Redox signalling regulates breast cancer metastasis via phenotypic and metabolic reprogramming due to p63 activation by HIF1α.

BACKGROUND: Redox signaling caused by knockdown (KD) of Glutathione Peroxidase 2 (GPx2) in the PyMT mammary tumour model promotes metastasis via phenotypic and metabolic reprogramming. However, the tumour cell subpopulations and transcriptional regulators governing these processes remained unknown. METHODS: We used single-cell transcriptomics to decipher the tumour cell subpopulations stimulated by GPx2 KD in the PyMT mammary tumour and paired pulmonary metastases. We analyzed the EMT spectrum across the various tumour cell clusters using pseudotime trajectory analysis and elucidated the transcriptional and metabolic regulation of the hybrid EMT state. RESULTS: Integration of single-cell transcriptomics between the PyMT/GPx2 KD primary tumour and paired lung metastases unraveled a basal/mesenchymal-like cluster and several luminal-like clusters spanning an EMT spectrum. Interestingly, the luminal clusters at the primary tumour gained mesenchymal gene expression, resulting in epithelial/mesenchymal subpopulations fueled by oxidative phosphorylation (OXPHOS) and glycolysis. By contrast, at distant metastasis, the basal/mesenchymal-like cluster gained luminal and mesenchymal gene expression, resulting in a hybrid subpopulation using OXPHOS, supporting adaptive plasticity. Furthermore, p63 was dramatically upregulated in all hybrid clusters, implying a role in regulating partial EMT and MET at primary and distant sites, respectively. Importantly, these effects were reversed by HIF1α loss or GPx2 gain of function, resulting in metastasis suppression. CONCLUSIONS: Collectively, these results underscored a dramatic effect of redox signaling on p63 activation by HIF1α, underlying phenotypic and metabolic plasticity leading to mammary tumour metastasis.

Redox signaling by glutathione peroxidase 2 links vascular modulation to metabolic plasticity of breast cancer.

In search of redox mechanisms in breast cancer, we uncovered a striking role for glutathione peroxidase 2 (GPx2) in oncogenic signaling and patient survival. GPx2 loss stimulates malignant progression due to reactive oxygen species/hypoxia inducible factor-α (HIF1α)/VEGFA (vascular endothelial growth factor A) signaling, causing poor perfusion and hypoxia, which were reversed by GPx2 reexpression or HIF1α inhibition. Ingenuity Pathway Analysis revealed a link between GPx2 loss, tumor angiogenesis, metabolic modulation, and HIF1α signaling. Single-cell RNA analysis and bioenergetic profiling revealed that GPx2 loss stimulated the Warburg effect in most tumor cell subpopulations, except for one cluster, which was capable of oxidative phosphorylation and glycolysis, as confirmed by coexpression of phosphorylated-AMPK and GLUT1. These findings underscore a unique role for redox signaling by GPx2 dysregulation in breast cancer, underlying tumor heterogeneity, leading to metabolic plasticity and malignant progression.

C-Src is Activated by the EGF Receptor in a Pathway that Mediates JNK and ERK Activation by Gonadotropin-Releasing Hormone in COS7 Cells.

The key participants in G-protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. The prototypical GPCR is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here, we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via Gαi and the EGF receptor, without the involvement of Hb-EGF or PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and PI3K. ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Beside the main pathway, the dissociated Gßγ and ß-arrestin may initiate additional (albeit minor) pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other GnRHR-bearing cells, indicating that GnRH can utilize various signaling mechanisms for MAPK activation. The unique pathway elucidated here, in which c-Src and PI3K are sequentially activated downstream of the EGF receptor, may serve as a prototype of signaling mechanisms by GnRHR and additional GPCRs in various cell types.

p21CIP1 Promotes Mammary Cancer-Initiating Cells via Activation of Wnt/TCF1/CyclinD1 Signaling.

Cancer stem cells (CSC) generate and sustain tumors due to tumor-initiating potential, resulting in recurrence or metastasis. We showed that knockout of the cell-cycle inhibitor, p21CIP1, in the PyMT mammary tumor model inhibits metastasis; however the mechanism remained unknown. Here, we show a pivotal role for p21 in potentiating a cancer stem-like phenotype. p21 knockout in PyMT mammary tumor cells caused dramatic suppression of CSC properties involving tumorsphere formation, ALDH1 activity, and tumor-initiating potential, which were in turn rescued by p21 overexpression into PyMT/p21 knockout cells. Interestingly, p21 knockout dramatically suppresses Wnt/ß-catenin signaling activity, leading to striking inhibition of LEF1 and TCF1 expression. TCF1 knockdown in PyMT cells suppressed tumorsphere formation due to Cyclin D1 attenuation. These data demonstrate that p21 promotes a CSC-like phenotype via activation of Wnt/TCF1/Cyclin D1 signaling. IMPLICATIONS: p21 is a strong promoter of mammary CSCs.

An Akt3 Splice Variant Lacking the Serine 472 Phosphorylation Site Promotes Apoptosis and Suppresses Mammary Tumorigenesis.

The Akt pathway is a well-known promoter of tumor malignancy. Akt3 is expressed as two alternatively spliced variants, one of which lacks the key regulatory serine 472 phosphorylation site. Whereas the function of full-length Akt3 isoform (Akt3/+S472) is well-characterized, that of Akt3/-S472 isoform remains unknown. Despite being expressed at a substantially lower level than Akt3/+S472 in triple-negative breast cancer cells, specific ablation of Akt3/-S472 enhanced, whereas overexpression, suppressed mammary tumor growth, consistent with a significant association with patient survival duration relative to Akt3/+S472. These effects were due to striking induction of apoptosis, which was mediated by Bim upregulation, leading to conformational activation of Bax and caspase-3 processing. Bim accumulation was caused by marked endocytosis of EGF receptors with concomitant ERK attenuation, which stabilizes BIM. These findings demonstrate an unexpected function of an endogenously expressed Akt isoform in promoting, as opposed to suppressing, apoptosis, underscoring that Akt isoforms may exert dissonant functions in malignancy.Significance: These results illuminate an unexpected function for an endogenously expressed Akt isoform in promoting apoptosis, underscoring the likelihood that different Akt isoforms exert distinct functions in human cancer. Cancer Res; 78(1); 103-14. ©2017 AACR.

Impact of high-fat diet on the proteome of mouse liver.

Chronic overnutrition, for instance, high-fat diet (HFD) feeding, is a major cause of rapidly growing incidence of metabolic syndromes. However, the mechanisms underlying HFD-induced adverse effects on human health are not clearly understood. HFD-fed C57BL6/J mouse has been a popular model employed to investigate the mechanisms. Yet, there is no systematic and comprehensive study of the impact of HFD on the protein profiles of the animal. Here, we present a proteome-wide study of the consequences of long-term HFD feeding. Utilizing a powerful technology, stable isotope labeling of mammals, we detected and quantitatively compared 965 proteins extracted from livers of chow-diet-fed and HFD-fed mice. Among which, 122 proteins were significantly modulated by HFD. Fifty-four percent of those 122 proteins are involved in metabolic processes and the majority participate in lipid metabolism. HFD up-regulates proteins that play important roles in fatty acid uptake and subsequent oxidation and are linked to the transcription factors PPARα and PGC-1α. HFD suppresses lipid biosynthesis-related proteins that play major roles in de novo lipogenesis and are linked to SREBP-1 and PPARγ. These data suggest that HFD-fed mice tend to develop enhanced fat utilization and suppressed lipid biosynthesis, understandably a self-protective mechanism to counteract to excessive fat loading, which causes liver steatosis. Enhanced fatty acid oxidation increases reactive oxygen species and inhibits glucose oxidation, which are associated with hyperglycemia and insulin resistance. This proteomics study provides molecular understanding of HFD-induced pathology and identifies potential targets for development of therapeutics for metabolic syndromes.

Inhibition of Prostaglandin Transporter (PGT) Promotes Perfusion and Vascularization and Accelerates Wound Healing in Non-Diabetic and Diabetic Rats.

Peripheral ischemia, resulting from diminished arterial flow and defective local vascularization, is one of the main causes of impaired wound healing in diabetes. Vasodilatory prostaglandins (PGs), including PGE2 and PGI2, regulate blood flow in peripheral tissues. PGs also stimulate angiogenesis by inducing vascular endothelial growth factor. However, PG levels are reduced in diabetes mainly due to enhanced degradation. We hypothesized that inhibition of the prostaglandin transporter (PGT) (SLCO2A1), which mediates the degradation of PGs, would increase blood flow and stimulate vascularization, thereby mitigating peripheral ischemia and accelerating wound healing in diabetes. Here we report that inhibiting PGT with intravenously injected PGT inhibitor, T26A, increased blood flow in ischemic hind limbs created in non-diabetic rats and streptozotocin induced diabetic rats. Systemic, or combined with topical, T26A accelerated closure of cutaneous wounds. Immunohistochemical examination revealed that inhibition of PGT enhanced vascularization (marked by larger numbers of vessels formed by CD34+ cells), and accelerated re-epithelialization of cutaneous wounds. In cultured primary human bone marrow CD34+ cells and human epidermal keratinocytes (HEKs) either inhibiting or silencing PGT increased migration in both cell lines. Thus PGT directly regulates mobilization of endothelial progenitor cells (EPCs) and HEKs, which could contribute to PGT-mediated vascularization and re-epithelialization. At the molecular level, systemic inhibition of PGT raised circulating PGE2. Taken together, our data demonstrate that PGT modulates arterial blood flow, mobilization of EPCs and HEKs, and vascularization and epithelialization in wound healing by regulating vasodilatory and pro-angiogenic PGs.

Medicinal properties of mangiferin, structural features, derivative synthesis, pharmacokinetics and biological activities.

The identification of biologically active and potentially therapeutically useful pharmacophores from natural products has been a long-term focus in the pharmaceutical industry. The recent emergence of a worldwide obesity and Type II diabetes epidemic has increased focus upon small molecules that can modulate energy metabolism, insulin sensitivity and fat biology. Interesting preliminary work done on mangiferin (MGF), the predominant constituent of extracts of the mango plant Mangifera indica L., portends potential for this pharmacophore as a novel parent compound for treating metabolic disorders. MGF is comprised of a C-glucosylated xanthone. Owing to the xanthone chemical structure, MGF has a redox active aromatic system and has antioxidant properties. MGF exerts varied and impressive metabolic effects in animals, improving metabolic disorders. For example we have discovered that MGF is a novel activator of the mammalian pyruvate dehydrogenase complex, leading to enhancement of carbohydrate utilization in oxidative metabolism, and leading to increased insulin sensitivity in animal models of obesity and insulin resistance. In addition, recent unbiased proteomics studies revealed that MGF upregulates proteins pivotal for mitochondrial bioenergetics and downregulates proteins controlling de novo lipogenesis in liver, helping to explain protective effects of MGF in prevention of liver steatosis. Several chemical studies have achieved synthesis of MGF, suggesting possible synthetic strategies to alter its chemical structure for development of structure-activity relationship (SAR) information. Ultimately, chemical derivatization studies could lead to the eventual development of novel therapeutics based upon the parent pharmacophore structure. Here we provide comprehensive review on chemical features of MGF, synthesis of its derivatives, its pharmacokinetics and biological activities.

Mangiferin stimulates carbohydrate oxidation and protects against metabolic disorders induced by high-fat diets.

Excessive dietary fat intake causes systemic metabolic toxicity, manifested in weight gain, hyperglycemia, and insulin resistance. In addition, carbohydrate utilization as a fuel is substantially inhibited. Correction or reversal of these effects during high-fat diet (HFD) intake is of exceptional interest in light of widespread occurrence of diet-associated metabolic disorders in global human populations. Here we report that mangiferin (MGF), a natural compound (the predominant constituent of Mangifera indica extract from the plant that produces mango), protected against HFD-induced weight gain, increased aerobic mitochondrial capacity and thermogenesis, and improved glucose and insulin profiles. To obtain mechanistic insight into the basis for these effects, we determined that mice exposed to an HFD combined with MGF exhibited a substantial shift in respiratory quotient from fatty acid toward carbohydrate utilization. MGF treatment significantly increased glucose oxidation in muscle of HFD-fed mice without changing fatty acid oxidation. These results indicate that MGF redirects fuel utilization toward carbohydrates. In cultured C2C12 myotubes, MGF increased glucose and pyruvate oxidation and ATP production without affecting fatty acid oxidation, confirming in vivo and ex vivo effects. Furthermore, MGF inhibited anaerobic metabolism of pyruvate to lactate but enhanced pyruvate oxidation. A key target of MGF appears to be pyruvate dehydrogenase, determined to be activated by MGF in a variety of assays. These findings underscore the therapeutic potential of activation of carbohydrate utilization in correction of metabolic syndrome and highlight the potential of MGF to serve as a model compound that can elicit fuel-switching effects.

Use of NMR saturation transfer difference spectroscopy to study ligand binding to membrane proteins.

Detection of weak ligand binding to membrane-spanning proteins, such as receptor proteins at low physiological concentrations, poses serious experimental challenges. Saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy offers an excellent way to surmount these problems. As the name suggests, magnetization transferred from the receptor to its bound ligand is measured by directly observing NMR signals from the ligand itself. Low-power irradiation is applied to a (1)H NMR spectral region containing protein signals but no ligand signals. This irradiation spreads quickly throughout the membrane protein by the process of spin diffusion and saturates all protein (1)H NMR signals. (1)H NMR signals from a ligand bound transiently to the membrane protein become saturated and, upon dissociation, serve to decrease the intensity of the (1)H NMR signals measured from the pool of free ligand. The experiment is repeated with the irradiation pulse placed outside the spectral region of protein and ligand, a condition that does not lead to saturation transfer to the ligand. The two resulting spectra are subtracted to yield the difference spectrum. As an illustration of the methodology, we review here STD-NMR experiments designed to investigate binding of ligands to the human sweet taste receptor, a member of the large family of G-protein-coupled receptors. Sweetener molecules bind to the sweet receptor with low affinity but high specificity and lead to a variety of physiological responses.

REEP2 enhances sweet receptor function by recruitment to lipid rafts.

Heterologously expressed sensory receptors generally do not achieve the ligand sensitivity observed in vivo, and may require specific accessory proteins to ensure optimal function. We searched for taste cell-expressed receptor transporting protein (RTP) and receptor expression enhancing protein (REEP) family members that might serve as accessory molecules to enhance gustatory receptor function. We determined that REEP2 is an integral membrane protein expressed in taste cells, physically associates with both subunits of the type 1 taste receptor 2 and type 1 taste receptor 3 sweet receptor and specifically enhances responses to tastants of heterologously expressed sweet and bitter taste receptors. Downregulation of endogenously expressed REEP2 in the chemosensory enteroendocrine GLUTag cell line dramatically reduced sensitivity of endogenous sweet receptors. In contrast to the observation that RTP1, RTP2, and REEP1 enhance function of olfactory receptors by promoting their transit to the cell surface, we found that REEP2 does not increase cell surface expression of sweet receptors but instead alters their spatial organization. REEP2 recruits sweet receptors into lipid raft microdomains localized near the taste cell's apical region, thereby improving G-protein-coupled receptor signaling and promoting receptor access to tastants arriving through the apical taste pore.

Direct NMR detection of the binding of functional ligands to the human sweet receptor, a heterodimeric family 3 GPCR.

We present a robust method for monitoring the binding of ligands to the heterodimeric (T1R2+T1R3) human sweet receptor (a family 3 GPCR receptor). The approach utilizes saturation transfer difference (STD) NMR spectroscopy with receptor proteins expressed on the surface of human epithelial kidney cells. The preparation investigated by NMR can contain either live cells or membranes isolated from these cells containing the receptor. We have used this approach to confirm the noncompetitive binding of alitame and cyclamate to the receptor and to determine that greatly reduced receptor binding affinity compared to wild-type brazzein explains the lack of sweetness of brazzein mutant A16C17. This approach opens new avenues for research on the mechanism of action of the sweet receptor and for the design of new noncalorigenic sweeteners.

Ggamma13 interacts with PDZ domain-containing proteins.

The G protein gamma13 subunit (Ggamma13) is expressed in taste and retinal and neuronal tissues and plays a key role in taste transduction. We identified PSD95, Veli-2, and other PDZ domain-containing proteins as binding partners for Ggamma13 by yeast two-hybrid and pull-down assays. In two-hybrid assays, Ggamma13 interacted specifically with the third PDZ domain of PSD95, the sole PDZ domain of Veli-2, and the third PDZ domain of SAP97, a PSD95-related protein. Ggamma13 did not interact with the other PDZ domains of PSD95. Coexpression of Ggamma13 with its Gbeta1 partner did not interfere with these two-hybrid interactions. The physical interaction of Ggamma13 with PSD95 in the cellular milieu was confirmed in pull-down assays following heterologous expression in HEK293 cells. The interaction of Ggamma13 with the PDZ domain of PSD95 was via the C-terminal CAAX tail of Ggamma13 (where AA indicates the aliphatic amino acid); alanine substitution of the CTAL sequence at the C terminus of Ggamma13 abolished its interactions with PSD95 in two-hybrid and pull-down assays. Veli-2 and SAP97 were identified in taste tissue and in Ggamma13-expressing taste cells. Coimmunoprecipitation of Ggamma13 and PSD95 from brain and of Ggamma13 and SAP97 from taste tissue indicates that Ggamma13 interacts with these proteins endogenously. This is the first demonstration that PDZ domain proteins interact with heterotrimeric G proteins via the CAAX tail of Ggamma subunits. The interaction of Ggamma13 with PDZ domain-containing proteins may provide a means to target particular Gbetagamma subunits to specific subcellular locations and/or macromolecular complexes involved in signaling pathways.

BRAF oncogenic mutations correlate with progression rather than initiation of human melanoma.

BRAF oncogenic mutations have been identified in significant numbers of melanocytic lesions. To correlate BRAF mutation and melanoma progression, we screened BRAF mutations in 65 melanocytic lesions, including nevi, radial growth phase (RGP), vertical growth phase (VGP) melanomas, and melanoma metastases, as well as 25 melanoma cell lines. PCR and direct sequencing were used to analyze DNA samples extracted from laser capture microdissected tissues. A similar high frequency (62-72%) of BRAF oncogenic mutations was identified in melanocytic nevi, VGP, metastatic melanomas, and melanoma cell lines [H. Davies et al., Nature (Lond.), 417: 949-954, 2002; P. M. Pollock et al., Nat. Genet., 33: 19-20, 2002; and M. S. Brose et al., Cancer Res., 62: 6997-7000, 2002]. In striking contrast, we found BRAF lesions in only 10% of the earliest stage or RGP melanomas. These findings imply that BRAF mutations cannot be involved in the initiation of the great majority of melanomas but instead reflect a progression event with important prognostic implications in the transition from the great majority of RGP melanomas to VGP and/or metastatic melanoma.

c-Src is activated by the epidermal growth factor receptor in a pathway that mediates JNK and ERK activation by gonadotropin-releasing hormone in COS7 cells.

Key participants in G protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. A prototypic GPCR that has been widely used to study these signaling mechanisms is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via G alpha i, EGF receptor without the involvement of Hb-EGF, and c-Src, but independently of PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and phosphatidylinositol 3-kinase (PI3K). ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Besides the main pathway, the dissociated G beta gamma and beta-arrestin may initiate additional, albeit minor, pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other cell lines bearing GnRHR, indicating that GnRH can utilize various signaling mechanisms for the activation of MAPK cascades. The unique pathway elucidated here in which c-Src and PI3K are sequentially activated downstream of the EGF receptor may serve as a prototype of signaling mechanisms by GnRHR and by additional GPCRs in various cell types.