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Vasculogenic Mimicry (VM) refers to the capacity to form a blood network from aggressive cancer cells in an independent way of endothelial cells, to provide nutrients and oxygen leading to enhanced microenvironment complexity and treatment failure. In a previous study, we demonstrated that VE-Cadherin and its phosphorylation at Y658 modulated kaiso-dependent gene expression (CCND1 and Wnt 11) through a pathway involving Focal Adhesion kinase (FAK). In the present research, using a proteomic approach, we have found that ß-catenin/TCF-4 is associated with nuclear VE-cadherin and enhances the capacity of malignant melanoma cells to undergo VM in cooperation with VE-Cadherin; in addition, preventing the phosphorylation of Y658 of VEcadherin upon FAK disabling resulted in VE-Cadherin/ß-catenin complex dissociation, increased ß-catenin degradation while reducing TCF-4-dependent genes transcription (C-Myc and Twist-1). Uveal melanoma cells knockout for VE-Cadherin loses ß-catenin expression while the rescue of VE-Cadherin (but not of the phosphorylation defective VE-Cadherin Y658F mutant) permits stabilization of ß-catenin and tumor growth reduction in vivo experiments. In vivo, the concomitant treatment with the FAK inhibitor PF-271 and the anti-angiogenic agent bevacizumab leads to a strong reduction in tumor growth concerning the single treatment. In conclusion, the anomalous expression of VE-Cadherin in metastatic melanoma cells (from both uveal and cutaneous origins), together with its permanent phosphorylation at Y658, favors the induction of the aggressive VM phenotype through the cooperation of ß-catenin with VE-Cadherin and by enhancing TCF-4 genes-dependent transcription.

Hyperoxia is used in order to counteract hypoxia effects in the TME (tumor microenvironment), which are described to boost the malignant tumor phenotype and poor prognosis. The reduction of tumor hypoxic state through the formation of a non-aberrant vasculature or an increase in the toxicity of the therapeutic agent improves the efficacy of therapies such as chemotherapy. Radiotherapy efficacy has also improved, where apoptotic mechanisms seem to be implicated. Moreover, hyperoxia increases the antitumor immunity through diverse pathways, leading to an immunopermissive TME. Although hyperoxia is an approved treatment for preventing and treating hypoxemia, it has harmful side-effects. Prolonged exposure to high oxygen levels may cause acute lung injury, characterized by an exacerbated immune response, and the destruction of the alveolar-capillary barrier. Furthermore, under this situation, the high concentration of ROS may cause toxicity that will lead not only to cell death but also to an increase in chemoattractant and proinflammatory cytokine secretion. This would end in a lung leukocyte recruitment and, therefore, lung damage. Moreover, unregulated inflammation causes different consequences promoting tumor development and metastasis. This process is known as protumor inflammation, where different cell types and molecules are implicated; for instance, IL-1ß has been described as a key cytokine. Although current results show benefits over cancer therapies using hyperoxia, further studies need to be conducted, not only to improve tumor regression, but also to prevent its collateral damage.

This volume provides detailed protocols for the identification and understanding of vasculogenic mimicry process in vitro and in vivo, in addition to protocols for microscopy and histology. Chapters guide readers through different materials, commercial and homemade scaffolds, Matrigel, cancer spheroids, 3D tissue constructs, vasculogenic processes, and mathematical model building. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols. Authoritative and cutting-edge, Vasculogenic Mimicry: Methods and Protocols aims to be a useful and practical guide to new researchers and experts looking to expand their knowledge.

The conditions of tumor cell growth are characterized by low oxygen supply (hypoxia) caused by insufficient blood delivery. Hypoxic cancers have a strong invasive potential, metastasis, and resistance to therapy. The key regulator of adaptation to tumor hypoxia is hypoxia inducible factor 1-¿ (HIF-1¿). Hyperoxia could be a clinically relevant treatment to fight tumors that present with hypoxia through the modification of tumor microenvironment.

The local conditions of tumor cell growth, known as the tumor microenvironment (TME), are characterized by low oxygen supply (hypoxia) caused by insufficient blood delivery. Hypoxia cancers have a strong invasive potential, metastasis, resistance to therapy and a poor clinical prognosis. The key regulator of adaptation to tumor hypoxia is hypoxia inducible factor 1-¿ (HIF-1¿). Despite its significance, the underlying transformations that cause this highly aggressive behaviour are poorly understood, specific pharmacological inhibition of HIF-1¿ activation in the tumor are not available. Our group has shown that PARP inhibitors can modulate HIF-1¿ levels and its activation. On the other hand, hyperoxia could be a treatment of medical interest to fight tumors that present with hypoxia; nevertheless, its use may involve clinically unacceptable lung damage. AIMS: In this study, we aim to demonstrate that the use of PARP inhibitors (1) will interfere with the adaptation of the tumor to the hypoxic microenvironment (which is recreated with the hypoxia-mimetic,CoCl2), in combination with the use with oxygen to induce hyperoxia and (2) will decrease the lung damage induced by reactive oxygen species. Therefore, the combined use of oxygen and PARP inhibitors in metastatic melanoma (expressing high levels of HIF-1) could delay metastasis and improve the efficacy of anti-tumor therapy.

Mechanism-based strategies to overcome resistance to PD-1 blockade therapy are urgently needed. We developed genetic acquired resistant models of JAK1, JAK2, and B2M loss-of-function mutations by gene knockout in human and murine cell lines. Human melanoma cell lines with JAK1/2 knockout became insensitive to IFN-induced antitumor effects, while B2M knockout was no longer recognized by antigen-specific T cells and hence was resistant to cytotoxicity. All of these mutations led to resistance to anti¿PD-1 therapy in vivo. JAK1/2-knockout resistance could be overcome with the activation of innate and adaptive immunity by intratumoral Toll-like receptor 9 agonist administration together with anti¿PD-1, mediated by natural killer (NK) and CD8 T cells. B2M-knockout resistance could be overcome by NK-cell and CD4 T-cell activation using the CD122 preferential IL2 agonist bempegaldesleukin. Therefore, mechanistically designed combination therapies can overcome genetic resistance to PD-1 blockade therapy. Significance: The activation of IFN signaling through pattern recognition receptors and the stimulation of NK cells overcome genetic mechanisms of resistance to PD-1 blockade therapy mediated through deficient IFN receptor and antigen presentation pathways. These approaches are being tested in the clinic to improve the antitumor activity of PD-1 blockade therapy.

Aberrant extra-vascular expression of VE-cadherin has been observed in metastasis associated with Vasculogenic Mimicry (VM); we have recently shown that in VM prone cells VE-cadherin is mainly in the form of phospho-VE-cadherin in Y658 allowing increased plasticity that potentiates VM development in malignant cells. In the current study, we present results to show that human malignant melanoma cells VM+, express the VE-cadherin phosphatase VE-PTP. VE-PTP forms a complex with VE-Cadherin and p120-catenin and the presence of this complex act as a safeguard to prevent VE-Cadherin protein degradation by autophagy. Indeed, VE-PTP silencing results in complete degradation of VE-cadherin with the features of autophagy. In summary, this study shows that VE-PTP is involved in VM formation and disruption of VE-PTP/VE-Cadherin/p120 complex results in enhanced autophagy in aggressive VM cells. Thus, we identify VE-PTP as a key player in VM development by regulating VE-cadherin protein degradation through autophagy.

Poly(ADP-ribose) polymerases (PARPs), represent a family of 17 proteins implicated in a variety of cell functions; some of them possess the enzymatic ability to synthesize and attach poly (ADP-ribose) (also known as PAR) to different protein substrates by a post-translational modification; PARPs are key components in the cellular response to stress with consequences for different physiological and pathological events, especially during neoplasia. In recent years, using PARP inhibitors as antitumor agents has raised new challenges in understanding their role in tumor biology. Notably, the function of PARPs and PAR in the dynamic of tumor microenvironment is only starting to be understood. In this review, we summarized the conclusions arising from recent studies on the interaction between PARPs, PAR and key features of tumor microenvironment such as hypoxia, autophagy, tumor initiating cells, angiogenesis and cancer-associated immune response.

Tankyrase 1 (TNKS1) and Tankyrase 2 (TNKS2) are two proteins that form a distinct subgroup inside the PARP family. Both TNKS share 82% of its sequence and have been linked to different cellular functions such as mitotic progression, glucose metabolism, stress granule formation and Wnt signaling. Furthermore, altered levels of TNKS1 and/or TNKS2 expression have been reported in several types of cancer such as colon, lung or brain. Both tankyrases synthesize linear chains of poly(ADP-ribose) (PAR) to produce posttranslational modifications of their target proteins and also itself through automodification. PARylation by TNKS appears to be tightly linked to ubiquitination by ubiquitin E3 ligases like RNF146. Deficient angiogenesis leads to tumor hypoxia resulting in increased aggressiveness and therapeutic resistance. The adaptation to this situation is carried out by the heterodimeric transcription factors hypoxia-inducible factor (HIF). In particular, the oxygen-dependent protein HIF-1/HIF-2 and the constitutively expressed protein HIF-1 are responsible for the induction of genes that allow the adaptation and survival of cells to hypoxia.

Aberrant extra-vascular expression of VE-cadherin (VEC) has been observed in metastasis associated with vasculogenic mimicry (VM); however, the ultimate reason why non-endothelial VEC favors the acquisition of this phenotype is not established. In this study, we show that human malignant melanoma cells have a constitutively high expression of phoshoVEC (pVEC) at Y658; pVEC is a target of focal adhesion kinase (FAK) and forms a complex with p120-catenin and the transcriptional repressor kaiso in the nucleus. FAK inhibition enabled kaiso to suppress the expression of its target genes and enhanced kaiso recruitment to KBS-containing promoters. Finally we have found that ablation of kaiso-repressed genes WNT11 and CCDN1 abolished VM. Thus, identification of pVEC as a component of the kaiso transcriptional complex establishes a molecular paradigm that links FAK-dependent phosphorylation of VEC as a major mechanism by which ectopical VEC expression exerts its function in VM.

Glioblastoma (GBM) is the most common and aggressive brain tumor and is associated with poor prognosis. GBM cells are frequently resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and finding new combinatorial therapies to sensitize glioma cells to TRAIL remains an important challenge. PIM kinases are serine/threonine kinases that promote cell survival and proliferation and are highly expressed in different tumors. In this work, we studied the role of PIM kinases as regulators of TRAIL sensitivity in GBM cells. Remarkably, PIM inhibition or knockdown facilitated activation by TRAIL of a TRAIL-R2/DR5-mediated and mitochondria-operated apoptotic pathway in TRAIL-resistant GBM cells. The sensitizing effect of PIM knockdown on TRAIL-induced apoptosis was mediated by enhanced caspase-8 recruitment to and activation at the death-inducing signaling complex (DISC). Interestingly, TRAIL-induced internalization of TRAIL-R2/DR5 was significantly reduced in PIM knockdown cells. Phospho-proteome profiling revealed a decreased phosphorylation of p62/SQSTM1 after PIM knockdown. Our results also showed an interaction between p62/SQSTM1 and the DISC that was reverted after PIM knockdown. In line with this, p62/SQSTM1 ablation increased TRAIL-R2/DR5 levels and facilitated TRAIL-induced caspase-8 activation, revealing an inhibitory role of p62/SQSTM1 in TRAIL-mediated apoptosis in GBM. Conversely, upregulation of TRAIL-R2/DR5 upon PIM inhibition and apoptosis induced by the combination of PIM inhibitor and TRAIL were abrogated by a constitutively phosphorylated p62/SQSTM1(S332E) mutant. Globally, our data represent the first evidence that PIM kinases regulate TRAIL-induced apoptosis in GBM and identify a specific role of p62/SQSTM1( Ser332) phosphorylation in the regulation of the extrinsic apoptosis pathway activated by TRAIL.

AMPK is a central energy sensor linking extracellular milieu fluctuations with the autophagic machinery. In the current study we uncover that Poly(ADP-ribosyl)ation (PARylation), a post-translational modification (PTM) of proteins, accounts for the spatial and temporal regulation of autophagy by modulating AMPK subcellular localisation and activation. More particularly, we show that the minority AMPK pool needs to be exported to the cytosol in a PARylation-dependent manner for optimal induction of autophagy, including ULK1 phosphorylation and mTORC1 inactivation. PARP-1 forms a molecular complex with AMPK in the nucleus in non-starved cells. In response to nutrient deprivation, PARP-1 catalysed PARylation, induced the dissociation of the PARP-1/AMPK complex and the export of free PARylated nuclear AMPK to the cytoplasm to activate autophagy. PARP inhibition, its silencing or the expression of PARylation-deficient AMPK mutants prevented not only the AMPK nuclear-cytosolic export but also affected the activation of the cytosolic AMPK pool and autophagosome formation. These results demonstrate that PARylation of AMPK is a key early signal to efficiently convey extracellular nutrient perturbations with downstream events needed for the cell to optimize autophagic commitment before autophagosome formation.

In this study, evidence is provided for the role of fructose-1,6-bisphosphatases (FBPases) in plant development and carbohydrate synthesis and distribution by analysing two Arabidopsis thaliana T-DNA knockout mutant lines, cyfbp and cfbp1, and one double mutant cyfbp cfbp1 which affect each FBPase isoform, cytosolic and chloroplastic, respectively. cyFBP is involved in sucrose synthesis, whilst cFBP1 is a key enzyme in the Calvin–Benson cycle. In addition to the smaller rosette size and lower rate of photosynthesis, the lack of cFBP1 in the mutants cfbp1 and cyfbp cfbp1 leads to a lower content of soluble sugars, less starch accumulation, and a greater superoxide dismutase (SOD) activity. The mutants also had some developmental alterations, including stomatal opening defects and increased numbers of root vascular layers. Complementation also confirmed that the mutant phenotypes were caused by disruption of the cFBP1 gene. cyfbp mutant plants without cyFBP showed a higher starch content in the chloroplasts, but this did not greatly affect the phenotype. Notably, the sucrose content in cyfbp was close to that found in the wild type. The cyfbp cfbp1 double mutant displayed features of both parental lines but had the cfbp1 phenotype. All the mutants accumulated fructose-1,6-bisphosphate and triose-phosphate during the light period. These results prove that while the lack of cFBP1 induces important changes in a wide range of metabolites such as amino acids, sugars, and organic acids, the lack of cyFBP activity in Arabidopsis essentially provokes a carbon metabolism imbalance which does not compromise the viability of the double mutant cyfbp cfbp1.

In this study, evidence is provided for the role of fructose-1,6-bisphosphatases (FBPases) in plant development and carbohydrate synthesis and distribution by analysing two Arabidopsis thaliana T-DNA knockout mutant lines, cyfbp and cfbp1, and one double mutant cyfbp cfbp1 which affect each FBPase isoform, cytosolic and chloroplastic, respectively. cyFBP is involved in sucrose synthesis, whilst cFBP1 is a key enzyme in the Calvin–Benson cycle. In addition to the smaller rosette size and lower rate of photosynthesis, the lack of cFBP1 in the mutants cfbp1 and cyfbp cfbp1 leads to a lower content of soluble sugars, less starch accumulation, and a greater superoxide dismutase (SOD) activity. The mutants also had some developmental alterations, including stomatal opening defects and increased numbers of root vascular layers. Complementation also confirmed that the mutant phenotypes were caused by disruption of the cFBP1 gene. cyfbp mutant plants without cyFBP showed a higher starch content in the chloroplasts, but this did not greatly affect the phenotype. Notably, the sucrose content in cyfbp was close to that found in the wild type. The cyfbp cfbp1 double mutant displayed features of both parental lines but had the cfbp1 phenotype. All the mutants accumulated fructose-1,6-bisphosphate and triose-phosphate during the light period. These results prove that while the lack of cFBP1 induces important changes in a wide range of metabolites such as amino acids, sugars, and organic acids, the lack of cyFBP activity in Arabidopsis essentially provokes a carbon metabolism imbalance which does not compromise the viability of the double mutant cyfbp cfbp1.