cancer cell-endothelium interaction. Using intravital imaging, we show that knockout of Ccn1 in endothelial cells inhibits melanoma cancer cell binding to the blood vessels, a critical step in cancer cell transit through the vasculature to metastasize. Targeting stiffness-induced changes in the vasculature, such as CCN1, is therefore a potential yet unappreciated mechanism to impair metastasis.

The impact of hypoxia on tumor-associated macrophages.

The role of tumor-associated macrophages (TAMs) in cancer is often correlated with poor prognosis, even though this statement should be interpreted with care, as the effects of macrophages primarily depend on their localization within the tumor. This versatile cell type orchestrates a broad spectrum of biological functions and exerts very complex and even opposing functions on cell death, immune stimulation or suppression, and angiogenesis, resulting in an overall pro- or antitumoral effect. We are only beginning to understand the environmental cues that contribute to transient retention of macrophages in a specific phenotype. It has become clear that hypoxia shapes and induces specific macrophage phenotypes that serve tumor malignancy, as hypoxia promotes immune evasion, angiogenesis, tumor cell survival, and metastatic dissemination. Additionally, TAMs in the hypoxic niches within the tumor are known to mediate resistance to several anticancer treatments and to promote cancer relapse. Thus, a careful characterization and understanding of this macrophage differentiation state is needed in order to efficiently tailor cancer therapy.

Factor-inhibiting HIF-1 (FIH-1) is required for human vascular endothelial cell survival.

Factor-inhibiting hypoxia-inducible factor (HIF)-1 (FIH-1) is an asparaginyl ß-hydroxylase enzyme that was initially found to hydroxylate the HIF-α, preventing its transcriptional activity and leading to adaptive responses to hypoxia. More recently, other substrates, such as neurogenic locus notch homolog (Notch), have been found to be alternative FIH targets, but the biologic relevance of this regulation was never investigated. Given the key function of Notch in angiogenesis, we here investigate the role of FIH/Notch signaling in endothelial cells. We report that FIH-1 silencing in HUVECs results in reduced growth and increased apoptosis. The knockdown of FIH is associated with increased Notch2 activity, leading to enhanced expression of the Notch target hairy/enhancer-of-split related with YRPW motif protein 1 (Hey-1). Consistent with recent findings showing that Notch2 suppresses survivin (a key inhibitor of apoptosis), FIH targeting in HUVECs leads to selective repression of survivin in endothelial cells, thus promoting cell apoptosis and growth arrest. Our data support the concept that FIH-1 may interact with Notch2 and repress its activity, thereby playing a critical role in controlling the survival of vascular endothelial cells. These findings might pave the way toward novel, antiangiogenic strategies in disorders that are characterized by excessive vascular growth, such as cancer and rheumatoid arthritis.

PHD3 regulates EGFR internalization and signalling in tumours.

Tumours exploit their hypoxic microenvironment to induce a more aggressive phenotype, while curtailing the growth-inhibitory effects of hypoxia through mechanisms that are poorly understood. The prolyl hydroxylase PHD3 is regulated by hypoxia and plays an important role in tumour progression. Here we identify PHD3 as a central regulator of epidermal growth factor receptor (EGFR) activity through the control of EGFR internalization to restrain tumour growth. PHD3 controls EGFR activity by acting as a scaffolding protein that associates with the endocytic adaptor Eps15 and promotes the internalization of EGFR. In consequence, loss of PHD3 in tumour cells suppresses EGFR internalization and hyperactivates EGFR signalling to enhance cell proliferation and survival. Our findings reveal that PHD3 inactivation provides a novel route of EGFR activation to sustain proliferative signalling in the hypoxic microenvironment.

Loss of PHD3 allows tumours to overcome hypoxic growth inhibition and sustain proliferation through EGFR.

Solid tumours are exposed to microenvironmental factors such as hypoxia that normally inhibit cell growth. However, tumour cells are capable of counteracting these signals through mechanisms that are largely unknown. Here we show that the prolyl hydroxylase PHD3 restrains tumour growth in response to microenvironmental cues through the control of EGFR. PHD3 silencing in human gliomas or genetic deletion in a murine high-grade astrocytoma model markedly promotes tumour growth and the ability of tumours to continue growing under unfavourable conditions. The growth-suppressive function of PHD3 is independent of the established PHD3 targets HIF and NF-κB and its hydroxylase activity. Instead, loss of PHD3 results in hyperphosphorylation of epidermal growth factor receptor (EGFR). Importantly, epigenetic/genetic silencing of PHD3 preferentially occurs in gliomas without EGFR amplification. Our findings reveal that PHD3 inactivation provides an alternative route of EGFR activation through which tumour cells sustain proliferative signalling even under conditions of limited oxygen availability.

PHD2 regulates arteriogenic macrophages through TIE2 signalling.

Occlusion of the main arterial route redirects blood flow to the collateral circulation. We previously reported that macrophages genetically modified to express low levels of prolyl hydroxylase domain protein 2 (PHD2) display an arteriogenic phenotype, which promotes the formation of collateral vessels and protects the skeletal muscle from ischaemic necrosis. However, the molecular mechanisms underlying this process are unknown. Here, we demonstrate that femoral artery occlusion induces a switch in macrophage phenotype through angiopoietin-1 (ANG1)-mediated Phd2 repression. ANG blockade by a soluble trap prevented the downregulation of Phd2 expression in macrophages and their phenotypic switch, thus inhibiting collateral growth. ANG1-dependent Phd2 repression initiated a feed-forward loop mediated by the induction of the ANG receptor TIE2 in macrophages. Gene silencing and cell depletion strategies demonstrate that TIE2 induction in macrophages is required to promote their proarteriogenic functions, enabling collateral vessel formation following arterial obstruction. These results indicate an indispensable role for TIE2 in sustaining in situ programming of macrophages to a proarteriogenic, M2-like phenotype, suggesting possible new venues for the treatment of ischaemic disorders.

Gene-targeting of Phd2 improves tumor response to chemotherapy and prevents side-toxicity.

The success of chemotherapy in cancer treatment is limited by scarce drug delivery to the tumor and severe side-toxicity. Prolyl hydroxylase domain protein 2 (PHD2) is an oxygen/redox-sensitive enzyme that induces cellular adaptations to stress conditions. Reduced activity of PHD2 in endothelial cells normalizes tumor vessels and enhances perfusion. Here, we show that tumor vessel normalization by genetic inactivation of Phd2 increases the delivery of chemotherapeutics to the tumor and, hence, their antitumor and antimetastatic effect, regardless of combined inhibition of Phd2 in cancer cells. In response to chemotherapy-induced oxidative stress, pharmacological inhibition or genetic inactivation of Phd2 enhances a hypoxia-inducible transcription factor (HIF)-mediated detoxification program in healthy organs, which prevents oxidative damage, organ failure, and tissue demise. Altogether, our study discloses alternative strategies for chemotherapy optimization.

Feedback regulators of hypoxia-inducible factors and their role in cancer biology.

Malignant tumors are characterized by regions of low oxygen concentration (hypoxia). The hypoxic tumor microenvironment contributes to tumor progression by activating a set of adaptive responses via the key transcriptional regulators HIF-1alpha and HIF-2alpha. These factors have been traditionally linked to an aggressive tumor phenotype by promoting processes essential for tumor growth, such as angiogenesis, glycolysis, metastasis and invasion, as well as differentiation and self renewal. Notably, the complex HIF pathway also initiates anti-tumorigenic mechanisms that lead to cell cycle arrest or cell death, indicating the need for a stringent control of the extent and the direction of the hypoxia response. The importance of this control for tumor cell survival is illustrated by the intricate regulation of HIF activity at the mRNA, protein and epigenetic level by a complex network of positive and negative feedback regulators. We propose that these feedback regulators help to flexibly adjust and adapt HIF activated responses to the fluctuating oxygen concentrations within tumors during acute and chronic hypoxia and to curtail the tumor-suppressing components of the HIF pathway. Moreover, feedback regulation of HIF induces a switch from HIF-1alpha to HIF-2alpha driven responses under chronic hypoxia which may have essential functions in the regulation of tumor cell differentiation and tumor stem cell maintenance. Given their central role in cancer biology, HIF feedback regulators may represent an attractive and novel anti-tumor therapy target to overcome cell death resistance in tumors.

A hypoxic niche regulates glioblastoma stem cells through hypoxia inducible factor 2 alpha.

Glioma growth and progression depend on a specialized subpopulation of tumour cells, termed tumour stem cells. Thus, tumour stem cells represent a critical therapeutic target, but the molecular mechanisms that regulate them are poorly understood. Hypoxia plays a key role in tumour progression and in this study we provide evidence that the hypoxic tumour microenvironment also controls tumour stem cells. We define a detailed molecular signature of tumour stem cell genes, which are overexpressed by tumour cells in vascular and perinecrotic/hypoxic niches. Mechanistically, we show that hypoxia plays a key role in the regulation of the tumour stem cell phenotype through hypoxia-inducible factor 2alpha and subsequent induction of specific tumour stem cell signature genes, including mastermind-like protein 3 (Notch pathway), nuclear factor of activated T cells 2 (calcineurin pathway) and aspartate beta-hydroxylase domain-containing protein 2. Notably, a number of these genes belong to pathways regulating the stem cell phenotype. Consistently, tumour stem cell signature genes are overexpressed in newly formed gliomas and are associated with worse clinical prognosis. We propose that tumour stem cells are maintained within a hypoxic niche, providing a functional link between the well-established role of hypoxia in stem cell and tumour biology. The identification of molecular regulators of tumour stem cells in the hypoxic niche points to specific signalling mechanisms that may be used to target the glioblastoma stem cell population.

Prolyl hydroxylases 2 and 3 act in gliomas as protective negative feedback regulators of hypoxia-inducible factors.

Adaptive responses to hypoxia in tumors are transcriptionally regulated by the hypoxia inducible factors (HIF-1alpha/HIF-2alpha), which are tightly controlled by the HIF-prolyl hydroxylases (PHD). Hypoxia induces expression of the PHD2 and PHD3 proteins in tumors but the pathobiological significance of these events is uncertain. Here, we show that PHD2 and PHD3 induction acts within a negative feedback loop to limit the hypoxic HIF response. In glioblastomas, PHD2 and PHD3 are hypoxia-inducible in vitro and expressed in hypoxic areas of tumors in vivo. Comparison with other PHDs revealed distinct cytoplasmatic and nuclear localization patterns of PHD2 and PHD3. Gain and loss of function experiments defined PHD2 and PHD3 as HIF target genes that remained operative even at low oxygen concentrations. We found that increased PHD levels could compensate for reduced oxygen availability to regulate the HIF response. This negative feedback loop protected tumor cells against hypoxia-induced cell death, functionally implicating this pathway in the control of the tumor-suppressive components of the HIF system in glioblastoma. Moreover, PHD inhibition facilitated cell death induction by staurosporine or tumor necrosis factor-related apoptosis-inducing ligand, hinting at a more general protective role of PHD in the regulation of cell viability. In summary, our findings recognize the PHD/HIF regulatory axis as a novel therapeutic target to disable a tumor's ability to adjust to hypoxic conditions and control cell survival, helping to potentially overcome therapeutic cell death resistance in glioblastomas.