Secreted CLIC3 drives cancer progression through its glutathione-dependent oxidoreductase activity

The secretome of cancer and stromal cells generates a microenvironment that contributes to tumour cell invasion and angiogenesis. Here we compare the secretome of human mammary normal and cancer-associated fibroblasts (CAFs). We discover that the chloride intracellular channel protein 3 (CLIC3) is an abundant component of the CAF secretome. Secreted CLIC3 promotes invasive behaviour of endothelial cells to drive angiogenesis and increases invasiveness of cancer cells both in vivo and in 3D cell culture models, and this requires active transglutaminase-2 (TGM2). CLIC3 acts as a glutathione-dependent oxidoreductase that reduces TGM2 and regulates TGM2 binding to its cofactors. Finally, CLIC3 is also secreted by cancer cells, is abundant in the stromal and tumour compartments of aggressive ovarian cancers and its levels correlate with poor clinical outcome. This work reveals a previously undescribed invasive mechanism whereby the secretion of a glutathione-dependent oxidoreductase drives angiogenesis and cancer progression by promoting TGM2-dependent invasion

Hypoxia Induces VEGF-C Expression in Metastatic Tumor Cells via a HIF-1α-Independent Translation-Mediated Mechanism

SummaryVarious tumors metastasize via lymph vessels and lymph nodes to distant organs. Even though tumors are hypoxic, the mechanisms of how hypoxia regulates lymphangiogenesis remain poorly characterized. Here, we show that hypoxia reduced vascular endothelial growth factor C (VEGF-C) transcription and cap-dependent translation via the upregulation of hypophosphorylated 4E-binding protein 1 (4E-BP1). However, initiation of VEGF-C translation was induced by hypoxia through an internal ribosome entry site (IRES)-dependent mechanism. IRES-dependent VEGF-C translation was independent of hypoxia-inducible factor 1α (HIF-1α) signaling. Notably, the VEGF-C IRES activity was higher in metastasizing tumor cells in lymph nodes than in primary tumors, most likely because lymph vessels in these lymph nodes were severely hypoxic. Overall, this transcription-independent but translation-dependent upregulation of VEGF-C in hypoxia stimulates lymphangiogenesis in tumors and lymph nodes and may contribute to lymphatic metastasis

Tumour hypoxia causes DNA hypermethylation by reducing TET activity

Hypermethylation of the promoters of tumour suppressor genes represses transcription of these genes, conferring growth advantages to cancer cells. How these changes arise is poorly understood. Here we show that the activity of oxygen-dependent ten-eleven translocation (TET) enzymes is reduced by tumour hypoxia in human and mouse cells. TET enzymes catalyse DNA demethylation through 5-methylcytosine oxidation. This reduction in activity occurs independently of hypoxia-associated alterations in TET expression, proliferation, metabolism, hypoxia-inducible factor activity or reactive oxygen species, and depends directly on oxygen shortage. Hypoxia-induced loss of TET activity increases hypermethylation at gene promoters in vitro. In patients, tumour suppressor gene promoters are markedly more methylated in hypoxic tumour tissue, independent of proliferation, stromal cell infiltration and tumour characteristics. Our data suggest that up to half of hypermethylation events are due to hypoxia, with these events conferring a selective advantage. Accordingly, increased hypoxia in mouse breast tumours increases hypermethylation, while restoration of tumour oxygenation abrogates this effect. Tumour hypoxia therefore acts as a novel regulator of DNA methylatio

The role of PHD2 in breast cancer metastasis.

Breast cancer (BC) is the most frequently diagnosed cancer in women in the US and Europe, affecting one in eight women. Despite improvements in radiotherapy, cytotoxic, hormonal, and targeted therapies, BC remains the second leading cause of cancer deaths in women, exceeded only by lung cancer. Metastatic relapse is a main cause of this high mortality, and occurs up to long periods of time after the removal of the primary tumor. Understanding the mechanisms that control metastasis is therefore pivotal for the design of improved and safe breast cancer treatment regimen. Hypoxia is a characteristic feature of most solid tumors, including BC, and is a strong stimulus of tumor cell invasion and metastasis. Hypoxia signaling regulates nearly every single step of the metastatic cascade, including epithelial-to-mesenchymal transition (EMT), intravasation, survival in the circulation, formation of the pre-metastatic niche, and growth from micro- to macro-metastatic lesions. Furthermore, hypoxic tumors display lower sensitivity to treatment, leading to poor prognosis. The hypoxia-inducible transcription factors (HIFs) mediate a variety of cellular adaptations to hypoxia. Prolyl-hydroxylases (PHD1-3) are oxygen sensors that regulate HIF levels in normoxia by targeting it for proteasomal degradation. Despite the crucial role of PHD2 as an oxygen sensor, its role in tumor growth and metastasis in general and of BC in particular, remains largely debated. Studies from our and other research teams on PHD2 in cancer yielded interesting results, highlighting different possible roles of PHD2 that may be cell-type dependent. On the one hand, the host lab demonstrated that haplodeficiency of PHD2 selectively in endothelial cells (ECs) reduced metastasis without affecting tumor growth, by normalizing the abnormal tumor vessels and reducing tumor cell intravasation, suggesting that PHD2 could be an anti-BC drug target. Using transplantable tumor models, others reported that silencing of PHD2 in cancer cells either increased or decreased tumor growth with different underlying mechanisms. Dissection of the role of PHD2 in conditions that allow the evaluation of cell-intrinsic effects as well as the impact of bidirectional tumor / stroma cross-talk, remains strongly warranted. This is particularly relevant in light of pharmacological PHD2 blockade, which would target PHD2 in all cells inside the tumor. Furthermore, the studies mentioned above only used transplantable tumor models. The role of PHD2 in breast cancer using a clinically more relevant spontaneously arising BC model thus remains undefined. In this study, we utilized the spontaneously arising PyMT-oncogene driven breast cancer model and intercrossed this transgenic line with mice with heterozygous gene deficiency of PHD2 (PHD2+/- mice; named PyMT+/- mice upon intercross with the the PyMT line). Tumor growth was unaffected, but metastasis and intravasation were reduced in PyMT+/- mice as compared to control mice (PyMT mice intercrossed with PHD2 wild type mice; named PyMT+/+ mice). Applying genetic strategies in vivo and in vitro, we show that this reduction in metastasis and intravasation can be ascribed to two independent mechanisms. First, we show that global “genetic targeting” of PHD2 in the entire tumor in PyMT+/- mice induces tumor vessel normalization (a.o. tighter endothelial lining, improved pericyte coverage, better perfused), similar to selective PHD2 haplodeficiency in ECs in xenograft models. Secondly, reduction in metastasis was also attributable to reduced activation of cancer-associated fibroblasts (CAFs). As compared to PyMT+/+ tumors, PyMT+/- tumors contained fewer activated CAFs, which deposited less cross-linked collagen matrix and contracted the collagen matrix less. These processes are known to induce cancer cell invasion. We showed that reduced CAF activation was independent of PHD2 level in fibroblasts, but reliant on the level of PHD2 in cancer cells. PHD2 haplodeficiency in cancer cells lowered the release of TGF-b1 and diminished the differentiation of normal fibroblasts to activated CAFs. Taken together, these results provide evidence that PHD2 is a potential therapeutical target; the inhibition of which can offer substantial anti-metastatic benefit. Additionally, improved vessel function in spontaneously developing tumor model by PHD2 haplodeficiency could increase chemotherapy delivery and thus provide an advantage during surgical tumor resection. Hypoxia is a characteristic feature of most solid tumors, including BC, and is a strong stimulus of tumor cell invasion and metastasis. Hypoxia signaling regulates nearly every single step of the metastatic cascade, including epithelial-to-mesenchymal transition (EMT), intravasation, survival in the circulation, formation of the pre-metastatic niche, and growth from micro- to macro-metastatic lesions. Furthermore, hypoxic tumors display lower sensitivity to treatment, leading to poor prognosis. The hypoxia-inducible transcription factors (HIFs) mediate a variety of cellular adaptations to hypoxia. Prolyl-hydroxylases (PHD1-3) are oxygen sensors that regulate HIF levels in normoxia by targeting it for proteasomal degradation. Despite the crucial role of PHD2 as an oxygen sensor, its role in tumor growth and metastasis in general and of BC in particular, remains largely debated. Studies from our and other research teams on PHD2 in cancer yielded interesting results, highlighting different possible roles of PHD2 that may be cell-type dependent. On the one hand, the host lab demonstrated that haplodeficiency of PHD2 selectively in endothelial cells (ECs) reduced metastasis without affecting tumor growth, by normalizing the abnormal tumor vessels and reducing tumor cell intravasation, suggesting that PHD2 could be an anti-BC drug target. Using transplantable tumor models, others reported that silencing of PHD2 in cancer cells either increased or decreased tumor growth with different underlying mechanisms. Dissection of the role of PHD2 in conditions that allow the evaluation of cell-intrinsic effects as well as the impact of bidirectional tumor / stroma cross-talk, remains strongly warranted. This is particularly relevant in light of pharmacological PHD2 blockade, which would target PHD2 in all cells inside the tumor. Furthermore, the studies mentioned above only used transplantable tumor models. The role of PHD2 in breast cancer using a clinically more relevant spontaneously arising BC model thus remains undefined. In this study, we utilized the spontaneously arising PyMT-oncogene driven breast cancer model and intercrossed this transgenic line with mice with heterozygous gene deficiency of PHD2 (PHD2+/- mice; named PyMT+/- mice upon intercross with the the PyMT line). Tumor growth was unaffected, but metastasis and intravasation were reduced in PyMT+/- mice as compared to control mice (PyMT mice intercrossed with PHD2 wild type mice; named PyMT+/+ mice). Applying genetic strategies in vivo and in vitro, we show that this reduction in metastasis and intravasation can be ascribed to two independent mechanisms. First, we show that global “genetic targeting” of PHD2 in the entire tumor in PyMT+/- mice induces tumor vessel normalization (a.o. tighter endothelial lining, improved pericyte coverage, better perfused), similar to selective PHD2 haplodeficiency in ECs in xenograft models. Secondly, reduction in metastasis was also attributable to reduced activation of cancer-associated fibroblasts (CAFs). As compared to PyMT+/+ tumors, PyMT+/- tumors contained fewer activated CAFs, which deposited less cross-linked collagen matrix and contracted the collagen matrix less. These processes are known to induce cancer cell invasion. We showed that reduced CAF activation was independent of PHD2 level in fibroblasts, but reliant on the level of PHD2 in cancer cells. PHD2 haplodeficiency in cancer cells lowered the release of TGF-B1 and diminished the differentiation of normal fibroblasts to activated CAFs. Taken together, these results provide evidence that PHD2 is a potential therapeutical target; the inhibition of which can offer substantial anti-metastatic benefit. Additionally, improved vessel function in spontaneously developing tumor model by PHD2 haplodeficiency could increase chemotherapy delivery and thus provide an advantage during surgical tumor resection.nrpages: 158status: publishe

The PHD2 oxygen sensor paves the way to metastasis

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How to teach an old dog new tricks: autophagy-independent action of chloroquine on the tumor vasculature

Chloroquine (CQ) is exploited in clinical trials as an autophagy blocker to potentiate anticancer therapy, but it is unknown if it solely acts by inhibiting cancer cell-autonomous autophagy. Our recent study shows that besides blocking cancer cell growth, CQ also affects endothelial cells (ECs) and promotes tumor vessel normalization. This vessel normalizing effect of CQ reduces tumor hypoxia, cancer cell intravasation, and metastasis, while improving the delivery and response to chemotherapy. By compromising autophagy in melanoma cells or using mice with a conditional knockout of ATG5 in ECs, we found that the favorable effects of CQ on the tumor vasculature do not rely on autophagy. CQ-induced vessel normalization relies mainly on altered endolysosomal trafficking and sustained NOTCH1 signaling in ECs. Remarkably these CQ-mediated effects are abrogated when tumors are grown in mice harboring EC-specific deletion of NOTCH1. The autophagy-independent vessel normalization by CQ leading to improved delivery and tumor response to chemotherapy further advocates its clinical use in combination with anticancer treatments.peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=kaup20status: publishe

Chloroquine anticancer activity is mediated by autophagy-independent effects on the tumor vasculature

Chloroquine is used clinically as an autophagy blocker to potentiate anticancer treatments. However, whether chloroquine acts solely through autophagy-dependent and cancer cell autonomous mechanisms has remained elusive. In a recent study we found that chloroquine reduced intratumoral hypoxia and metastasis, while improving chemotherapy response, largely through an autophagy-independent, NOTCH1-reliant mechanism of tumor vessel normalization.peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=kmco20status: publishe

Metabolism of stromal and immune cells in health and disease

Cancer cells have been at the centre of cell metabolism research, but the metabolism of stromal and immune cells has received less attention. Nonetheless, these cells influence the progression of malignant, inflammatory and metabolic disorders. Here we discuss the metabolic adaptations of stromal and immune cells in health and disease, and highlight how metabolism determines their differentiation and function.status: publishe

Emerging novel functions of the oxygen-sensing prolyl hydroxylase domain enzymes

Oxygen-sensing prolyl hydroxylase domain enzymes (PHDs) target hypoxia-inducible factor (HIF)-α subunits for proteasomal degradation in normoxia through hydroxylation. Recently, novel mechanisms of PHD activation and function have been unveiled. Interestingly, PHD3 can unexpectedly amplify HIF signaling through hydroxylation of the glycolytic enzyme pyruvate kinase (PK) muscle isoform 2 (PKM2). Recent studies have also yielded insight into HIF-independent PHD functions, including the control of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking in synaptic transmission and the activation of transient receptor potential cation channel member A1 (TRPA1) ion channels by oxygen levels in sensory nerves. Finally, PHD activation has been shown to involve the iron chaperoning function of poly(rC) binding protein (PCBP)1 and the (R)-enantiomer of 2-hydroxyglutarate (2-HG). The intersection of these regulatory pathways and interactions highlight the complexity of PHD regulation and function.Reviewstatus: publishe

Synthesis, cytotoxicity testing, and structure-activity relationships of novel 6-chloro-7-(4-phenylimino-4H-3,1-benzoxazin-2-yl)-3-(substituted)-1,4,2-benzodithiazine 1,1-dioxides

A new series of 16 6-chloro-1,1-dioxo-7-{4-[(4-R(1)-phenyl)imino]-4H-3,1-benzoxazin-2-yl}-3-(substituted amino)-1,4,2-benzodithiazines 7-22 was prepared in order to evaluate the cytotoxic activity against six human cancer cell lines. The structures of the new compounds were confirmed by IR, (1)H-, and (13)C-NMR, elemental analysis and in the cases of 11 and 31 by X-ray crystal structure analysis. This analysis showed that contrary to our earlier report the structures contain a benzoxazine ring instead of the proposed quinazolinone ring. The bioassay indicated that the benzodithiazine derivatives 7-22 possess cancer cell growth-inhibitory properties. Some compounds showed a high level of selectivity for certain cell lines. The most active compounds 11, 12, 16, 19, 21, and 22 exhibited potency higher or comparable to cisplatin. The compounds were particularly effective in LCLC-103H and MCF-7 cell lines with IC(50) values of 0.49-1.60 µM. Quantitative structure activity relationships (QSAR) revealed that a chloro substituent R(1) in the phenyl ring as well as the shape of the substituted amino group at R(2) (e.g., unsaturation is beneficial) are important for potency.status: publishe