Down-regulation Of CXCR4 Expression Research Articles

The magnitude of CXCR4 signaling regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1

CXCR4 antagonists sensitize FLT3/ITD+ AML cells to FLT3 inhibitors; however, CXCR4 signaling can induce apoptosis in AML cells, raising the question of whether CXCR4 signaling exerts divergent effects on FLT3/ITD+ cells. The present study investigated the paradoxical function of CXCR4 in resistance to FLT3 inhibitors. The FLT3 inhibitor quizartinib significantly decreased the number of FLT3/ITD+ Ba/F3 cells, whereas 1 ng/ml CXCL12 showed a significant protective effect against quizartinib. In contrast, CXCL12 over 100 ng/ml significantly decreased FLT3/ITD+ cell viability with concomitant downregulation of Runx1. Moreover, the survival of FLT3/ITD+ Ba/F3 or MOLM13 cells with low surface CXCR4 expression incubated with quizartinib was significantly enhanced by 100 ng/ml CXCL12; however, this protective effect of CXCL12 against quizartinib was barely detected in cells with high surface CXCR4 expression. Although silencing Runx1 downregulated CXCR4 expression, RUNX1 expression levels were significantly higher in CXCR4LOW FLT3/ITD+ Ba/F3 cells incubated with 100 ng/ml CXCL12 than in CXCR4HIGH cells, coincident with an increase in FLT3 phosphorylation. Silencing RUNX1 partially abrogated resistance to quizartinib in CXCR4LOW cells incubated with CXCL12, whereas ectopic RUNX1 significantly restored resistance in CXCR4HIGH cells. These results indicate that CXCR4 signaling of different magnitudes paradoxically regulates resistance to quizartinib in FLT3/ITD+ cells via RUNX1.

Retraction Note to: Inhibition of breast cancer metastasis with microRNA-302a by downregulation of CXCR4 expression

Retraction Note to: Inhibition of breast cancer metastasis with microRNA-302a by downregulation of CXCR4 expression

MiR-9a-5p alleviates ventilator-induced lung injury in rats by inhibiting the activation of the MAPK signaling pathway via CXCR4 expression downregulation

BackgroundGlobally, Mechanical ventilation is the most commonly used short-term life support technology. Ventilator-induced lung injury (VILI) is an inflammatory injury caused by mechanical ventilation. MicroRNAs (miRNAs) are considered as new gene regulators that play an important role in lung injury and inflammation. However, the role and mechanism of action of miR-9a-5p in VILI remain unclear. MethodsHerein, a rat model of VILI was established. To determine the expression levels of miR-9a-5p and CXCR4 mRNA, real-time quantitative polymerase chain reactions (qRT-PCR) were conducted. As well as western blot (WB) and immunofluorescence analyses, we determined the expression of CXCR4, SDF-1 and MAPK signaling pathway-related kinases. Hematoxylin and eosin (H&E) staining and the wet-dry ratio of the lung tissue were used to evaluate organ injury. An enzyme-linked immunosorbent assay (Elisa) and myeloperoxidase (MPO) activity measurements were performed to evaluate the inflammatory response. In addition, double luciferase reporter assays were used to verify the association between miR-9a-5p and CXCR4. ResultsThe expression of miR-9a-5p was low, whereas that of CXCR4 was high in the lung tissues of VILI rats. The overexpression of miR-9a-5p alleviated the degree of pathological injury in the lung tissues of rats with VILI, downregulating inflammatory cytokine expression and MPO activity. In the VILI rat model, miR-9a-5p targeted the negative regulation of CXCR4, and CXCR4 overexpression to reverse the lung-protective and anti-inflammatory effects of miR-9a-5p overexpression in VILI rats. miR-9a-5p also inhibited the phosphorylation of extracellular signal receptor-activated kinase (ERK), a protein related to the MAPK signaling pathway, by downregulating CXCR4 expression. ConclusionmiR-9a-5p can hinder the activation of the MAPK/ERK signaling pathway and reduce inflammatory reactions and lung injury in VILI rats through the targeted regulation of CXCR4 expression. Therefore, miR-9a-5p could serve as an intervention target to supply a new strategy for the care of VILI.

ATP Inhibits Breast Cancer Migration and Bone Metastasis through Down-Regulation of CXCR4 and Purinergic Receptor P2Y11.

Simple SummaryThe skeleton is the most frequent metastatic site for advanced breast cancer, and complications resulting from breast cancer metastasis are a leading cause of death in patients. Therefore, the discovery of new targets for the treatment of breast cancer bone metastasis is of great significance. ATP released by bone osteocytes is shown to activate purinergic signaling and inhibit the metastasis of breast cancer cells in the bone. The aim of our study was to unveil the underlying molecular mechanism of ATP and purinergic signaling in inhibiting the bone metastasis of breast cancer cells. We demonstrated that CXCR4 and P2Y11 are key factors in regulating this process, and understanding of this important mechanism will aid in identifying new targets and developing first-in-class therapeutics.ATP released by bone osteocytes is shown to activate purinergic signaling and inhibit the metastasis of breast cancer cells into the bone. However, the underlying molecular mechanism is not well understood. Here, we demonstrate the important roles of the CXCR4 and P2Y11 purinergic receptors in mediating the inhibitory effect of ATP on breast cancer cell migration and bone metastasis. Wound-healing and transwell migration assays showed that non-hydrolysable ATP analogue, ATPγS, inhibited migration of bone-tropic human breast cancer cells in a dose-dependent manner. BzATP, an agonist for P2X7 and an inducer for P2Y11 internalization, had a similar dose-dependent inhibition on cell migration. Both ATPγS and BzATP suppressed the expression of CXCR4, a chemokine receptor known to promote breast cancer bone metastasis, and knocking down CXCR4 expression by siRNA attenuated the inhibitory effect of ATPγS on cancer cell migration. While a P2X7 antagonist A804598 had no effect on the impact of ATPγS on cell migration, antagonizing P2Y11 by NF157 ablated the effect of ATPγS. Moreover, the reduction in P2Y11 expression by siRNA decreased cancer cell migration and abolished the impact of ATPγS on cell migration and CXCR4 expression. Similar to the effect of ATPγS on cell migration, antagonizing P2Y11 inhibited bone-tropic breast cancer cell migration in a dose-dependent manner. An in vivo study using an intratibial bone metastatic model showed that ATPγS inhibited breast cancer growth in the bone. Taken together, these results suggest that ATP inhibits bone-tropic breast cancer cells by down-regulating the P2Y11 purinergic receptor and the down-regulation of CXCR4 expression.

Antifade Carbon Dots on a Plasmonic Substrate for Enhanced Protein Detection in Immunotherapy.

Fluorescence microscopic analysis of checkpoint protein expression is capable of predicting clinical outcomes for checkpoint blockade immunotherapy. However, accurate detection of their expression levels is hindered by fluorophore photobleaching and cell autofluorescence. We now develop a sensitive and robust fluorescence microscopy method that uses antifade graphite-structured carbon dots (GCDs) on a plasmonic Ag substrate (named ACPAS) for the accurate detection of checkpoint proteins in immunotherapy. In ACPAS, a Ag substrate is used to enhance the fluorescence of GCDs while a continuous illumination is implemented to quench cell autofluorescence, thus enabling a dramatic improvement in the signal-to-background ratio by up to 33-fold. We use ACPAS to monitor programmed death ligand-1 (PD-L1) expression levels on various tumor cells and finely differentiate their microscopic changes in combination with chemokine receptor CXCR4-targeted treatments. ACPAS analysis reveals for the first time that CXCR4 agonist (SDF-1α) and antagonist (AMD3100) can potentiate PD-L1 expression by down-regulating CXCR4 expression on tumor cells, which provides valuable information on the development of anti-PD-L1 and anti-CXCR4 combination therapy. We envision that ACPAS will become a broadly useful tool for protein expression studies in biomedicine and life sciences.

Saikosaponin A Inhibits Triple-Negative Breast Cancer Growth and Metastasis Through Downregulation of CXCR4.

Purpose: Due to a lack of recognized molecular targets for therapy, patients with triple-negative breast cancer (TNBC), unlike other subtypes of breast cancers, generally have not benefited from the advances made with targeted agents. The CXCR4/SDF-1 axis is involved in tumor growth and metastasis of TNBC. Therefore, down-regulation of the expression of CXCR4 in cancer cells is a potential therapeutic strategy for inhibiting primary tumor growth and metastasis of TNBC. In order to identify bioactive compounds that inhibit the expression of CXCR4 in traditional Chinese medicines, we investigated the capacity of saikosaponin A (SSA), one of the active ingredients isolated from Radix bupleuri, to affect CXCR4 expression and function in TNBC cells.Methods: Analyses of cell growth, migration, invasion, and protein expression were performed. Knockdowns by small interfering RNA (siRNA) and non-invasive bioluminescence were also used.Results: SSA reduced proliferation and colony formation of SUM149 and MDA-MB-231 cells. SSA inhibited migration and invasion of TNBC cells. For mice, SSA inhibited primary tumor growth and reduced lung metastasis of highly metastatic, triple-negative 4T1-luc cells. SSA inhibited CXCR4 expression but did not regulate CXCR7 expression in vitro and in vivo. The inhibitory effects on the migration and invasion of TNBC cells were reversed by down-regulation of CXCR4 expression. In addition, SSA inactivated the Akt/mTOR signaling pathway and inhibited MMP-9 and MMP-2 expression.Conclusions: The results show that SSA exerts an anti-TNBC effect through the inhibition of CXCR4 expression and thus has the potential to be a candidate therapeutic agent for TNBC patients.

Circulating myocardial microRNAs from infarcted hearts are carried in exosomes and mobilise bone marrow progenitor cells

Myocardial microRNAs (myo-miRs) are released into the circulation after acute myocardial infarction (AMI). How they impact remote organs is however largely unknown. Here we show that circulating myo-miRs are carried in exosomes and mediate functional crosstalk between the ischemic heart and the bone marrow (BM). In mice, we find that AMI is accompanied by an increase in circulating levels of myo-miRs, with miR-1, 208, and 499 predominantly in circulating exosomes and miR-133 in the non-exosomal component. Myo-miRs are imported selectively to peripheral organs and preferentially to the BM. Exosomes mediate the transfer of myo-miRs to BM mononuclear cells (MNCs), where myo-miRs downregulate CXCR4 expression. Injection of exosomes isolated from AMI mice into wild-type mice downregulates CXCR4 expression in BM-MNCs and increases the number of circulating progenitor cells. Thus, we propose that myo-miRs carried in circulating exosomes allow a systemic response to cardiac injury that may be leveraged for cardiac repair.

Tetramethylpyrazine (TMP) ameliorates corneal neovascularization via regulating cell infiltration into cornea after alkali burn

In the present study, we investigated the underlying mechanism of tetramethylpyrazine (TMP)-medicated inhibition of corneal neovascularization (CNV). Our data showed that TMP could effectively downregulate the expression levels of CXCR4 mRNA and protein, as well as inhibit HUVECs, endothelial cells, tubule formation in vitro. In vivo, alkali burn (1 M NaOH) could remarkably upregulate CXCR4 expression and increase the migration of TNF-α-positive cells to corneal stroma. TMP drops could significantly downregulate CXCR4 expression in cornea, compared to the control. However, there was no difference in the downregulation of CXCR4 between TMP and FK506, an immunosuppressive drug. Moreover, the immunofluorescent staining of CD45 showed TMP and FK506 could significantly restrain the bone marrow (BM)-derived infiltration while the F4/80 staining reflects the suppression of macrophage aggregation. Meanwhile TMP could regulate the Interleukin 10 (IL-10) and FK506 could restrain the Interleukin 2 (IL-2). Furthermore, TMP and FK506 significantly ameliorate corneal opacity and neovascularization. Clinical assessment detected an obvious improvement in TMP and FK506 treatment groups, compared to controls in vivo. Thus, TMP had similar effects in inhibition of immune response and CNV by suppressing BM-infiltrating cells into cornea as FK506. TMP could be a potential agent in eye-drop therapy for cornea damaged by Alkali Burn.

High CD123 levels enhance proliferation in response to IL-3, but reduce chemotaxis by downregulating CXCR4 expression.

High expression of the α chain of the interleukin-3 receptor (IL-3Rα; CD123) is a hallmark of acute myeloid leukemia (AML) leukemic stem cells (LSCs). Elevated CD123 expression is part of the diagnostic immunophenotyping of myeloid leukemia, and higher expression is associated with poor prognosis. However, the biological basis of the poorer prognosis is unclear, and may include heightened IL-3 signaling and non-cell autonomous interactions with the bone marrow (BM) microenvironment. We used TF-1 cells expressing different levels of CD123 and found elevated CD123 levels amplified the proliferative response to exogenous IL-3 and maintained viability in reducing IL-3 concentrations. This was associated with stronger activation of STAT5, Akt, and extracellular signal-regulated kinase 1/2 in vitro. Surprisingly, in vivo e14.5 fetal liver cells transduced with retroviral constructs to express high CD123 failed to engraft in syngeneic recipients. In exploring the underlying mechanism for this, we found that CXCR4, a key molecule involved in LSC/BM interactions, was specifically downregulated in CD123 overexpressing cells in a manner dependent on IL-3 signaling. CXCR4 downregulation was sufficient to alter the chemotactic response of hematopoietic cells to stromal derived factor-1 (SDF-1). Thus, we propose that the overexpression of CD123 in AML LSC dictates their location by altering CXCR4/SDF-1 interaction in the BM, raising the possibility that this mechanism underpins the egress of BM AML LSC and more mature cells into the circulation.

COUP-TFI mitotically regulates production and migration of dentate granule cells and modulates hippocampal Cxcr4 expression.

Development of the dentate gyrus (DG), the primary gateway for hippocampal inputs, spans embryonic and postnatal stages, and involves complex morphogenetic events. We have previously identified the nuclear receptor COUP-TFI as a novel transcriptional regulator in the postnatal organization and function of the hippocampus. Here, we dissect its role in DG morphogenesis by inactivating it in either granule cell progenitors or granule neurons. Loss of COUP-TFI function in progenitors leads to decreased granule cell proliferative activity, precocious differentiation and increased apoptosis, resulting in a severe DG growth defect in adult mice. COUP-TFI-deficient cells express high levels of the chemokine receptor Cxcr4 and migrate abnormally, forming heterotopic clusters of differentiated granule cells along their paths. Conversely, high COUP-TFI expression levels downregulate Cxcr4 expression, whereas increased Cxcr4 expression in wild-type hippocampal cells affects cell migration. Finally, loss of COUP-TFI in postmitotic cells leads to only minor and transient abnormalities, and to normal Cxcr4 expression. Together, our results indicate that COUP-TFI is required predominantly in DG progenitors for modulating expression of the Cxcr4 receptor during granule cell neurogenesis and migration.

Neuropilin-1 Is an Important Niche Component and Exerts Context-Dependent Effects on Hematopoietic Stem Cells.

Marrow adipocytes pose a significant problem in post-transplant regeneration of hematopoiesis owing to their negative effects on regeneration of hematopoiesis. However, the precise mechanism operative in this negative regulation is not clear. In this study, we show that marrow adipocytes express neuropilin-1 (NRP1) as a function of differentiation and inhibit regeneration of hematopoiesis by three principal mechanisms: one, by inducing apoptosis in hematopoietic stem/progenitor cells (HSPCs) through the death receptor-mediated pathway; two, by downregulating CXCR4 expression on the HSPCs through ligand-mediated internalization; and three, by secreting copious amounts of transforming growth factor β1 (TGFβ1), a known inhibitor of hematopoiesis. Silencing of NRP1 in these adipocytes rescued the apoptosis of cocultured HSPCs and boosted the CXCR4 surface expression on them, showing an active role of NRP1 in these processes. However, such silencing had no effect on TGFβ1 secretion and consequent inhibition of hematopoiesis by them, showing that secretion of TGFβ1 by adipocytes is independent of NRP1 expression by them. Surprisingly, mesenchymal stromal cells modified with NRP1 supported expansion of HSPCs having enhanced functionality, suggesting that NRP1 exerts a context-dependent effect on hematopoiesis. Our data demonstrate that NRP1 is an important niche component and exerts context-dependent effects on HSPCs. Based on these data, we speculate that antibody- or peptide-mediated blocking of NRP1-HSC interactions coupled with a pharmacological inhibition of TGFβ1 signaling may help in combating the negative regulation of post-transplant regeneration of hematopoiesis in a more effective manner.

Contribution and underlying mechanisms of CXCR4 overexpression in patients with systemic lupus erythematosus.

Aberrant expression of CXCR4 has been indicated to play a role in the pathogenesis of systemic lupus erythematosus (SLE), but the mechanism of CXCR4 dysregulation in SLE is unclear. This study is aimed to explore the clinical significance and possible mechanisms of abnormal CXCR4 expression on B cells from patients with untreated SLE. Expression of CXCR4 on peripheral B cells was determined by flow cytometry and western blotting. Freshly isolated B cells were cultured with exogenous interleukin 21(IL-21) in the presence or absence of CD40 ligand (CD40L) plus anti-IgM antibody (aIgM), and changes in CXCR4 expression were detected. Involvement of phosphatidylinositol 3 kinase (PI3K)/Akt and Janus kinase/Signal transducer and activator of transcription (JAK/STAT) signaling pathways was assessed by adding blocking agents Ly294002 and AG490. Since CD63 is reported to mediate endosomal recruitment of CXCR4 and BCL6 is capable of silencing CD63 gene transcription, we also measured BCL6 and CD63 gene transcription with real-time PCR. It was shown that CXCR4 expression on B cells was significantly upregulated in SLE patients, especially in those with lupus nephritis, and was positively correlated with SLE Disease Activity Index scores and negatively with the serum complement 3 levels (P<0.05). Downregulation of CXCR4 by IL-21 was intact. In contrast, a similar effect of aIgM plus CD40L in downregulating CXCR4 expression was defective in SLE patients but was restored by co-stimulation with IL-21 in vitro. Both Ly294002 and AG490 promoted downregulation of surface CXCR4 expression on B cells from SLE patients (P=0.078 and P=0.064). Furthermore, B cells from SLE patients exhibited diminished CD63 mRNA and enhanced BCL6 mRNA expression (both P<0.05). To sum up, CXCR4 was overexpressed on SLE B cells, positively correlating with disease activity and kidney involvement. Overactivation of the PI3K/Akt and JAK/STAT pathways as well as defective CD63 synthesis may contribute to CXCR4 dysregulation in SLE.

Abstract 4123: Thymoquinone inhibits bone metastasis in a breast cancer mouse model by modulating CXCR4/CXCL12 signaling axis

Abstract Several lines of evidence(s) indicate that CXCR4 overexpression has been correlated with distant site metastasis and poor overall survival rate in patient with breast cancer. The tumor metastasis promoting molecule CXCR4 is considered as a potential therapeutic target for inhibiting breast cancer metastasis. Thus, novel agents that can down-regulate CXCR4 expression have potential against breast cancer metastasis. In the present report we investigated the effect of thymoquinone (TQ), derived from the seeds of medicinal plant Nigella sativa, on the expression and regulation of CXCR4 in breast cancer cells. In addition, we evaluated the effect of TQ in a metastasis mouse model established by intracardiac injection of luciferase tagged MDA-MB-231 breast cancer cells that metastasize to the bones. We observed that TQ could inhibit the expression of CXCR4 in MCF-7 and MDA-MB-231 cells in a dose and time dependent manner. TQ (2 mg/kg or 4 mg/kg) treatment for four weeks significantly inhibited tumor growth and significantly reduced metastasis to multiple vital organs, including lungs, brain and bone. Immuno-histochemical analysis of the lung and brain tissue showed significant reduction in CXCR4, Ki67, MMP9, VEGFR2 and COX2 expression compared to control tissues. TQ treatment also reduced the overall bone tumor burden. Overall, our results show that TQ exerts its antitumor and anti-metastatic effects by downregulation of CXCR4 expression both in vitro and in vivo thus may have possible potential for the treatment of breast cancer. Acknowledgement: This work was supported by NUHS Basic Seed Research grant to Prof. Benny Tan. Citation Format: Muthu K. Shanmugam, Annie Hsu, Kam Man Hui, Benny K.H. Tan, Gautam Sethi. Thymoquinone inhibits bone metastasis in a breast cancer mouse model by modulating CXCR4/CXCL12 signaling axis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4123.

Abstract A19: Epigenetic plasticity of the CXCR4 locus contributes to cell state transitions in Ewing sarcoma

Abstract Purpose of the Study: Ewing sarcoma (ES) is the second most common bone cancer in children. Despite improved survival for patients with localized disease, the outcome for patients with metastases remains dismal. In order to improve outcomes for ES patients, there is an imminent need to further understand the molecular and cellular biology driving ES metastasis. CXCR4 is the most commonly expressed chemokine receptor in human cancer and high expression contributes to metastasis. We have recently demonstrated that CXCR4 is dynamic and that its expression is induced in response to microenvironmental cues leading to transition of cells from CXCR4- to CXCR4+ states. In the current study we investigated the potential contribution of epigenetic regulation to the CXCR4 cell-state transitions in response to changes in the microenvironment. Experimental Procedures: Immunohistochemistry of 93 diagnostic tumor sections was performed and frequency of CXCR4+ cells assessed. Chromatin Immunoprecipitation (ChIP) PCR was used to analyze histone modifications at the CXCR4 locus in ES cell lines. mRNA levels were quantified using qRT-PCR. Chemotactic migration and invasion to CXCL12 was evaluated using the x-CELLigence system in the presence and absence of the bromodomain inhibitor JQ1. Data Summary: CXCR4+ tumor cells were identified in 31 cases. Frequency ranged from &amp;lt;5% to 100%. CXCR4+ cells were more often detected in patients whose tumor had metastasized (p=0.008). ChIP of ES cells in ambient culture showed enrichment of both the activating, H3K4me3, and repressive, H3K27me3m marks at the CXCR4 promoter. ChIP-re-ChIP of the same chromatin confirmed that the promoter exists in a bivalent state in these conditions. Levels of CXCR4 directly correlated with H3K4me3 and inversely correlated with H3K27me3 marks suggesting that transcription of CXCR4 is, at least in part, regulated by the competing actions of these polycomb- and trithorax-dependent histone modifications. This was confirmed in studies of FACS-sorted CXCR4+ and CXCR4- cells. Specifically, we detected preferential enrichment of H3K4me3 in CXCR4+ and of H3K27me3 in CXCR4- cells. Significantly, exposure of ES cells to serum-deprivation or hypoxia, resulted in loss of bivalency in both bulk populations as well as FACS-sorted CXCR4- cells, as evidenced by loss of H3K27me3 with retention of H3K4me3 modifications. Loss of bivalency was accompanied by upregulation of CXCR4. Finally, we noted that an active enhancer exists upstream of the CXCR4 locus in HeLa cells, which express constitutively high levels of CXCR4. Therefore, we hypothesized that this enhancer may also contribute to CXCR4 regulation in ES. In support of this, JQ1 treatment of ES cells resulted in marked down-regulation of CXCR4 expression in ambient conditions. In addition, JQ1 treatment blocked up-regulation of CXCR4 in response to both serum-deprivation and hypoxia. Interestingly, enrichment of activating marks increased at the enhancer under conditions of stress. In addition, chemotactic migration of ES cells to CXCL12 was impeded by JQ1. Conclusions: In summary, we have demonstrated that stress-dependent induction of CXCR4 in ES is, in part, epigenetically regulated. In particular, the CXCR4 promoter in ES cells resides in a bivalent state, which permits rapid and reversible transitions between CXCR4- and CXCR4+ cell states. Further, enhancer-dependent regulation of CXCR4 transcription opens the door to the potential of bromodomain inhibitors, such as JQ1, as novel therapeutics that could be used to antagonize metastasis-promoting cell state transitions. Citation Format: Melanie A. Krook, Raj Patel, David Lucas, Rashmi Chugh, Elizabeth R. Lawlor. Epigenetic plasticity of the CXCR4 locus contributes to cell state transitions in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A19.

Tetramethylpyrazine (TMP), an Active Ingredient of Chinese Herb Medicine Chuanxiong, Attenuates the Degeneration of Trabecular Meshwork through SDF-1/CXCR4 Axis.

BackgroundA traditional Chinese medicine, Tetramethylpyrazine (TMP), has been prescribed as a complementary treatment for glaucoma to improve patient prognosis. However, the pharmacological mechanism of action of TMP is poorly understood. In previous studies, we demonstrated that TMP exerts potent inhibitory effects on neovascularization, suppresses the tumorigenic behavior of glioma cells, and protects neural cells by regulating CXCR4 expression. Here, we further investigated whether the SDF-1/CXCR4 pathway is also involved in the TMP-mediated activity in trabecular meshwork cells.Methodology/Principal FindingsCXCR4 expression was examined by quantitative real-time PCR in trabecular and iris specimens from 54 primary open-angle glaucoma (POAG) patients who required surgery and 19 non-glaucomatous donors. Our data revealed markedly elevated CXCR4 expression in the trabecular meshwork of POAG patients compared with that of controls. Consistently, CXCR4 expression was much higher in glaucomatous trabecular meshwork cells than in normal trabecular meshwork cells. Using RT-PCR and western blot assays, we determined that glaucoma-related cytokines and dexamethasone (DEX) also significantly up-regulated CXCR4 expression in primary human trabecular meshwork (PHTM) cells. Moreover, the TGF-β1-mediated induction of CXCR4 expression in PHTM cells was markedly down-regulated by TMP compared with control treatment (PBS) and the CXCR4 antagonist AMD3100. In addition, TMP could counteract the TGF-β1-induced effects on stress fiber accumulation and expansion of PHTM cells. TMP markedly suppressed the migration of PHTM cells stimulated by TGF-β1 in transwell and scratch wound assays. TMP also suppressed the extracellular matrix (ECM) accumulation induced by TGF-β2.ConclusionsOur findings demonstrate that CXCR4 might be involved in the pathogenetic changes in the trabecular meshwork of patients with POAG. Additionally, TMP might exert its beneficial effects in POAG patients by down-regulating CXCR4 expression.

Abstract 1906: CXCR4 is a novel target of cancer chemopreventative isothiocyanates in prostate cancer cells

Abstract Isothiocyanates (ITCs) derived from cruciferous vegetables, including phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit in vivo activity against prostate cancer in xenograft and transgenic mouse models, and thus are appealing for chemoprevention of this disease. Watercress constituent PEITC and SFN-rich broccoli sprout extract are under clinical investigation but the molecular mechanisms underlying their cancer chemopreventive effects are not fully understood. The present study demonstrates that chemokine receptor CXCR4 is a novel target of ITCs in prostate cancer cells. Exposure of prostate cancer cells (LNCaP, 22Rv1, C4-2, and PC-3) to pharmacologically applicable concentrations of PEITC, benzyl isothiocyanate (BITC), and SFN (2.5 and 5 μmol/L) resulted in downregulation of CXCR4 expression. None of the isothiocyanates affected secretion of CXCR4 ligand (stromal-derived factor-1). In vivo inhibition of PC-3 xenograft growth upon PEITC treatment was associated with a significant decrease in CXCR4 protein level. A similar trend was discernible in the tumors from SFN-treated TRAMP mice compared with those of control mice, but the difference was not significant. Stable overexpression of CXCR4 in PC-3 cells conferred significant protection against wound healing, cell migration, and cell viability inhibition by ITCs. Inhibition of cell migration resulting from PEITC and BITC exposure was significantly augmented by RNA interference of CXCR4. This study demonstrates, for the first time, that cancer chemopreventive ITCs suppress CXCR4 expression in prostate cancer cells in vitro as well as in vivo. These results suggest that CXCR4 downregulation may be an important pharmacodynamic biomarker of cancer chemopreventative ITCs in prostate adenocarcinoma. Citation Format: Ali I. Amjad, Kozue Sakao, Avani R. Vyas, Sreenivasa R. Chinni, Rahul Parikh, Shivendra V. Singh. CXCR4 is a novel target of cancer chemopreventative isothiocyanates in prostate cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1906. doi:10.1158/1538-7445.AM2015-1906

Abstract 498: A bivalent promoter regulates stress dependent induction of CXCR4 in Ewing sarcoma

Abstract CXCR4 is the most commonly expressed chemokine receptor in human cancer and high expression contributes to metastasis. We recently showed that CXCR4 expression is dynamic in Ewing sarcoma (ES) and that its expression is induced by cell stress. In the current study we investigated the potential contribution of epigenetic regulation to this stress response. Chromatin Immunoprecipitation (ChIP) was used to analyze histone modifications at the CXCR4 locus. CXCR4 mRNA levels were quantified using qRT-PCR. Chemotactic migration to CXCL12 was evaluated using transwell assays in the presence and absence of the bromodomain inhibitor JQ1. ChIP showed enrichment of both H3K4me3 and H3K27me3 marks at the CXCR4 promoter in unstressed ES cells. ChIP-re-ChIP of the same chromatin confirmed that the promoter exists in a bivalent state in ambient conditions. Levels of CXCR4 directly correlated with H3K4me3 and inversely correlated with H3K27me3 marks suggesting that transcription of CXCR4 is, at least in part, regulated by the competing actions of these polycomb- and trithorax-dependent histone modifications. This was confirmed using studies of CXCR4+ and CXCR4- cells that had been isolated by FACS prior to ChIP. In these sorted populations we detected preferential enrichment of H3K4me3 in CXCR4+ and H3K27me3 in CXCR4- cells. Significantly, exposure of ES cells to stress, either serum-deprivation or hypoxia, resulted in loss of bivalency as demonstrated by diminished enrichment of H3K27me3 in the continued presence of high-levels of H3K4me3. Loss of bivalency was accompanied by upregulation of CXCR4 thereby confirming the key contribution of the promoter's chromatin state to transcriptional regulation. Finally, we noted that an active enhancer exists upstream of the CXCR4 locus in HeLa cells, which express constitutively high levels of CXCR4. Therefore, we hypothesized that this enhancer may also contribute to CXCR4 regulation in ES. In support of this, JQ1 treatment of ES cells resulted in marked down-regulation of CXCR4 expression in ambient conditions. In addition, JQ1 treated cells failed to up-regulate CXCR4 in response to both serum-deprivation and hypoxia. Significantly, chemotactic migration of ES cells to CXCL12 was impeded by JQ1. The effects of JQ1 on basal and stress-dependent induction of CXCR4 were reproduced in both HeLa and neuroblastoma cells indicating that epigenetic regulation of CXCR4 extends to other cancer types. In summary, we have demonstrated that stress-dependent induction of CXCR4 in ES is, in part, epigenetically regulated. In particular, the CXCR4 promoter in ES cells resides in a bivalent state which permits rapid and reversible transitions between CXCR4- and CXCR4+ states. Further, enhancer-dependent regulation of CXCR4 transcription opens the door to the potential of bromodomain inhibitors as novel therapeutics that could be used to antagonize metastasis-promoting cell state transitions. Citation Format: Melanie A. Krook, Elizabeth R. Lawlor. A bivalent promoter regulates stress dependent induction of CXCR4 in Ewing sarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 498. doi:10.1158/1538-7445.AM2015-498

Abstract 436: SDF1α / CXCR4 axis might be associated with growth-interaction between cancer-associated fibroblasts and gastric cancer cells in hypoxic tumor microenvironment

Abstract Background: Cancer-associated fibroblasts (CAF) have been suggested to be associated with the progression of gastric cancer cells. Hypoxic region was frequently found in cancer microenvironment of gastric cancer. The aim of our study was to clarify the interaction between CAF and gastric cancer cells under hypoxia. We found that hypoxia up-regulated the growth and interaction between the CAF and gastric cancer cells through SDF1α/CXCR4 dependent mechanism. Materials and Methods: Six gastric cancer cell lines and three fibroblasts cell lines were used. The expression level of CXCR4 under hypoxia was examined by RT-PCR, Western blot. SDF1 production from CAF under hypoxia wad examined by ELISA. The proliferation of diffuse type gastric cancer cells that were co-culture with CAF was examined under hypoxia, in comparison with that under normoxia. Proliferation activity of diffuse type gasitrc cancer cells was examined in the presence of CAF, CXCR4 inhibitor, FGFR2 inhibitor, HIF1α siRNA or CXCR4 siRNA under hypoxia, in comparison with that under normoxia. Results: The proliferation of diffuse type gastric cancer cells was significantly increased in the presence of CAF. The proliferation - stimulating effects by CAF were evident in hypoxia, in compared with normoxia. CXCR4 expression of diffuse type gastric cancer cells was significantly increased under hypoxia, but decreased FGFR2 expression under hypoxia. HIF1α siRNA significantly down-regulated CXCR4 expression level in hypoxia. SDF1 from CAF was increased under hypoxia, and was also decreased by HIF1α knockdown. FGFR2 inhibitor significantly decreased he proliferation-stimulating activity of CAF under normoxia, but not under hypoxia. Although, CXCR4 inhibitor significantly decreased the proliferation-stimulating activity of CAF under hypoxia, but not under normoxia. These findings suggested that the key signaling associated with the growth-interaction between CAF and gastric cancer cells in hypoxic condition might be different from those in normoxic condition. Conclusion: Hypoxic microenvironment switch the driver signaling from FGF7/FGFR2 to SDF-1/CXCR4 in diffuse type of gastric cancer. CXCR4/SDF1 axis might play an important role for the proliferation of gastric cancer in hypoxic tumor microenvironment. Citation Format: Haruhito Kinoshita, Masakazu Yashiro, Hiroaki Kasashima, Go Masuda, Tamami Morisaki, Tatsunari Fukuoka, Katsunobu Sakurai, Takahiro Toyokawa, Kenjiro Kimura, Naoshi Kubo, Hiroaki Tanaka, Kazuya Muguruma, Masaichi Ohira, Kosei Hirakawa. SDF1α / CXCR4 axis might be associated with growth-interaction between cancer-associated fibroblasts and gastric cancer cells in hypoxic tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 436. doi:10.1158/1538-7445.AM2015-436

Numb inhibits proliferation and migration of human bladder cancer cell line BIU-87 by negative modulating CXCR4

Objective To study the effect of Numb gene on CXCR4 expression and proliferation and migration of bladder cancer. Methods Human bladder cancer cells were divided into 3 groups: experimental group (transfected with Numb-ORF plasmid), negative-control group (transfected with blank vector), and blank-control group (no DNA transfected in). The expressions of Numb and CXCR4 were detected by real-time PCR and Western blotting respectively. Cell proliferation and migration were measured by MTS and Transwell assay respectively. Results Numb expressions in experimental group, negative-control group and blank-control group were 31.044±3.350, 4.401±0.567 and 4.287±0.341 respectively, with a statistical significance (F=183.418, P=0.000). CXCR4 levels in experimental group, negative-control group and blank-control group were 0.344±0.167, 0.996±0.148 and 1.010±0.106 respectively, with a statistical significance (F=21.355, P=0.002). In MTS assay, the absorb values in experimental group, negative-control group and blank-control group were 0.615±0.057, 0.987±0.063 and 0.957±0.066, with a statistical difference (F=33.210, P=0.001). In Transwell assay, the numbers of migratory cells in experimental group, negative-control group and blank-control group were 164.667±19.858, 670.133±38.760 and 667.533±27.610, with a statistical significance (F=286.788, P=0.000). Conclusion Overexpression of Numb can suppress the ability of proliferation and migration of the BIU-87 cells through down-regulation of CXCR4 expression in human bladder cancer. Key words: Urinary bladder neoplasms; Receptors, CXCR4; Cell proliferation; Cell migration assays

CXCR4 is a novel target of cancer chemopreventative isothiocyanates in prostate cancer cells.

Isothiocyanates (ITCs) derived from cruciferous vegetables, including phenethyl isothiocyanate (PEITC) and sulforaphane (SFN), exhibit in vivo activity against prostate cancer in a xenograft and transgenic mouse model, and thus are appealing for chemoprevention of this disease. Watercress constituent PEITC and SFN-rich broccoli sprout extract are under clinical investigations but the molecular mechanisms underlying their cancer chemopreventive effects are not fully understood. The present study demonstrates that chemokine receptor CXCR4 is a novel target of ITCs in prostate cancer cells. Exposure of prostate cancer cells (LNCaP, 22Rv1, C4-2, and PC-3) to pharmacologically applicable concentrations of PEITC, benzyl isothiocyanate (BITC), and SFN (2.5 and 5 μmol/L) resulted in downregulation of CXCR4 expression. None of the ITCs affected secretion of CXCR4 ligand (stromal-derived factor-1). In vivo inhibition of PC-3 xenograft growth upon PEITC treatment was associated with a significant decrease in CXCR4 protein level. A similar trend was discernible in the tumors from SFN-treated TRAMP mice compared with those of control mice, but the difference was not significant. Stable overexpression of CXCR4 in PC-3 cells conferred significant protection against wound healing, cell migration, and cell viability inhibition by ITCs. Inhibition of cell migration resulting from PEITC and BITC exposure was significantly augmented by RNAi of CXCR4. This study demonstrates, for the first time, that cancer chemopreventive ITCs suppress CXCR4 expression in prostate cancer cells in vitro as well as in vivo. These results suggest that CXCR4 downregulation may be an important pharmacodynamic biomarker of cancer chemopreventative ITCs in prostate adenocarcinoma.