Hypoxia Promotes Invadosome Formation by Lung Fibroblasts.

Lung parenchymal hypoxia has emerged as a cardinal feature of idiopathic pulmonary fibrosis (IPF). Hypoxia promotes cancer cell invasion and metastasis through signaling that is dependent upon the lysophosphatidic acid (LPA) receptor, LPA1 (LPAR1). Abundant data indicate that LPA1-dependent signaling also enhances lung fibrogenesis in IPF. We recently reported that fibroblasts isolated from the lungs of individuals with IPF have an increased capacity to form subcellular matrix-degradative structures known as invadosomes, an event that correlates with the degree of lung fibrosis. We therefore hypothesized that hypoxia promotes invadosome formation in lung fibroblasts through LPA1-dependent signaling. Here, it is demonstrated that invadosome formation by fibroblasts from the lungs of individuals with advanced IPF is inhibited by both the tyrosine receptor kinase inhibitor nintedanib and inhibition of LPA1. In addition, exposure of normal human lung fibroblasts to either hypoxia or LPA increased their ability to form invadosomes. Mechanistically, the hypoxia-induced invadosome formation by lung fibroblasts was found to involve LPA1 and PDGFR-Akt signaling. We concluded that hypoxia increases the formation of invadosomes in lung fibroblasts through the LPA1 and PDGFR-Akt signaling axis, which represents a potential target for suppressing lung fibrosis.

The development of a rapid patient-derived xenograft model to predict chemotherapeutic drug sensitivity/resistance in malignant glial tumors.

BACKGROUND: High-grade gliomas (HGG) are aggressive brain tumors associated with short median patient survival and limited response to therapies, driving the need to develop tools to improve patient outcomes. Patient-derived xenograft (PDX) models, such as mouse PDX, have emerged as potential Avatar platforms for personalized oncology approaches, but the difficulty for some human grafts to grow successfully and the long time required for mice to develop tumors preclude their use for HGG. METHODS: We used a rapid and efficient ex-ovo chicken embryo chorioallantoic membrane (CAM) culture system to evaluate the efficacy of oncologic drug options for HGG patients. RESULTS: Implantation of fresh glioma tissue fragments from 59 of 60 patients, that include difficult-to-grow IDH-mutated samples, successfully established CAM tumor xenografts within 7 days, with a tumor take rate of 98.3%. These xenografts faithfully recapitulate the histological and molecular characteristics of the primary tumor, and the ability of individual fragments to form tumors was predictive of poor patient prognosis. Treatment of drug-sensitive or drug-resistant xenografts indicates that the CAM-glioma assay enables testing tumor sensitivity to temozolomide and carboplatin at doses consistent with those administered to patients. In a proof-of-concept study involving 14 HGG patients, we observed a correlation of 100% between the CAM xenograft response to temozolomide or carboplatin and the clinical response of patients. CONCLUSION: The CAM-glioma model is a fast and reliable assay that has the potential to serve as a complementary model to drug discovery and a real-time Avatar platform to predict the best treatment for HGG patients.

Establishment of a ccRCC patient-derived chick chorioallantoic membrane model for drug testing.

Clear cell renal cell carcinoma (ccRCC) is an aggressive subtype of renal cell carcinoma accounting for the majority of deaths in kidney cancer patients. Advanced ccRCC has a high mortality rate as most patients progress and develop resistance to currently approved targeted therapies, highlighting the ongoing need for adequate drug testing models to develop novel therapies. Current animal models are expensive and time-consuming. In this study, we investigated the use of the chick chorioallantoic membrane (CAM), a rapid and cost-effective model, as a complementary drug testing model for ccRCC. Our results indicated that tumor samples from ccRCC patients can be successfully cultivated on the chick chorioallantoic membrane (CAM) within 7 days while retaining their histopathological characteristics. Furthermore, treatment of ccRCC xenografts with sunitinib, a tyrosine kinase inhibitor used for the treatment of metastatic RCC, allowed us to evaluate differential responses of individual patients. Our results indicate that the CAM model is a complementary in vivo model that allows for rapid and cost-effective evaluation of ccRCC patient response to drug therapy. Therefore, this model has the potential to become a useful platform for preclinical evaluation of new targeted therapies for the treatment of ccRCC.

Hypoxia Selectively Increases a SMAD3 Signaling Axis to Promote Cancer Cell Invasion.

Transforming growth factor ß (TGFß) plays a paradoxical role in cancer, first inhibiting then promoting its progression, a duality that poses a real challenge for the development of effective TGFß-targeted therapies. The major TGFß downstream effectors, SMAD2 and SMAD3, display both distinct and overlapping functions and accumulating evidence suggests that their activation ratio may contribute to the dual effect of TGFß. However, the mechanisms responsible for their selective activation remain poorly understood. Here, we provide experimental evidence that hypoxia induces the pro-invasive arm of TGFß signaling through a selective increase in SMAD3 interaction with SMAD-Anchor for Receptor Activation (SARA). This event relies on HDAC6-dependent SMAD3 bioavailability, as well as increased SARA recruitment to EEA1+ endosomes. A motility gene expression study indicated that SMAD3 selectively increased the expression of ITGB2 and VIM, two genes that were found to be implicated in hypoxia-induced cell invasion and associated with tumor progression and metastasis in cohorts of cancer patients. Furthermore, CAM xenograft assays show the significant benefit of selective inhibition of the SMAD3 signaling pathway as opposed to global TGFß inhibition in preventing tumor progression. Overall, these results suggest that fine-tuning of the pro-invasive HDAC6-SARA-SMAD3 axis could be a better strategy towards effective cancer treatments.

The Chicken Chorioallantoic Membrane Tumor Assay as a Relevant In Vivo Model to Study the Impact of Hypoxia on Tumor Progression and Metastasis.

Hypoxia in the tumor microenvironment is a negative prognostic factor associated with tumor progression and metastasis, and therefore represents an attractive therapeutic target for anti-tumor therapy. To test the effectiveness of novel hypoxia-targeting drugs, appropriate preclinical models that recreate tumor hypoxia are essential. The chicken ChorioAllantoic Membrane (CAM) assay is increasingly used as a rapid cost-effective in vivo drug-testing platform that recapitulates many aspects of human cancers. However, it remains to be determined whether this model recreates the hypoxic microenvironment of solid tumors. To detect hypoxia in the CAM model, the hypoxic marker pimonidazole was injected into the vasculature of tumor-bearing CAM, and hypoxia-dependent gene expression was analyzed. We observed that the CAM model effectively supports the development of hypoxic zones in a variety of human tumor cell line-derived and patient's tumor fragment-derived xenografts. The treatment of both patient and cell line-derived CAM xenografts with modulators of angiogenesis significantly altered the formation of hypoxic zones within the xenografts. Furthermore, the changes in hypoxia translated into modulated levels of chick liver metastasis as measured by Alu-based assay. These findings demonstrate that the CAM xenograft model is a valuable in vivo platform for studying hypoxia that could facilitate the identification and testing of drugs targeting this tumor microenvironment.

Activation of TRPV4 channels leads to a consistent tocolytic effect on human myometrial tissues.

BACKGROUND: Human myometrium is a therapeutic target for labor induction and preterm labor. OBJECTIVE: This study aimed to assess the physiological role of alternative calcium conductance on contractions triggered by uterotonic drugs in human myometrium. Membrane conductances, supported by TRPV channels, may provide alternative pathways to control either free intracellular and/or submembrane Ca2+-concentration, which in turn will modulate membrane polarization and contractile responses. STUDY DESIGN: Uterine biopsies were obtained from consenting women undergoing elective caesarean delivery at term without labor (N = 22). Isometric tension measurements were performed on uterine smooth muscle strips (n = 132). Amplitude, frequency, and area under the curve (AUC) of phasic contractions, as well as resting tone, were measured under various experimental conditions. Immuno histo- and cyto-chemistry, as well as Western blot analyses, have been performed with specific antibodies against TRPV1, TRPV3, and TRPV4 proteins. TRPV4 agonists; GSK1016790A, 4αPDD, and 5,6-EET were used to assess the role of TRPV4 channels on rhythmic activity triggered by 30-300 nM oxytocin. 5 µM of ruthenium red was used as an efficient blocker of ionic current through TRPV4 channels. Nanomolar concentrations of iberiotoxin (IbTX) were also used to confirm the downstream involvement of BKCa channels in controlling uterine reactivity and contractility. RESULTS: The expression of TRPV3 and TRPV4 isoforms has now been demonstrated in human myometrial tissue and cell culture. Nanomolar concentrations of the TRPV4 agonists, (either GSK1016790A or 4αPDD) abolished the rhythmic contractions, resulting in a rapid and consistent tocolytic effect. While 5 µM of ruthenium reversed this tocolytic effect. The addition of IbTX (a BKCa channel blocker) reversed the effects of GSK1016790A. Carvacrol, a TRPV3 agonist, had similar tocolytic effects on rhythmic contractions albeit at higher concentrations. This inhibitory effect was also reversed by ruthenium red. CONCLUSION: Collectively, these data suggest that activation of TRPV4 leads to a Ca2+ entry and subsequent BKCa channel activation (increase in open state probability), which in turn hyperpolarizes the myometrial cell membrane, inactivating L-type Ca2+ channels and efficiently abrogates contractile activity. Consequently, alternative Ca2+ conductance supported by TRPV4 plays a physiological role in the modulation of myometrial reactivity.

Hypoxia Downregulates LPP3 and Promotes the Spatial Segregation of ATX and LPP1 During Cancer Cell Invasion.

Hypoxia is a common characteristic of advanced solid tumors and a potent driver of tumor invasion and metastasis. Recent evidence suggests the involvement of autotaxin (ATX) and lysophosphatidic acid receptors (LPARs) in cancer cell invasion promoted by the hypoxic tumor microenvironment; however, the transcriptional and/or spatiotemporal control of this process remain unexplored. Herein, we investigated whether hypoxia promotes cell invasion by affecting the main enzymes involved in its production (ATX) and degradation (lipid phosphate phosphatases, LPP1 and LPP3). We report that hypoxia not only modulates the expression levels of lysophosphatidic acid (LPA) regulatory enzymes but also induces their significant spatial segregation in a variety of cancers. While LPP3 expression was downregulated by hypoxia, ATX and LPP1 were asymmetrically redistributed to the leading edge and to the trailing edge, respectively. This was associated with the opposing roles of ATX and LPPs in cell invasion. The regulated expression and compartmentalization of these enzymes of opposing function can provide an effective way to control the generation of an LPA gradient that drives cellular invasion and migration in the hypoxic zones of tumors.

The Hypoxic Tumor Microenvironment Promotes Invadopodia Formation and Metastasis through LPA1 Receptor and EGFR Cooperation.

Hypoxia, a common feature of solid tumors, has been critically involved in cell invasion and metastasis, but the underlying mechanisms remain poorly understood. Previously, it has been observed that the lysophosphatidic acid receptor 4 (LPA4) signaling axis mediates production of the degradative subcellular structures invadopodia, which are known to be required for metastasis. Here, it is demonstrated that LPA1 (LPAR1) is a common and major receptor used for hypoxia-induced invadopodia production in various cancer cell lines. The widespread use of LPA1 was not due to increased LPA1 expression but rather relied on Src-mediated cross-talk with EGFR. LPA1-mediated phosphorylation of Y845-EGFR under hypoxia led to PI3K/Akt activation, an event that increases the ability of cells to produce invadopodia. Moreover, phospho-Y845-EGFR was upregulated in hypoxic zones of tumors and a combination of EGFR and LPA1 inhibition synergistically suppressed metastasis in vivo Implications: This study uncovers an LPA1-EGFR signaling axis that is used for cell invasion in hypoxia and suggests a potential target to impede cancer metastasis. Mol Cancer Res; 16(10); 1601-13. ©2018 AACR.

HDAC6 deacetylase activity is required for hypoxia-induced invadopodia formation and cell invasion.

Despite significant progress in the cancer field, tumor cell invasion and metastasis remain a major clinical challenge. Cell invasion across tissue boundaries depends largely on extracellular matrix degradation, which can be initiated by formation of actin-rich cell structures specialized in matrix degradation called invadopodia. Although the hypoxic microenvironment within solid tumors has been increasingly recognized as an important driver of local invasion and metastasis, little is known about how hypoxia influences invadopodia biogenesis. Here, we show that histone deacetylase 6 (HDAC6), a cytoplasmic member of the histone deacetylase family, is a novel modulator of hypoxia-induced invadopodia formation. Hypoxia was found to enhance HDAC6 tubulin deacetylase activity through activation of the EGFR pathway. Activated HDAC6, in turn, triggered Smad3 phosphorylation resulting in nuclear accumulation. Inhibition of HDAC6 activity or knockdown of the protein inhibited both hypoxia-induced Smad3 activation and invadopodia formation. Our data provide evidence that hypoxia influences invadopodia formation in a biphasic manner, which involves the activation of HDAC6 deacetylase activity by EGFR, resulting in enhanced Smad phosphorylation and nuclear accumulation. The identification of HDAC6 as a key participant of hypoxia-induced cell invasion may have important therapeutic implications for the treatment of metastasis in cancer patients.

Hypoxia-induced invadopodia formation involves activation of NHE-1 by the p90 ribosomal S6 kinase (p90RSK).

The hypoxic and acidic microenvironments in tumors are strongly associated with malignant progression and metastasis, and have thus become a central issue in tumor physiology and cancer treatment. Despite this, the molecular links between acidic pH- and hypoxia-mediated cell invasion/metastasis remain mostly unresolved. One of the mechanisms that tumor cells use for tissue invasion is the generation of invadopodia, which are actin-rich invasive plasma membrane protrusions that degrade the extracellular matrix. Here, we show that hypoxia stimulates the formation of invadopodia as well as the invasive ability of cancer cells. Inhibition or shRNA-based depletion of the Na(+)/H(+) exchanger NHE-1, along with intracellular pH monitoring by live-cell imaging, revealed that invadopodia formation is associated with alterations in cellular pH homeostasis, an event that involves activation of the Na(+)/H(+) exchange rate by NHE-1. Further characterization indicates that hypoxia triggered the activation of the p90 ribosomal S6 kinase (p90 RSK), which resulted in invadopodia formation and site-specific phosphorylation and activation of NHE-1. This study reveals an unsuspected role of p90RSK in tumor cell invasion and establishes p90RS kinase as a link between hypoxia and the acidic microenvironment of tumors.

Adiponectin action from head to toe.

Adiponectin, the most abundant protein secreted by white adipose tissue, is known for its involvement in obesity-related disorders such as insulin resistance, type 2 diabetes mellitus and atherosclerosis. Moreover, modulation of the circulating adiponectin concentration is observed in pathologies that are more or less obesity-related, such as cancer and rheumatoid arthritis. The wide distribution of adiponectin receptors in various organs and tissues suggests that adiponectin has pleiotropic effects on numerous physiological processes. Besides its well-known insulin-sensitizing, anti-inflammatory and antiatherosclerotic properties, accumulating evidence suggests that adiponectin may also have anticancer properties and be cardioprotective. A beneficial effect of adiponectin on female reproductive function was also suggested. Since adiponectin has numerous beneficial biological functions, its use as a therapeutic agent has been suggested. However, the use of adiponectin or its receptors as therapeutic targets is complicated by the presence of different adiponectin oligomeric isoforms and production sites, by multiple receptors with differing affinities for adiponectin isoforms, and by cell-type-specific effects in different tissues. In this review, we discuss the known and potential roles of adiponectin in various tissues and pathologies. The therapeutic promise of administration of adiponectin and the use of its circulating levels as a diagnostic biomarker are further discussed based on the latest experimental studies.

The Promyelocytic Leukemia Zinc Finger (PLZF ) gene is a novel transcriptional target of the CCAAT-Displacement-protein (CUX1) repressor.

The CCAAT-Displacement-Protein (CUX1) can transcriptionally repress sucrase­isomaltase gene expression, a specific product of enterocytes that becomes re-expressed during human colonic polyposis. Little is known of the gene repertoire that is directly affected by CUX1 in the intestinal epithelial context. This article identifies the Promyelocytic Leukemia Zinc Finger (PLZF) gene as a transcriptional target for the CUX1 repressor. CUX1 interacts in vivo with multiple DNA-binding sites in the 5'-UTR and promoter of the PLZF gene in colorectal cancer cells, a region that is functionally targeted by CUX1 in cotransfection assays. PLZF was found to be induced in colorectal cancer cell lines, correlating with a low detectable level of CUX1, a pattern that was reversed in normal human colonocytes. Reduction of p200CUX1 expression by RNAi in the Caco-2/15 cell line increased PLZF gene transcript expression. Because of the implication of Plzf in the regulation of stem cell maintenance, as well as Wnt and Ras signaling, in other systems, our observations suggest that the novel genetic relationship between CUX1 and PLZF could be of relevance to human diseases, such as leukemia, and open up a new field of investigation for the implication of these regulators during intestinal polyposis and cancer.