Glycolysis Inhibition of Autophagy Drives Malignancy in Ovarian Cancer: Exacerbation by IL-6 and Attenuation by Resveratrol.

Cancer cells drive the glycolytic process towards the fermentation of pyruvate into lactate even in the presence of oxygen and functioning mitochondria, a phenomenon known as the "Warburg effect". Although not energetically efficient, glycolysis allows the cancer cell to synthesize the metabolites needed for cell duplication. Autophagy, a macromolecular degradation process, limits cell mass accumulation and opposes to cell proliferation as well as to cell migration. Cancer cells corrupt cancer-associated fibroblasts to release pro-inflammatory cytokines, which in turn promote glycolysis and support the metastatic dissemination of cancer cells. In mimicking in vitro this condition, we show that IL-6 promotes ovarian cancer cell migration only in the presence of glycolysis. The nutraceutical resveratrol (RV) counteracts glucose uptake and metabolism, reduces the production of reactive oxygen species consequent to excessive glycolysis, rescues the mitochondrial functional activity, and stimulates autophagy. Consistently, the lack of glucose as well as its metabolically inert analogue 2-deoxy-D-glucose (2-DG), which inhibits hexokinase 2 (HK2), trigger autophagy through mTOR inhibition, and prevents IL-6-induced cell migration. Of clinical relevance, bioinformatic analysis of The Cancer Genome Atlas dataset revealed that ovarian cancer patients bearing mutated TP53 with low expression of glycolytic markers and IL-6 receptor, together with markers of active autophagy, display a longer overall survival and are more responsive to platinum therapy. Taken together, our findings demonstrate that RV can counteract IL-6-promoted ovarian cancer progression by rescuing glycolysis-mediated inhibition of autophagy and support the view that targeting Warburg metabolism can be an effective strategy to limit the risk for cancer metastasis.

Deciphering a GPCR-lncrna-miRNA nexus: Identification of an aberrant therapeutic target in ovarian cancer.

Ovarian cancer ranks as a leading cause of mortality among gynecological malignancies, primarily due to the lack of early diagnostic tools, effective targeted therapy, and clear understanding of disease etiology. Previous studies have identified the pivotal role of Lysophosphatidic acid (LPA)-signaling in ovarian cancer pathobiology. Our earlier transcriptomic analysis identified Urothelial Carcinoma Associated-1 (UCA1) as an LPA-stimulated long non-coding RNA (lncRNA). In this study, we elucidate the tripartite interaction between LPA-signaling, UCA1, and let-7 miRNAs in ovarian cancer progression. Results show that the elevated expression of UCA1 enhances cell proliferation, invasive migration, and therapy resistance in high-grade serous ovarian carcinoma cells, whereas silencing UCA1 reverses these oncogenic phenotypes. UCA1 expression inversely correlates with survival outcomes and therapy response in ovarian cancer clinical samples, underscoring its prognostic significance. Mechanistically, UCA1 sequesters let-7 miRNAs, effectively neutralizing their tumor-suppressive functions involving key oncogenes such as Ras and c-Myc. More significantly, intratumoral delivery of UCA1-specific siRNAs inhibits the growth of cisplatin-refractory ovarian cancer xenografts, demonstrating the therapeutic potential of targeting LPAR-UCA1-let-7 axis in ovarian cancer. Thus, our results identify LPAR-UCA1-let-7 axis as a novel avenue for targeted treatment strategies.

Optical coherence tomography for multicellular tumor spheroid category recognition and drug screening classification via multi-spatial-superficial-parameter and machine learning.

Optical coherence tomography (OCT) is an ideal imaging technique for noninvasive and longitudinal monitoring of multicellular tumor spheroids (MCTS). However, the internal structure features within MCTS from OCT images are still not fully utilized. In this study, we developed cross-statistical, cross-screening, and composite-hyperparameter feature processing methods in conjunction with 12 machine learning models to assess changes within the MCTS internal structure. Our results indicated that the effective features combined with supervised learning models successfully classify OVCAR-8 MCTS culturing with 5,000 and 50,000 cell numbers, MCTS with pancreatic tumor cells (Panc02-H7) culturing with the ratio of 0%, 33%, 50%, and 67% of fibroblasts, and OVCAR-4 MCTS treated by 2-methoxyestradiol, AZD1208, and R-ketorolac with concentrations of 1, 10, and 25 µM. This approach holds promise for obtaining multi-dimensional physiological and functional evaluations for using OCT and MCTS in anticancer studies.

Targeting Oncometabolites in Peritoneal Cancers: Preclinical Insights and Therapeutic Strategies.

Peritoneal cancers present significant clinical challenges with poor prognosis. Understanding the role of cancer cell metabolism and cancer-promoting metabolites in peritoneal cancers can provide new insights into the mechanisms that drive tumor progression and can identify novel therapeutic targets and biomarkers for early detection, prognosis, and treatment response. Cancer cells dynamically reprogram their metabolism to facilitate tumor growth and overcome metabolic stress, with cancer-promoting metabolites such as kynurenines, lactate, and sphingosine-1-phosphate promoting cell proliferation, angiogenesis, and immune evasion. Targeting cancer-promoting metabolites could also lead to the development of effective combinatorial and adjuvant therapies involving metabolic inhibitors for the treatment of peritoneal cancers. With the observed metabolomic heterogeneity in cancer patients, defining peritoneal cancer metabolome and cancer-promoting metabolites holds great promise for improving outcomes for patients with peritoneal tumors and advancing the field of precision cancer medicine. This review provides an overview of the metabolic signatures of peritoneal cancer cells, explores the role of cancer-promoting metabolites as potential therapeutic targets, and discusses the implications for advancing precision cancer medicine in peritoneal cancers.

Optical Coherence Tomography of Tumor Spheroids Identifies Candidates for Drug Repurposing in Ovarian Cancer.

OBJECTIVE: Multicellular tumor spheroids (MCTs) are indispensable models for evaluating drug efficacy for precision cancer therapeutic strategies as well as for repurposing FDA-approved drugs for ovarian cancer. However, current imaging techniques cannot provide effective monitoring of pathological responses due to shallow penetration and experimentally operative destruction. We plan to utilize a noninvasive optical imaging tool to achieve in vivo longitudinal monitoring of the growth of MCTs and therapeutic responses to repurpose three FDA-approved drugs for ovarian cancer therapy. METHODS: A swept-source optical coherence tomography (SS-OCT) system was used to monitor the volume growth of MCTs over 11 days. Three inhibitors of 2-Methoxyestradiol (2-ME), AZD1208, and R-Ketorolac (R-keto) with concentrations of 1, 10, and 25 µM were employed to treat ovarian MCTs on day 5. Three-dimensional (3D), intrinsic optical attenuation contrast, and degree of uniformity were applied to analyze the therapeutic effect of these inhibitors on ovarian MCTs. RESULTS: We found that 2-ME, AZD1208, and R-keto with concentration of 10 and 25 µM significantly inhibited the volume growth of ovarian MCTs. There was no effect to necrotic tissues from all concentrations of 2-ME, AZD1208, and R-keto inhibitors from our OCT results. 2-ME and AZD1208 inhibited the growth of high uniformity tissues within MCTs and higher concentrations provided more significant inhibitory effects. CONCLUSION: Our results indicated that OCT was capable and reliable to monitor the therapeutic effect of inhibitors to ovarian MCTs and it can be used for the rapid characterization of novel therapeutics for ovarian cancers in the future.

Identification of GNA12-driven gene signatures and key signaling networks in ovarian cancer.

With the focus on defining the oncogenic network stimulated by lysophosphatidic acid (LPA) in ovarian cancer, the present study sought to interrogate the oncotranscriptome regulated by the LPA-mediated signaling pathway. LPA, LPA-receptor (LPAR) and LPAR-activated G protein 12 α-subunit, encoded by G protein subunit α 12 (GNA12), all serve an important role in ovarian cancer progression. While the general signaling mechanism regulated by LPA/LPAR/GNA12 has previously been characterized, the global transcriptomic network regulated by GNA12 in ovarian cancer pathophysiology remains largely unknown. To define the LPA/LPAR/GNA12-orchestrated oncogenic networks in ovarian cancer, transcriptomic and bioinformatical analyses were conducted using SKOV3 cells, in which the expression of GNA12 was silenced. Array analysis was performed in Agilent SurePrint G3 Human Comparative Genomic Hybridization 8×60 microarray platform. The array results were validated using Kuramochi cells. Gene and functional enrichment analyses were performed using Database for Annotation, Visualization and Integrated Discovery, Search Tool for Retrieval of Interacting Genes and Cytoscape algorithms. The results indicated a paradigm in which GNA12 drove ovarian cancer progression by upregulating a pro-tumorigenic network with AKT1, VEGFA, TGFB1, BCL2L1, STAT3, insulin-like growth factor 1 and growth hormone releasing hormone as critical hub and/or bottleneck nodes. Moreover, GNA12 downregulated a growth-suppressive network involving proteasome 20S subunit (PSM) ß6, PSM α6, PSM ATPase 5, ubiquitin conjugating enzyme E2 E1, PSM non-ATPase 10, NDUFA4 mitochondrial complex-associated, NADH:ubiquinone oxidoreductase subunit B8 and anaphase promoting complex subunit 1 as hub or bottleneck nodes. In addition to providing novel insights into the LPA/LPAR/GNA12-regulated oncogenic networks in ovarian cancer, the present study identified several potential nodes in this network that could be assessed for targeted therapy.

GNAi2/gip2-Regulated Transcriptome and Its Therapeutic Significance in Ovarian Cancer.

Increased expression of GNAi2, which encodes the α-subunit of G-protein i2, has been correlated with the late-stage progression of ovarian cancer. GNAi2, also referred to as the proto-oncogene gip2, transduces signals from lysophosphatidic acid (LPA)-activated LPA-receptors to oncogenic cellular responses in ovarian cancer cells. To identify the oncogenic program activated by gip2, we carried out micro-array-based transcriptomic and bioinformatic analyses using the ovarian cancer cell-line SKOV3, in which the expression of GNAi2/gip2 was silenced by specific shRNA. A cut-off value of 5-fold change in gene expression (p < 0.05) indicated that a total of 264 genes were dependent upon gip2-expression with 136 genes coding for functional proteins. Functional annotation of the transcriptome indicated the hitherto unknown role of gip2 in stimulating the expression of oncogenic/growth-promoting genes such as KDR/VEGFR2, CCL20, and VIP. The array results were further validated in a panel of High-Grade Serous Ovarian Carcinoma (HGSOC) cell lines that included Kuramochi, OVCAR3, and OVCAR8 cells. Gene set enrichment analyses using DAVID, STRING, and Cytoscape applications indicated the potential role of the gip2-stimulated transcriptomic network involved in the upregulation of cell proliferation, adhesion, migration, cellular metabolism, and therapy resistance. The results unravel a multi-modular network in which the hub and bottleneck nodes are defined by ACKR3/CXCR7, IL6, VEGFA, CYCS, COX5B, UQCRC1, UQCRFS1, and FYN. The identification of these genes as the critical nodes in GNAi2/gip2 orchestrated onco-transcriptome establishes their role in ovarian cancer pathophysiology. In addition, these results also point to these nodes as potential targets for novel therapeutic strategies.

Unraveling Autocrine Signaling Pathways through Metabolic Fingerprinting in Serous Ovarian Cancer Cells.

Focusing on defining metabolite-based inter-tumoral heterogeneity in ovarian cancer, we investigated the metabolic diversity of a panel of high-grade serous ovarian carcinoma (HGSOC) cell-lines using a metabolomics platform that interrogate 731 compounds. Metabolic fingerprinting followed by 2-dimensional and 3-dimensional principal component analysis established the heterogeneity of the HGSOC cells by clustering them into five distinct metabolic groups compared to the fallopian tube epithelial cell line control. An overall increase in the metabolites associated with aerobic glycolysis and phospholipid metabolism were observed in the majority of the cancer cells. A preponderant increase in the levels of metabolites involved in trans-sulphuration and glutathione synthesis was also observed. More significantly, subsets of HGSOC cells showed an increase in the levels of 5-Hydroxytryptamine, γ-aminobutyrate, or glutamate. Additionally, 5-hydroxytryptamin synthesis inhibitor as well as antagonists of γ-aminobutyrate and glutamate receptors prohibited the proliferation of HGSOC cells, pointing to their potential roles as oncometabolites and ligands for receptor-mediated autocrine signaling in cancer cells. Consistent with this role, 5-Hydroxytryptamine synthesis inhibitor as well as receptor antagonists of γ-aminobutyrate and Glutamate-receptors inhibited the proliferation of HGSOC cells. These antagonists also inhibited the three-dimensional spheroid growth of TYKNU cells, a representative HGSOC cell-line. These results identify 5-HT, GABA, and Glutamate as putative oncometabolites in ovarian cancer metabolic sub-type and point to them as therapeutic targets in a metabolomic fingerprinting-based therapeutic strategy.

G(alpha)12/13 inhibition enhances the anticancer effect of bortezomib through PSMB5 downregulation.

Bortezomib is a proteasome inhibitor approved for anticancer therapy. However, variable sensitivity of tumor cells exists in this therapy probably due to differences in the expression of proteasome subunits. G(alpha)(12/13) serves modulators or signal transducers in diverse pathways. This study investigated whether cancer cells display differential sensitivity to bortezomib with reference to G(alpha)(12/13) expression, and if so, whether G(alpha)(12/13) affects the expression of proteasome subunits and their activities. Bortezomib treatment exhibited greater sensitivities in Huh7 and SNU886 cells (epithelial type) than SK-Hep1 and SNU449 cells (mesenchymal type) that exhibited higher levels of G(alpha)(12/13). Overexpression of an active mutant of G(alpha)(12) (Galpha(12)QL) or G(alpha)(13) (G(alpha)(13)QL) diminished the ability of bortezomib to induce cytotoxicity in Huh7 cells. Moreover, transfection with the minigene that disturbs G protein-coupled receptor-G protein coupling (CT12 or CT13) increased it in SK-Hep1 cells. Consistently, MiaPaCa2 cells transfected with CT12 or CT13 exhibited a greater sensitivity to bortezomib. Evidence of G(alpha)(12/13)'s antagonism on the anticancer effect of bortezomib was verified in the reversal by G(alpha)(12)QL or G(alpha)(13)QL of the minigenes' enhancement of cytotoxity. Real-time polymerase chain reaction assay enabled us to identify PSMB5, multicatalytic endopeptidase complex-like-1, and proteasome activator subunit-1 repression by CT12 or CT13. Furthermore, G(alpha)(12/13) inhibition enhanced the ability of bortezomib to repress PSMB5, as shown by immunoblotting and proteasome activity assay. Moreover, this inhibitory effect on PSMB5 was attenuated by G(alpha)G(alpha)(12)QL or G(alpha)(13)QL. In conclusion, the inhibition of G(alpha)(12/13) activities may enhance the anticancer effect of bortezomib through PSMB5 repression, providing insight into the G(alpha)(12/13) pathway for the regulation of proteasomal activity.

The G12 family proteins upregulate matrix metalloproteinase-2 via p53 leading to human breast cell invasion.

Although mounting evidence suggests a role for G(12) proteins, G(α12) and G(α13), in tumor progression, a direct role of G(12) proteins has not been determined. This study aims to elucidate the molecular mechanism for a tumorigenic and invasive potential of G(α12) and G(α13) in MCF10A human breast epithelial cells. Here, we report, for the first time, that G(α12) and G(α13) induce upregulation of matrix metalloproteinase (MMP)-2 leading to the invasive and migratory phenotypes in MCF10A cells. We further show that p53 is an important transcription factor for induction of MMP-2 transcriptional activation by G(α12/13). G(α12/13)-induced MMP-2 upregulation, invasion, and migration are dependent on the activation of Ras, Rac1, MKK3/6, p38, and Akt. Using human breast tissue samples, we demonstrate that the expression levels of G(α12) and MMP-2 are strongly correlated with the pathogenically diagnosed cancer (P < 0.0001). Moreover, the expression of G(α12) shows a strong correlation with that of MMP-2 in human breast cancer tissues, implicating the in vivo tumorigenic potential of G(α12). Taken together, this study elucidated the role of G(12) proteins in regulating processes for MMP-2 expression and malignant phenotypic conversion of MCF10A human breast epithelial cells, providing a molecular basis for the promoting role of G(α12) and G(α13) in breast cell invasion.

Chemopreventive and Anticancer Effects of Thymoquinone: Cellular and Molecular Targets.

Thymoquinone (TQ) is a bioactive component derived from the seeds of Nigella sativa that are commonly as black cumin. Evidences indicate that the medicinal properties of TQ have been recognized for more than 2000 years. TQ has been shown to possess potent chemopreventive properties that include anti-inflammatory and anti-neoplastic activities. Recent studies have unraveled the multiple mechanisms through which TQ exerts its chemopreventive and anticancer activity in different cancer cells in a contextual manner. The present review aims to provide a brief compendium on the molecular mechanisms through which TQ inhibits signaling pathways underlying cancer genesis, progression, and metastasis.

Differential effects of thymoquinone on lysophosphatidic acid-induced oncogenic pathways in ovarian cancer cells.

Thymoquinone, a therapeutic phytochemical derived from Nigella sativa, has been shown to have a potent anticancer activity. However, it has been identified that the tumor microenvironment (TME) can attenuate the anticancer effects of thymoquinone (TQ) in ovarian cancer. Lysophosphatidic acid (LPA), a lipid growth factor present in high concentration in the TME of ovarian cancer, has been shown to regulate multiple oncogenic pathways in ovarian cancer. Taking account of the crucial role of LPA in the genesis and progression of ovarian cancer, the present study is focused on assessing the efficacy of TQ in inhibiting LPA-stimulated oncogenic pathways in ovarian cancer cells. Our results indicate that TQ is unable to attenuate LPA-stimulated proliferation or metabolic reprogramming in ovarian cancer cells. However, TQ potently inhibits the basal as well as LPA-stimulated migratory responses of the ovarian cancer cells. Furthermore, TQ abrogates the invasive migration of ovarian cancer cells induced by Gαi2, through which LPA stimulates cell migration. TQ also attenuates the activation of JNK, Src, and FAK, the downstream signaling nodes of LPA-LPAR-Gαi2 signaling pathway. In addition to establishing the differential effects of TQ in ovarian cancer cells, our results unravel the antitherapeutic role of LPA in the ovarian cancer TME could override the inhibitory effects of TQ on cell proliferation and metabolic reprogramming of ovarian cancer cells. More importantly, the concomitant finding that TQ could still sustain its inhibitory effect on LPA-stimulated invasive cell migration, points to its potential use as a response-specific therapeutic agent in ovarian cancer.

Transactivation of platelet-derived growth factor receptor alpha by the GTPase-deficient activated mutant of Galpha12.

The GTPase-deficient, activated mutant of Galpha12 (Galpha12Q229L, or Galpha12QL) induces neoplastic growth and oncogenic transformation of NIH 3T3 cells. Using microarray analysis, we have previously identified a role for platelet-derived growth factor receptor alpha (PDGFRalpha) in Galpha12-mediated cell growth (R. N. Kumar et al., Cell Biochem. Biophys. 41:63-73, 2004). In the present study, we report that Galpha12QL stimulates the functional expression of PDGFRalpha and demonstrate that the expression of PDGFRalpha by Galpha12QL is dependent on the small GTPase Rho. Our results indicate that it is cell type independent as the transient expression of Galpha12QL or the activation of Galpha12-coupled receptors stimulates the expression of PDGFRalpha in NIH 3T3 as well as in human astrocytoma 1321N1 cells. Furthermore, we demonstrate the presence of an autocrine loop involving PDGF-A and PDGFRalpha in Galpha12QL-transformed cells. Analysis of the functional consequences of the Galpha12-PDGFRalpha signaling axis indicates that Galpha12 stimulates the phosphatidylinositol 3-kinase (PI3K)-AKT signaling pathway through PDGFR. In addition, we show that Galpha12QL stimulates the phosphorylation of forkhead transcription factor FKHRL1 via AKT in a PDGFRalpha- and PI3K-dependent manner. Since AKT promotes cell growth by blocking the transcription of antiproliferative genes through the inhibitory phosphorylation of forkhead transcription factors, our results describe for the first time a PDGFRalpha-dependent signaling pathway involving PI3K-AKT-FKHRL1, regulated by Galpha12QL in promoting cell growth. Consistent with this view, we demonstrate that the expression of a dominant negative mutant of PDGFRalpha attenuated Galpha12-mediated neoplastic transformation of NIH 3T3 cells.

Ovarian cancer cell-derived lysophosphatidic acid induces glycolytic shift and cancer-associated fibroblast-phenotype in normal and peritumoral fibroblasts.

Cancer-associated fibroblasts (CAFs) play a critical role in cancer progression, metastasis, and therapy resistance. Molecular events that confer CAF-phenotype to predecessor-cells are not fully understood. We demonstrate here that the ovarian cancer cell-conditioned medium (OCC-CM) induces CAF-phenotype in MRC5 lung-fibroblasts and it can be mimicked by LPA. While OCC-CM and LPA stimulated the expression of cellular CAF-markers by 3-days, they induced aerobic glycolysis, a metabolic marker for CAF, by 6 hrs. OCC-CM/LPA-induced glycolysis in lung (MRC5) as well as ovarian fibroblasts (NOF151) was inhibited by the LPA-receptor antagonist, Ki16425. Ovarian cancer patient-derived ascitic fluid-induced aerobic glycolysis in both NFs and Ovarian CAFs and it was inhibited by Ki16425. Further analysis indicated that LPA upregulated HIF1α-levels and the silencing of HIF1α attenuated LPA-induced glycolysis in both NOFs and CAFs. These results establish LPA-induced glycolytic-shift as the earliest, potentially priming event, in NF to CAF-transition. These findings also identify a role for LPA-LPAR-HIF1α signaling-hub in the maintenance of the glycolytic-phenotype in CAFs. Our results provide evidence that targeted inhibition of LPA-mediated metabolic reprogramming in CAFs may represent an adjuvant therapy in ovarian cancer.

LPA Induces Metabolic Reprogramming in Ovarian Cancer via a Pseudohypoxic Response.

Although hypoxia has been shown to reprogram cancer cells toward glycolytic shift, the identity of extrinsic stimuli that induce metabolic reprogramming independent of hypoxia, especially in ovarian cancer, is largely unknown. In this study, we use patient-derived ovarian cancer cells and high-grade serous ovarian cancer cell lines to demonstrate that lysophosphatidic acid (LPA), a lipid growth factor and GPCR ligand whose levels are substantially increased in ovarian cancer patients, triggers glycolytic shift in ovarian cancer cells. Inhibition of the G protein α-subunit Gαi2 disrupted LPA-stimulated aerobic glycolysis. LPA stimulated a pseudohypoxic response via Rac-mediated activation of NADPH oxidase and generation of reactive oxygen species, resulting in activation of HIF1α. HIF1α in turn induced expression of glucose transporter-1 and the glycolytic enzyme hexokinase-2 (HKII). Treatment of mice bearing ovarian cancer xenografts with an HKII inhibitor, 3-bromopyruvate, attenuated tumor growth and conferred a concomitant survival advantage. These studies reveal a critical role for LPA in metabolic reprogramming of ovarian cancer cells and identify this node as a promising therapeutic target in ovarian cancer.Significance: These findings establish LPA as a potential therapeutic target in ovarian cancer, revealing its role in the activation of HIF1α-mediated metabolic reprogramming in this disease. Cancer Res; 78(8); 1923-34. ©2018 AACR.

Heterodimeric Fc-fused IL12 shows potent antitumor activity by generating memory CD8+ T cells.

Interleukin-12 (IL12) (p35/p40 complex) is a heterodimeric cytokine with potent anti-tumor activity. However, its short serum half-life and high dose-related toxicities limit its clinical efficacy. Here, we constructed heterodimeric immunoglobulin Fc-fused mouse IL12 (mIL12) in a monovalent binding format (mono-mIL12-Fc) to generate long-acting mIL12 in the naturally occurring heterodimeric form. Mono-mIL12-Fc exhibited a much longer plasma half-life than recombinant mIL12, enabling twice-weekly systemic injections to remove established tumors in syngeneic mouse models. Mono-mIL12-Fc was more potent than wild-type Fc-based bivalent-binding IL12-Fc (bi-mIL12-Fc) for eradicating large established immunogenic tumors without noticeable toxicities by enhancing interferon-γ production and the proliferation of immune effector cells in tumors. More importantly, mono-mIL12-Fc triggered weaker IL12 signaling than bi-mIL12-Fc, favoring the generation of functional and protective memory CD8+ T cells. Our results demonstrate that heterodimeric-Fc-fused IL12 is a suitable format for augmenting adaptive CD8+ T cell immune responses, providing a practical alternative to the systemic administration of IL12 for antitumor therapy.

Corrigendum: Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins.

[This corrects the article on p. 394 in vol. 7, PMID: 27766096.].

Immunoglobulin Fc Heterodimer Platform Technology: From Design to Applications in Therapeutic Antibodies and Proteins.

The monospecific and bivalent characteristics of naturally occurring immunoglobulin G (IgG) antibodies depend on homodimerization of the fragment crystallizable (Fc) regions of two identical heavy chains (HCs) and the subsequent assembly of two identical light chains (LCs) via disulfide linkages between each HC and LC. Immunoglobulin Fc heterodimers have been engineered through modifications to the CH3 domain interface, with different mutations on each domain such that the engineered Fc fragments, carrying the CH3 variant pair, preferentially form heterodimers rather than homodimers. Many research groups have adopted different strategies to generate Fc heterodimers, with the goal of high heterodimerization yield, while retaining biophysical and biological properties of the wild-type Fc. Based on their ability to enforce heterodimerization between the two different HCs, the established Fc heterodimers have been extensively exploited as a scaffold to generate bispecific antibodies (bsAbs) in full-length IgG and IgG-like formats. These have many of the favorable properties of natural IgG antibodies, such as high stability, long serum half-life, low immunogenicity, and immune effector functions. As of July 2016, more than seven heterodimeric Fc-based IgG-format bsAbs are being evaluated in clinical trials. In addition to bsAbs, heterodimeric Fc technology is very promising for the generation of Fc-fused proteins and peptides, as well as cytokines (immunocytokines), which can present the fusion partners in the natural monomeric or heterodimeric form rather than the artificial homodimeric form with wild-type Fc. Here, we present relevant concepts and strategies for the generation of heterodimeric Fc proteins, and their application in the development of bsAbs in diverse formats for optimal biological activity. In addition, we describe wild-type Fc-fused monomeric and heterodimeric proteins, along with the difficulties associated with their preparations, and discuss the use of heterodimeric Fc as an alternative scaffold of wild-type Fc for naturally monomeric or heterodimeric proteins, to create Fc-fusion proteins with novel therapeutic modality.

Lysophosphatidic acid stimulates epithelial to mesenchymal transition marker Slug/Snail2 in ovarian cancer cells via Gαi2, Src, and HIF1α signaling nexus.

Recent studies have identified a critical role for lysophosphatidic acid (LPA) in the progression of ovarian cancer. Using a transcription factor activation reporter array, which analyzes 45 distinct transcription factors, it has been observed that LPA observed robustly activates the transcription factor hypoxia-induced factor-1α (HIF1α) in SKOV3.ip ovarian cancer cells. HIF1α showed 150-fold increase in its activation profile compared to the untreated control. Validation of the array analysis indicated that LPA stimulates a rapid increase in the levels of HIF1α in ovarian cancer cells, with an observed maximum level of HIF1α-induction by 4 hours. Our report demonstrates that LPA stimulates the increase in HIF1α levels via Gαi2. Consistent with the role of HIF1α in epithelial to mesenchymal transition (EMT) of cancer cells, LPA stimulates EMT and associated invasive cell migration along with an increase in the expression levels N-cadherin and Slug/Snail2. Using the expression of Slug/Snail2 as a marker for EMT, we demonstrate that the inhibition of Gαi2, HIF1α or Src attenuates this response. In line with the established role of EMT in promoting invasive cell migration, our data demonstrates that the inhibition of HIF1α with the clinically used HIF1α inhibitor, PX-478, drastically attenuates LPA-stimulates invasive migration of SKOV3.ip cells. Thus, our present study demonstrates that LPA utilizes a Gαi2-mediated signaling pathway via Src kinase to stimulate an increase in HIF1α levels and downstream EMT-specific factors such as Slug, leading to invasive migration of ovarian cancer cells.

Extracellular Release of CD11b by TLR9 Stimulation in Macrophages.

CpG-DNA upregulates the expression of pro-inflammatory cytokines, chemokines and cell surface markers. Investigators have shown that CD11b (integrin αM) regulates TLR-triggered inflammatory responses in the macrophages and dendritic cells. Therefore, we aimed to identify the effects of CpG-DNA on the expression of CD11b in macrophages. There was no significant change in surface expression of CD11b after CpG-DNA stimulation. However, CD11b was released into culture supernatants after stimulation with phosphorothioate-backbone modified CpG-DNA such as PS-ODN CpG-DNA 1826(S). In contrast, MB-ODN 4531 and non-CpG-DNA control (regardless of backbone type and liposome-encapsulation) failed to induce release of CD11b. Therefore, the context of the CpG-DNA sequence and phosphorothioate backbone modification may regulate the effects of CpG-DNA on CD11b release. Based on inhibitor studies, CD11b release is mediated by p38 MAP kinase activation, but not by the PI3K and NF-κB activation. CD11b release is mediated by lysosomal degradation and by vacuolar acidification in response to CpG-DNA stimulation. The amount of CD11b in the exosome precipitant was significantly increased by CpG-DNA stimulation in vivo and in vitro depending on TLR9. Our observations perhaps give more insight into understanding of the mechanisms involved in CpG-DNA-induced immunomodulation in the innate immunity.