Proliferation Signaling Research Articles

CXCL12 restricts tumor growth by suppressing the Ras, ERK1/2, c-Myc, and the immune checkpoint PD-L1 pathways

Cytokines constitute a family of proteins that modulate the immune system and are secreted by many cells. CXCL12, along with its receptor CXCR4, are essential players in numerous processes. Dysregulation of their function underlie the mechanism(s) of several pathologies, including malignancies. Here, we demonstrate an unexpected effect of the cytokine and its receptor: In both cells and animal models, CXCL12 restricts tumorigenicity of the human glioblastoma cells U87-MG and U-118, and of a cell line derived from PyMT mouse breast cancer. Overexpression of CXCL12 inhibits activation of the oncogene Ras which results in downregulation of its proliferative signals, such as reduced phosphorylation of the extracellular signal-regulated kinase 1/2 (ERK1/2), inhibition of c-Myc expression, and subsequent inhibition of cell cycle. Furthermore, CXCL12 induces downregulation of the growth differentiation factor 15 (GDF15), insulin-like growth factor-binding protein 6 (IGFBP6), and matrix metalloproteinase-3 (MMP3), which are implicated in sending metastases. Indeed, monitoring cell migration in vitro and generation of metastases in mice demonstrate that CXCL12 slows the migration of U87-MG and PyMT cells. Remarkably, overexpression of CXCL12 also downregulates the cell surface immune checkpoint protein programmed cell death-ligand 1 (PD-L1), resulting in recruitment of cytotoxic CD8 T cells into xenografts accompanied by their shrinkage. Overall, CXCL12 inhibits tumor growth through several distinct mechanisms: inhibition of cell cycle and migration, as well as impairment of immune checkpoint, thereby stimulating a strong host's immune response. The mechanism(s) that renders CXCL12 a tumor-promoting factor in certain cells and a suppressor in others has remained elusive.

Interleukin-22 promotes endometrial carcinoma cell proliferation and cycle progression via ERK1/2 and p38 activation.

Endometrial carcinoma (EC) is one of the most common gynecological malignant tumors, but its underlying pathogenic mechanisms are largely obscure. Interleukin-22 (IL-22), one cytokine in the tumor immune microenvironment, was reported to be associated with carcinoma progression. Here, we aimed to investigate the regulation of IL-22 in endometrial carcinoma. Enzyme-linked immunosorbent assay (ELISA) analysis of IL-22 was done in 27 controls and 51 patients with EC. We examined the proliferative potential, cycle progression, and signaling pathways modulated by IL-22 in EC cells. Western blot analysis was performed to investigate the expression of proliferative and cycle-related proteins in EC cells. The effect of IL-22 mediated by interleukin-22 receptor alpha 1 (IL-22RA1) was examined using cell transfection with small interfering RNA (siRNA). In addition, a xenograft tumor model was performed to assess the effect of IL-22 in vivo. We demonstrated significant up-regulation of serum IL-22 concentrations in EC patients (42.59 ± 23.72pg/mL) compared to the control group (27.47 ± 8.29pg/mL). High levels of IL-22 concentrations appear to correlate with malignant clinicopathological features of EC. Treatment with IL-22 promoted cell proliferation and G1/S phase progression in Ishikawa and HEC-1B cells. Western blot analysis revealed that c-Myc, cyclin E1, cyclin-dependent kinase (CDK)2, cyclin D1, CDK4, CDK6, p-extracellular signal-regulated kinase1/2 (p-ERK1/2), and p-p38 were highly expressed in EC cells exposed to IL-22. Moreover, in the EC mice model, we found that giving exogenous IL-22 increased tumor volume and weight. Immunohistochemistry showed that intra-tumor Ki-67 expression was up-regulated upon IL-22 treatment. The IL-22-mediated changes in cell proliferation, cycle progression, and protein expression can be effectively inhibited by the ERK1/2 inhibitor U0126 and the p38 inhibitor SB202190. In addition, the role of IL-22 in EC is receptor-dependent. Our findings suggest that IL-22 promotes endometrial carcinoma cell proliferation and G1/S phase progression by activating ERK1/2 and p38 signaling. Therefore, IL-22 may represent a potential therapeutic target for the treatment of endometrial carcinoma.

PARP1 Inhibitors: An Important Part in Cancer Treatment

Abstract: Cancer is a highly fatal disease that is typified by aberrant cells proliferating out of control. The properties of acquired cells, including as genomic instability and mutations, cellular death evasion, and proliferative signaling sustenance, are evidenced by the hallmarks of cancer and contribute to the establishment of malignant tumors. A protein called poly(ADP-ribose) polymerase- 1 (PARP1) is essential for both cell survival and DNA damage repair, two processes that affect cellular control. Since homologous recombination deficient cells exhibit cellular death upon suppression of PARP1, the PARP protein has gained attention as a potential target for anticancer treatments. The Food and Drug Administration (FDA) has already approved a number of effective PARP1 inhibitors, including olaparib and niraparib. The final chemical has a 1,3,4- thiadiazole core in its structure. In fact, heterocyclic moieties have gained attention due to their numerous medicinal advantages, which include their capacity to combat cancer. The compounds derived from these substances have been studied as PARP1 inhibitors, with promising results. Thus, the goal of this study is to go over the importance of PARP1 in cancer as well as its role in cell regulation. It also attempts to offer a comprehensive assessment of the literature that has been published in the previous fifteen years about PARP1 inhibitors, including the different scaffolds, with an emphasis on features of the structure-activity relationship. This will provide vital information for the creation of fresh, more effective PARP1 inhibitors.

Comparative liver transcriptome analysis in hamsters infected with food-borne trematodes Opisthorchis felineus, Opisthorchis viverrini, or Clonorchis sinensis.

Epidemiologically important food-borne trematodes Opisthorchis viverrini and Clonorchis sinensis are recognized as biological carcinogens of Group 1A, while Opisthorchis felineus is in Group 3 as noncarcinogenic to humans. Mechanisms of the biological carcinogenesis are still elusive. Some studies highlight chronic inflammation as a key factor and common pathway for cancer initiation and progression. Nonetheless, the chronic inflammation alone does not explain why these three species differ in carcinogenicity. We focused this study on genome-wide landscapes of liver gene expression and activation of cellular pathways in Mesocricetus auratus golden hamsters infected with C. sinensis (South Korea), O. viverrini (Thailand), or O. felineus (Russia) at 1 and 3 months after infection initiation. Liver transcriptomes of golden hamsters (HiSeq Illumina, 2X150 bp) were sequenced at 1 and 3 months postinfection. Data processing was carried out using the following bioinformatic and experimental approaches: analysis of differential gene expression, estimates of proportions of affected liver cell types, liver histopathology, and examination of weighted gene coexpression networks. All infections caused enrichment with inflammatory response signaling pathways, fibrogenesis and cell proliferation, and IL2-STAT5, TNF-NF-κB, TGF-β, Hippo, MAPK, and PI3K-Akt signaling pathways. Nevertheless, species-specific responses to each infection were noted too. We also identified species-specific responses of liver cell types, differentially expressed gene clusters, and cellular pathways associated with structural liver damage (such as periductal fibrosis, epithelial neoplasia, and inflammation). This is the first comparative analysis of gene expression landscapes in the liver of experimental animals infected with O. viverrini, O. felineus, or C. sinensis. The trematodes have species-specific effects on the hepatobiliary system by triggering signaling pathways, thereby leading to differences in the severity of hepatobiliary structural lesions and contributing to the pathogenicity of closely related foodborne trematodes.

DAB2IP loss in luminal a breast cancer leads to NF-κB-associated aggressive oncogenic phenotypes.

Despite proven therapy options for estrogen receptor-positive (ER+) breast tumors, a substantial number of patients with ER+ breast cancer exhibit relapse with associated metastasis. Loss of expression of RasGAPs leads to poor outcomes in several cancers, including breast cancer. Mining the The Cancer Genome Atlas (TCGA) breast cancer RNA-Seq dataset revealed that low expression of the RasGAP DAB2IP was associated with a significant decrease in relapse-free survival in patients with Luminal A breast cancer. Immunostaining demonstrated that DAB2IP loss occurred in grade 2 tumors and higher. Consistent with this, genes upregulated in DAB2IP-low Luminal A tumors were shared with more aggressive tumor subtypes and were associated with proliferation, metastasis, and altered ER signaling. Low DAB2IP expression in ER+ breast cancer cells was associated with increased proliferation, enhanced stemness phenotypes, and activation of IKK, the upstream regulator of the transcription factor NF-κB. Integrating cell-based ChIP-Seq with motif analysis and TCGA RNA-Seq data, we identified a set of candidate NF-κB target genes upregulated with loss of DAB2IP linked with several oncogenic phenotypes, including altered RNA processing. This study provides insight into mechanisms associated with aggressiveness and recurrence within a subset of the typically less aggressive Luminal A breast cancer intrinsic subtype.

Thiazolidinedione derivatives in cancer therapy: exploring novel mechanisms, therapeutic potentials, and future horizons in oncology.

Thiazolidinedione derivatives have shown significant potential as targeted cancer therapies by leveraging their various mechanisms of action. These include suppressing cell proliferation, triggering apoptosis, and influencing signaling pathways associated with tumor development. Their multifaceted effects make them promising candidates for advancing cancer treatment strategies. They have shown significant promise as anti-cancer agents, particularly through their ability to inhibit lipogenesis pathways and apoptosis essential for cancer cell survival and proliferation. This review comprehensively examines the anti-cancer potential of thiazolidinedione derivatives by targeting key aspects of lipid metabolism, apoptosis, and various mechanistic pathways. This review provides an in-depth examination of the anti-cancer potential of TZD derivatives, focusing on their mechanisms of action, therapeutic applications, and future directions in oncology. The anti-tumor effects of TZDs primarily involve the stimulation of peroxisome proliferator-activated receptor gamma (PPAR-γ), suppressing cell proliferation, induction of apoptosis, and inhibition of angiogenesis. Moreover, recent evidence highlights their ability to modulate non-PPAR-γ pathways, such as PI3K/Akt, NF-κB, and MAPK, further expanding their role in overcoming drug resistance and enhancing therapeutic outcomes. This review explores the preclinical (in vitro and in vivo) and clinical research investigating TZD derivatives efficacy in various cancer types. The insights underscore the significance of targeting lipogenesis as a novel anti-cancer strategy, positioning thiazolidinedione derivatives as potent candidates for future cancer therapeutics. As the oncology landscape evolves, TZD derivatives (rosiglitazone, pioglitazone, inolitazone, troglitazone, and 2,4-thiazolidinedione derivatives) represent a promising class of agents with the potential to contribute meaningfully to cancer treatment. By integrating existing knowledge with recent advancements, this study provides valuable insights into the role of thiazolidinedione derivatives in cancer treatment, paving the way for further research and clinical applications.

The Rho effector ARHGAP18 coordinates a Hippo pathway feedback loop through YAP and Merlin to regulate the cytoskeleton and epithelial cell polarity.

The organization of the cell's cytoskeletal filaments is coordinated through a complex symphony of signaling cascades originating from internal and external cues. Two major actin regulatory pathways are signal transduction through Rho family GTPases and growth and proliferation signaling through the Hippo pathway. These two pathways act to define the actin cytoskeleton, controlling foundational cellular attributes such as morphology and polarity. In this study, we use human epithelial cells to investigate the interplay between the Hippo and Rho Family signaling pathways, which have predominantly been characterized as independent actin regulatory mechanisms. We identify that the RhoA effector, ARHGAP18, forms a complex with the Hippo pathway transcription factor YAP to address a long-standing enigma in the field. Using super resolution STORM microscopy, we characterize the changes in the actin cytoskeleton, on the single filament level, that arise from CRISPR/Cas9 knockout of ARHGAP18. We report that the loss of ARHGAP18 results in alterations of the cell that derive from both aberrant RhoA signaling and inappropriate nuclear localization of YAP. These findings indicate that the Hippo and Rho family GTPase signaling cascades are coordinated in their temporal and spatial control of the actin cytoskeleton.

PIEZO1 overexpression in hereditary hemorrhagic telangiectasia arteriovenous malformations.

Hereditary hemorrhagic telangiectasia (HHT) is an inherited vascular disorder characterized by arteriovenous malformations (AVMs). Loss-of-function mutations in Activin receptor-like kinase 1 (ALK1) cause type 2 HHT and Alk1 knockout (KO) mice develop AVMs due to overactivation of VEGFR2/PI3K/AKT signaling pathways. However, the full spectrum of signaling alterations in Alk1 mutants remains unknown and means to combat AVM formation in patients are yet to be developed. Single-cell RNA sequencing of endothelial-specific Alk1 KO mouse retinas and controls identified a cluster of endothelial cells (ECs) that was unique to Alk1 mutants and that overexpressed fluid shear stress (FSS) signaling signatures including upregulation of the mechanosensitive ion channel PIEZO1. PIEZO1 overexpression was confirmed in human HHT lesions, and genetic and pharmacological PIEZO1 inhibition was tested in Alk1 KO mice, as well as downstream PIEZO1 signaling. Pharmacological PIEZO1 inhibition, and genetic Piezo1 deletion in Alk1 -deficient mice effectively mitigated AVM formation. Furthermore, we identified that elevated VEGFR2/AKT, ERK5-p62-KLF4, hypoxia and proliferation signaling were significantly reduced in Alk1 - Piezo1 double ECKO mice. PIEZO1 overexpression and signaling is integral to HHT2, and PIEZO1 blockade reduces AVM formation and alleviates cellular and molecular hallmarks of ALK1-deficient cells. This finding provides new insights into the mechanistic underpinnings of ALK1-related vascular diseases and identifies potential therapeutic targets to prevent AVMs.

Glycogen synthase kinase-3 inhibition and insulin enhance proliferation and inhibit maturation of human iPSC-derived cardiomyocytes via TCF and FOXO signaling.

Embryonic signaling pathways exert stage-specific effects during cardiac development, yet the precise signals for proliferation or maturation remain elusive. To uncover the cues for proliferation, we performed a combinatory cell-cycle screen for insulin and glycogen synthase kinase-3 (GSK3) inhibition in spontaneously beating human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our analysis for proliferation, and subsequential downstream sarcomere development, gene expression analysis, and molecular interventions identified a temporal interplay between insulin/Akt/FOXO and CHIR99021/Wnt/GSK3/TCF signaling. Combined pathway activation led to proliferation of immature hiPSC-CMs with low sarcomere and mitochondria content, while, in the absence of pathway activators, cardiomyocytes rapidly exited the cell cycle and fetched higher organization of sarcomeres and mitochondria. Our data demonstrate two important pathways, which enhance proliferation and inhibit maturation, and provide molecular mechanistic understanding of these cell fate decisions in immature hiPSC-CMs.

Unveiling the Role of Mechanistic Target of Rapamycin Kinase (MTOR) Signaling in Cancer Progression and the Emergence of MTOR Inhibitors as Therapeutic Strategies.

The mechanistic target of rapamycin kinase (MTOR) is pivotal for cell growth, metabolism, and survival. It functions through two distinct complexes, mechanistic TORC1 and mechanistic TORC2 (mTORC1 and mTORC2). These complexes function in the development and progression of cancer by regulating different cellular processes, such as protein synthesis, lipid metabolism, and glucose homeostasis. The mTORC1 complex senses nutrients and initiates proliferative signals, and mTORC2 is crucial for cell survival and cytoskeletal rearrangements. mTORC1 and mTORC2 have therefore emerged as potential targets for cancer treatment. Several mTOR inhibitors, including rapamycin and its analogs (rapalogs), primarily target mTORC1 and are effective for specific cancer types. However, these inhibitors often lead to resistance and limited long-term advantages due to the activation of survival pathways through feedback mechanisms. Researchers have created next-generation inhibitors targeting mTORC1 and mTORC2 and dual PI3K/mTOR inhibitors to address these difficulties. These inhibitors demonstrate enhanced anti-tumor effects by simultaneously disrupting multiple signaling pathways and show promise for improved and long-lasting therapies. However, development of resistance and adverse side effects remain a significant obstacle. Recent additions known as RapaLinks have emerged as a boon to counter drug-resistant cancer cells, as they are more potent and provide a more comprehensive blockade of mTOR signaling pathways. This Review combines current research findings and clinical insights to enhance our understanding of the crucial role of mTOR signaling in cancer biology and highlights the evolution of mTOR inhibitors as promising therapeutic approaches.

GCC2 promotes non-small cell lung cancer progression by maintaining Golgi apparatus integrity and stimulating EGFR signaling pathways

Fundamental changes in intracellular processes, such as overactive growth signaling pathways, are common in carcinomas and are targets of many cancer therapeutics. GRIP and coiled-coil containing 2 (GCC2) is a trans-Golgi network (TGN) golgin maintaining Golgi apparatus structure and regulating vesicle transport. Here, we found an aberrant overexpression of GCC2 in non-small cell lung cancer (NSCLC) and conducted shRNA-mediated gene knockdown to investigate the role of GCC2 in NSCLC progression. shRNA-mediated GCC2 knockdown suppressed NSCLC cell growth, migration, stemness, and epithelial-mesenchymal transition (EMT) in vitro and tumor growth in vivo. In addition, GCC2 knockdown suppressed cancer cell exosome secretion and the oncogenic capacity of cancer cell-derived exosomes. Mechanistically, GCC2 inhibition decreased epidermal growth factor receptor (EGFR) expression and downstream growth and proliferation signaling. Furthermore, GCC2 inhibition compromised Golgi structural integrity in cancer cells, indicating a functional role of GCC2 in regulating intracellular trafficking and signaling to promote lung cancer progression. Together, these findings suggest GCC2 as a potential therapeutic target for the treatment of NSCLC.

Tapinarof, an Aryl Hydrocarbon Receptor Ligand, Mitigates Fibroblast Activation in Thyroid Eye Disease: Implications for Novel Therapy.

In thyroid eye disease (TED), activation and proliferation of orbital fibroblasts (OFs) promotes remodeling and causes an increase in the volume of orbital tissue. Platelet-derived growth factors (PDGFs) are elevated in TED and promote OF activation. The aryl hydrocarbon receptor (AHR), a ligand activated nuclear receptor, is important in regulating OF activation. AHR ligands have been evaluated as therapeutic agents for inflammatory diseases. Here, we hypothesize that AHR ligands will block PDGF-induced signaling in TED OFs. OFs from both non-TED and TED patients were treated with PDGFβ, with or without the AHR ligands 6-Formylindolo[3,2-b]carbazole (FICZ) or tapinarof. Cell viability was measured by the Alamar Blue assay. Cell proliferation was quantified using the BrdU assay. Cell lysates were collected and analyzed by Western blotting and real-time quantitative PCR (RT-qPCR) to measure PDGF and AHR signaling. Scratch assays were used to measure OF migration. PDGFβ induced proliferation in TED OFs significantly more than in non-TED OFs. Additionally, PDGFβ increased phosphorylation of AKT and expression of thymidylate synthase (TYMS). PDGFβ dependent proliferation and downstream signaling were attenuated by FICZ or tapinarof. TYMS and other PDGF target genes were upregulated by PDGFβ and reduced by AHR activation. PDGFβ induced TED OF migration while both FICZ and tapinarof diminished this effect. PDGF signaling led to increased proliferation and activation of TED OFs. Treatment of TED OFs with the AHR ligands, FICZ and tapinarof, mitigated PDGF induced effects. These studies support the concept that AHR and PDGF signaling could form the basis for new TED therapeutics.

Identifying potential inhibitors against galectin-3-binding protein (LGALS3BP) for therapeutic targeting of glioma

Glioblastoma is one of the most lethal types of Gliomas, and its treatment greatly depends on the stage of its detection. Galactin-3-binding protein (LGALS3BP) serves as a novel circulatory biomarker for detecting glioma at an early stage. This protein is also responsible for metastasis, proliferative signaling, angiogenesis, and immune system evasion in the case of brain tumors. Inhibition of LGALS3BP can help in the reduction of metastasis and progression of the disease. Currently, no effective drug is available that can completely treat glioma. In this study, we have virtually screened the National Cancer Institute (NCI) drug databank to discover potential inhibitors of LGALS3BP. Based on the binding free energy calculations using MMPBSA, three compounds, 627861 (−16.69 kcal/mol), 329090 (−13.66 kcal/mol), and 627855 (−10.01 kcal/mol), were selected as potent inhibitors. 200 ns MD simulation studies further complemented this study. Finally, we recommend three molecules, 627861, 329090, and 627855, can be potential inhibitors of LGAL3SBP. The structural scaffolds of these molecules can also lead to the optimization of better inhibitors of LGALS3BP and be implicated in the therapeutic management of glioma after desired experimental validations.

Inhibition of NPC1 suppresses cell proliferation and β-catenin signaling activation of liver cancer.

Niemann-Pick Type C1 (NPC1) plays a significant role in the development of liver diseases and liver cancer. Our objective was to investigate the involvement of NPC1 in regulating liver cancer development. We observed that high levels of NPC1 expression in tumor tissues from patients with liver cancer were associated with a poor prognosis. Through in vitro experiments, we found that inhibiting NPC1 expression reduced the proliferation, invasion, and migration of liver cancer cells, while also inducing apoptosis in these cells. Additionally, the inhibition of NPC1 led to decreased activation of Wnt/β-catenin signaling. In vivo studies further supported our findings by demonstrating that the suppression of liver cancer cell growth was effectively achieved through the inhibition of NPC1. Overall, our results strongly indicate that the inhibition of NPC1 suppresses liver cancer cell proliferation. Targeting NPC1 is a promising potential therapeutic strategy for liver cancer.

Abstract 4145829: Fatty Acid Binding Protein 4 as Potential Indicator for Gestational Diabetes Mellitus-Induced Fetal Endothelial Dysfunction

Introduction/Background: Gestational diabetes mellitus (GDM) is the most common complication during pregnancy. GDM is leading to an elevated risk for the development of cardiovascular diseases both in the mother and the child in later life. Previous studies have identified fatty acid-binding protein 4 (FABP4) as a prime candidate involved in the pathophysiology of GDM. Indeed, women with GDM exhibit elevated blood levels of FABP4, suggesting its potential role as a biomarker for endothelial dysfunction and cardiovascular risk assessment in GDM. Research Question/Hypothesis: Does FABP4 affect the function of human endothelial cells from patients with GDM? Goals/Aim: Our aim is a better understanding of the role of FABP4 in fetal endothelial dysfunction of patients with GDM and the development of potential therapeutic strategies. Methods/Approach: Primary human umbilical vein endothelial cells (HUVECs) were isolated from umbilical cords obtained from normoglycemic and GDM pregnancies. Total mRNA from HUVECs was used for bulk RNA sequencing to compare gene expression pattern. HUVECs were subjected to functional analyses assessing proliferation, migration, NO and insulin signaling. Finally, human endothelial cells were exposed to high laminar flow (30 dyn/cm 2 ) or simvastatin (10 nM) treatment to investigate their impact on FABP4 expression and endothelial function. Results/Data: Bulk RNA sequencing data analysis revealed FABP4 as a gene overexpressed in GDM HUVECs compared to normoglycemic controls. Gene set enrichment analysis showed in addition an enrichment of genes involved in angiogenesis and Notch signaling pathways in human endothelial cells from GDM patients. GDM HUVECs had a significantly higher wound healing capacity compared to normoglycemic controls. The PI3K/AKT/mTOR pathway was enriched in GDM HUVECs. GDM HUVECs displayed impaired activation of AKT and eNOS (expression and phosphorylation) after stimulation with insulin, suggesting a possible insulin resistance. Finally, subjecting HUVECs to high laminar shear stress or simvastatin treatment caused a reduction of FABP4 mRNA expression and an increase of eNOS expression, indicating potential therapeutic strategies to improve endothelial function. Conclusions: We provided evidence that FABP4 could be involved in fetal GDM-induced endothelial dysfunction. High laminar shear stress and medications activating eNOS signaling could protect the endothelium against the negative impact of FABP4 in GDM.

BIOM-41. HMGB2 IS A CANDIDATE PROGNOSTIC BIOMARKER IN BOTH ADULT AND PEDIATRIC DIFFUSE GLIOMAS

Abstract Few prognostic molecular markers are common to pediatric- and adult diffuse gliomas (PDG and ADG). HMGB2 is a protein expressed in human brain development and in de-differentiated high-grade gliomas (HGG), which we have recently described as a putative prognostic and predictive biomarker in ADG, associated with proliferative activity and EIF2 signaling pathway upregulation. Additionally, we showed that HMGB2 shRNA knockdown (HMGB2-KD) reduced cell proliferation and colony formation, and radio-sensitized ADG cell lines. Aims: To assess the prognostic potential of HMGB2 in PDG and its association with stemness. METHODS 1) We singled out HMGB2 from proteomic profiles of an independent PDG cohort previously associated with EIF2 and MYCN signaling upregulation for survival analysis. 2) PDG cells SF188 were de-differentiated with substitution of DMEM by a neurobasal (NB) medium devoid of serum for stemness association studies. 3) We generated HMGB2-KD clones of PDG cell lines SF188 (grown in DMEM and NB) and KNS42 with 3 different shRNAs. RESULTS HMGB2 expression was significantly related to histological grade in PDG (grade 3 vs. grade 2 and grade 4 vs. grade 2: fold change= 8.16 and 9.39; p=0.0045 and <0.001; FDR= 0.04 and 0.0006, respectively) and survival (p=0.004, FDR=0.142 for PFS and p=0.03, FDR=0.291 for OS), in a Cox model including age and grade as covariables. Compared with cells growing in DMEM, SF188 cells growing in NB medium had a morphological change from stellate cells to round cells growing in spheres, with 2-fold increase in C-MYC and HMGB2 expression by Western analysis. HMGB2-KD resulted in >90% reduction of HMGB2 expression in SF188 and >70% in KNS42. CONCLUSION Collectively, our preliminary results suggest that HMGB2 is associated with stemness, has similar roles in PDG and ADG, and may be validated as prognostic/predictive biomarker in both adult and pediatric populations. Mechanistic studies of radiation response are underway.

CNSC-27. INTRATUMORAL CANCER CELL NETWORKS IN BRAIN METASTASES

Abstract Recently, we have shown that primary brain tumors form highly organized, small-world scale-free networks, reminiscent of early networks in neurodevelopment and highly resistant to therapy. Such putative cancer-cell intrinsic neural mechanisms are becoming an increasing focus in the field of Cancer Neuroscience, but it has not yet been shown whether tumors of non-CNS origin also have the ability to form multicellular networks which sustain proliferative signaling and increase resistance to therapy. We observed synchronized calcium activity using longitudinal microscopy both in vivo and in vitro that were reminiscent of gap junction coupled networks. Indeed, dye transfer experiments verified functional coupling of brain metastases cells in vitro. Similarly, inhibition of gap junctions with different pharmacological agents significantly reduced calcium oscillations and tumour proliferation in vitro. Furthermore, gap junctions are significantly upregulated in brain-tropic sublines compared to parental cells. To understand the functional relevance of these gap-junction coupled tumor cell networks, we performed bulk RNA-Sequencing of two melanoma metastasis models in monoculture under gap junction inhibition and control: We observed reduced calcium communication and a concordant downregulation of neurodevelopmental and synaptic pathways within the tumor cells upon network disconnection, highlighting a potential recapitulation of neural-like features in metastases to the brain. To investigate the therapeutic potential of targeting these networks, we treated mice with brain metastases with two brain-penetrant gap junction blockers and observed significantly reduced tumor burden. Finally, we investigated the presence of heterotypic networks between brain metastases cells and the brain microenvironment with our SR101 dye transfer pipeline. Contrary to our findings in primary brain tumors, no gap junction connections to cells of the brain microenvironment in those models with reduced tumor growth upon network inhibition could be detected, supporting a cancer cell intrinsic gap junction mediated network and highlighting a selectivity of these homogenous tumor networks and potential therapeutic vulnerability.

CSIG-11. TUMOR-CELL INTRINSIC CALCIUM OSCILLATIONS DRIVE BRAIN METASTASIS

Abstract Calcium is a ubiquitous secondary messenger which regulates many cellular processes. Recently, we have shown that a subpopulation of glioblastoma cells can intrinsically generate calcium oscillations which drive proliferative signalling. Here, we set out to investigate whether such cancer cell autonomous oscillations are also present in secondary brain tumours. Employing longitudinal intravital two-photon microscopy in awake mice, we demonstrate that brain metastases of melanoma and lung cancer exhibit spontaneous calcium oscillations in vivo along the metastatic cascade. These oscillations are already observed during intravascular arrest and in in vitro monocultures, suggesting a tumour-cell intrinsic origin. We developed a fully automatic image analysis pipeline to accurately quantify calcium oscillations both in vivo and in vitro. Mathematical modelling of cytosolic and ER calcium stores can predict stable calcium oscillations with similar frequency and peak characteristics as those experimentally observed. We discovered that these oscillations are tightly regulated by an interplay of calcium-induced calcium release and store-operated calcium entry (SOCE). Similarly, SOCE-related ion channels are enriched in tissue from human melanoma brain metastases compared to healthy brain controls and calcium activity can also be observed ex vivo in patient-derived brain metastasis resections. Finally, by correlating calcium activity and EdU incorporation we discovered that the presence of calcium oscillations predicts their proliferative potential in vitro. Importantly, by intravital two-photon microscopy we show that calcium activity correlates with metastasis size in preclinical models, suggesting a potential new therapeutic angle from which to target brain metastases.

Investigating the relationship between β-carotene intake from diet and supplements, smoking, and lung cancer risk

β-carotene is a naturally occurring and safe dietary source of vitamin A that is associated with cancer risk reductions when consumed in typical dietary amounts. However, two clinical trials reported increased incidence of lung cancer and total mortality among heavy smokers taking β-carotene supplements (20 or 30 mg/day). Based on these findings, the Joint FAO/WHO Expert Committee on Food Additives withdrew Acceptable Daily Intake values for β-carotene (0–5 mg/kg bw). We evaluated relevant epidemiological and toxicological literature to assess the biological plausibility of this relationship and identified three mechanisms involving cellular proliferation signaling, a mode of action for cancer promotion. The overall weight of evidence consistently demonstrated typical dietary doses of β-carotene decreased cellular proliferation signaling via these mechanisms, even in the presence of smoke, while co-exposure to smoke and higher, supplemental doses increased cellular proliferation signaling through these same pathways. The production of excessive oxidative β-carotene metabolites via reactions with smoke constituents may be a key event underlying this relationship. Consistent with previous findings, our evaluation indicated consumption of up to 50 mg/day β-carotene does not present safety concerns for the non-smoking general population. Heavy smokers consuming less than 15 mg β-carotene/day are not expected to be at an increased risk of lung cancer.

Single-cell RNA sequencing analysis reveals the role of TXNDC5 in keloid formation.

Thioredoxin domain-containing protein 5 (TXNDC5) is associated with fibrosis in a variety of organs, but its mechanism of action in keloid is unclear. In this study, we aimed to investigate the mechanism of TXNDC5 in keloid. Single-cell RNA sequencing data of keloid and normal scar samples obtained from public databases were normalized and clustered using the Seurat package. Pathway enrich analysis was conducted using biological process enrichment analysis and Gene Set Enrichment Analysis (GSEA). In addition, TXNDC5 expression and its effects on migration and invasion of keloid fibroblasts (KFs) were validated based on cell function experiments. A total of five cell types were obtained. The KF clusters were further clustered into two fibroblast subtypes (Fibroblast cells 1 and Fibroblast cells 2). Biological process enrichment analysis showed that transforming growth factor beta (TGF-β) signaling pathway was enriched in the two fibroblast subtypes. GSEA analysis demonstrated that genes in TGF-β signaling pathway were mainly enriched in Fibroblast cells 1, and that genes involved in cell proliferation, migration, and the TGF-β signaling pathway were all high-expressed in fibroblast cells 1. TXNDC5 was positively correlated with fibroblast proliferation, migration and TGF-β signaling pathway, and AUCell score. The cellular experiment confirmed that the messenger RNA and protein levels of TXNDC5 and TGF-β1 were high-expressed in KFs cells (P<0.001), and that knockdown of TXNDC5 downregulated TGF-β1 expression and inhibited migration and invasion of KFs (P<0.0001). Our study indicated that TGF-β signaling pathway was enriched in fibroblast cells, and TXNDC5 was positively correlated with proliferation, migration, and TGF-β signaling pathway. Cellular experiment demonstrated that knocking down TXNDC5 downregulated TGF-β1 expression, and suppressed migration and invasion of KFs. The current discoveries provided a new therapeutic strategy for the treatment of keloid.