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

Cancer associated fibroblasts: the architects of stroma remodelling

Fibroblasts have exceptional phenotypic plasticity and capability to secrete vast amount of soluble factors, ECM components and extracellular vesicles. While in physiological conditions this makes fibroblasts master regulators of tissue homeostasis and healing of injured tissues, in solid tumours cancer-associated fibroblasts (CAFs) co-evolve with the disease, and alter the biochemical and physical structure of the tumour microenvironment, as well as the behaviour of the surrounding stromal and cancer cells. Thus CAFs are fundamental regulators of tumour progression and influence response to therapeutic treatments. Increasing efforts are devoted to better understand the biology of CAFs to bring insights to develop complementary strategies to target this cell type in cancer. Here we highlight components of the tumour microenvironment that play key roles in cancer progression and invasion, and provide an extensive overview of past and emerging understanding of CAF biology as well as the contribution that mass spectrometry (MS)-based proteomics has made to this field

Emerging tumor spheroids technologies for 3D in vitro cancer modeling

"Article in Press, Available online 31 October 2017" ; "S0163-7258(17)30268-1"Cancer is a leading cause of mortality and morbidity worldwide. Around 90% of deaths are caused by metastasis and just 10% by primary tumor. The advancement of treatment approaches is not at the same rhythm of the disease; making cancer a focal target of biomedical research. To enhance the understanding and promts the therapeutic delivery; concepts of tissue engineering are applied in the development of in vitro models that can bridge between 2D cell culture and animal models, mimicking tissue microenvironment. Tumor spheroid represents highly suitable 3D organoid-like framework elucidiating the intra and inter cellular signaling of cancer, like that formed in physiological niche. However, spheroids are of limited value in studying critical biological phenomenon such as tumor-stroma interactons involving extra cellular matrix or immune system. Therefore, a compelling need of tailoring spheroid technologies with physiologically relevant biomaterials or in silico models, is ever emerging. The diagnostic and prognostic role of spheroids rearrangements within biomaterials or microfluidic channel is indicative of patient management; particularly for the decision of targated therapy. Fragmented information on available in vitro spheroid models and lack of critical analysis on transformation aspects of these strategies; pushes the urge to comprehensively overview the recent technological advancements (e.g. bioprinting, micro-fluidic technologies or use of biomaterials to attain the third dimension) in the shed of tranlationable cancer research. In present article, relationships between current models and their possible exploitation in clinical success is explored with the highlight of existing challenges in defining therapeutic targets and screening of drug efficacy.The authors are thankful to European Union (Horizon 2020) funded project FoReCaST (No. 668983), the FCT fellowship to J. Silva-Correia (Grant No. SFRH/BPD/100590/2014), distinctions to J.M.O. under the Investigator FCT program (IF/00423/2012) and V.M.C. under the Investigator FCT program (IF/01214/2014) for supporting this work financially.info:eu-repo/semantics/publishedVersio

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

Various 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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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

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