CXCR4 Promoter Research Articles

Macrophage Migration Inhibitory Factor (MIF) Upregulates CXCR7 and Contributes to Chemotherapy Resistance in Colorectal Cancer.

Colorectal cancer is one of the most common malignant tumors worldwide, with high incidence and mortality rates making it a focus of research. Chemotherapy is a primary treatment modality for colon cancer, but chemotherapy resistance severely impacts treatment efficacy. MIF has been found to promote tumor progression and resistance in various cancers. This study aims to investigate the role of MIF in chemotherapy resistance in colon cancer and its potential mechanisms, particularly through the upregulation of CXCR7 expression, affecting the metabolism and drug sensitivity of colon cancer cells. The expression levels of MIF in colon cancer tissues and its association with patient prognosis were evaluated by analyzing TCGA and HPA data. Subsequently, the expression levels of MIF in colon cancer cell lines and resistant cell lines were detected by qRT-PCR and immunohistochemistry, and the effect of MIF on oxaliplatin sensitivity was assessed. The impact of MIF on the metabolic activity of colon cancer cells was measured using a cellular energy metabolism analyzer. Further experiments explored the mechanism by which MIF affects the metabolic activity of colon cancer cells through the upregulation of CXCR7 expression, and the role of CTCF in regulating CXCR7 transcription was validated by silencing CTCF. Finally, the effect of MIF on drug sensitivity of colon cancer cells was verified in a mouse xenograft tumor model. In this study, we found that the expression of MIF in colon cancer tissues was significantly higher than in normal tissues, and high MIF expression was associated with poor prognosis in patients. The expression levels of MIF in resistant colon cancer cell lines were significantly higher than in parental cell lines, and MIF overexpression significantly increased the resistance of colon cancer cells to oxaliplatin. Conversely, silencing MIF significantly reduced the IC50 value of resistant cells and increased apoptosis. MIF overexpression significantly increased the ECAR and OCR levels of colon cancer cells, while MIF knockdown significantly reduced these metabolic indicators. Further studies indicated that MIF affects the metabolic activity of colon cancer cells by upregulating CXCR7 expression. CTCF binding peaks at the CXCR7 promoter region and luciferase activity assays indicated that CTCF regulates CXCR7 transcription, and silencing CTCF significantly enhanced the sensitivity of colon cancer cells to oxaliplatin. In vivo experiments in mice showed that MIF silencing combined with oxaliplatin treatment significantly inhibited tumor growth and increased the necrotic area of tumor tissues. In conclusion, this study reveals the crucial role of MIF in chemotherapy resistance in colon cancer through the upregulation of CXCR7 expression, with CTCF playing an important regulatory role in this process. Our findings provide new theoretical insights and potential therapeutic targets for overcoming chemotherapy resistance in colon cancer. Future research should further explore the roles of MIF and CXCR7 in other types of cancers and the potential of MIF and CXCR7 as therapeutic targets.

Abstract 7655: The CXCR4/CXCR7/CXCL12 axis and PBRM1 are involved in everolimus resistance in human renal cancer cell lines

Abstract Purpose: Renal cell carcinoma (RCC) is one of the most common malignancies in the genitourinary system. Combinations of Immunocheckpoints inhibitors plus TKIs represents the actual standard of therapy for metastatic RCC patients, while mTOR inhibitors play a role in the second-line treatment of mRCC in combination with lenvatinib. Thus characterizing resistance mechanism to mTOR inhibitors is unmet need. Experimental Design: A498 RAD-resistant renal cell carcinoma (RCC) cells were generated through increasing doses of RAD001 (RAD1-5-10 μM). Gene expression was assessed by qRT-PCR. pERK and transwell migration were evaluated in A498, RAD10 and RAD10shP. PBRM1sh was obtained in RAD10 with lentiviral particles while CXCR4 transfection in RAD10 was realized using pCMV6-AC-h-CXCR4-GFP. ChIP assay was assessed using Chromata ChIP kit. Results: Gene expression analysis revealed reduction in CXCR4/CXCR7 expression in A498 RAD 1-5-10-resistant cells (RAD1-5-10) (CXCR4:32.8, 37, 40.7-fold and CXCR7:2.3, 2.3, 2-fold, respectively), while PBRM1 and CXCL12 were overexpressed as compared to A498 (PBRM1: 2.2, 2.34 and 3.32-fold, and CXCL12: 7.2-, 5.6-, and 10-fold, respectively). The axis CXCL12-CXCR4 was functionally inhibited in RAD10 as demonstrated by inhibition in CXCL12- pERK and migration. YY1, already described as CXCR4 and CXCR7 negative regulator, was upregulated in RAD10 cells. ChIP confirmed higher binding of YY1 on CXCR4 and CXCR7 promoters in RAD10 as compared to A498. To address CXCR4 role, human CXCR4 was transfected in RAD10 cells (RAD10 CXCR4+) and RAD001 cytotoxicity was evaluated; interestingly, RAD10 CXCR4+ entirely restored RAD001 sensitivity in RAD10 cells. PBRM1 silencing in RAD10 (RAD10shP) downregulated mTOR and 4EBP1 (1.54 and 3-fold, compared to RAD10). As described, CXCR4 decreased in RAD10 and PBRM1 silencing did not further affect it, while CXCR7 increased (20-fold) in RAD10shP, indicating that PBRM1 regulates CXCR7 expression rather than CXCR4 in RAD001 resistant cells. RAD10shP migrated more towards CXCL11 than CXCL12 according to the overexpression of CXCR7 in RAD10shP cells. YY1 expression in RAD10 was higher than WT (2.6-fold) and was reduced in RAD10shP compared to RAD10 (1.6-fold) displaying a possible role of PBRM1 in regulating CXCR7 in RAD001-resistant RCC through YY1. CXCR4/CXCL12 signaling in RAD10shP cells more actively induced pERK and pAKT following CXCL12 or CXCL11-mediated as compared to RAD10 demonstrating that PBRM1 silencing restored a sensitive-like phenotype; however, PBRM1 silencing partially restored RAD001 sensitivity in RAD10 (RAD10 were 48-fold more resistant than A498, RAD10shP were 8.8-fold more resistant than A498). Conclusion: Taken together, these results demonstrate that both PBRM1 and CXCR4 are responsible for RAD001 resistance with PBRM1 regulating CXCR7 and mTOR shedding light on the new mechanistic roles of PBRM1 in RCC Citation Format: Federica Auletta, Caterina Ieranò, Giuseppina Rea, Maria Napolitano, Francesca Galdiero, Anna Maria Bello, Pierfrancesco Tassone, Maria Teresa Di Martino, Stefania Scala. The CXCR4/CXCR7/CXCL12 axis and PBRM1 are involved in everolimus resistance in human renal cancer cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7655.

Histone Methyltranferase SETD8 Stimulates Proliferation and Anti-Apoptotic Effect in Acute Myeloid Leukemia through Epigenetically Upregulation of CXCR4

Acute myeloid leukemia (AML) is the most common type of leukemia., characterized by the rapid and uncontrolled growth of abnormal cells. Epigenetic modifications control gene expression without affecting its DNA sequence. H4K20me1 is the mono-methylation occurring on lysine 20 of histone H4, acting as an epigenetic mark that promotes gene expression. SETD8 is responsible for catalyzing the mono-methylation of lysine 20 on histone H4. While SETD8's role and mechanisms have been studied in various types of cancer, its function in AML remains unclear. This study is based on the screening of a small molecule compound library, which identified the inhibitor UNC0379 targeting the histone methyltransferase SETD8, showing clear inhibitory effects on three non-M3 AML cell lines. Bioinformatical analysis revealed elevated SETD8 expression in AML patients compared to normal individuals and higher SETD8 expression corresponded with lower overall survival rates (OS). Further investigations using AML cell lines and normal peripheral blood mononuclear cells (PBMCs) demonstrated that SETD8 knockdown or UNC0379 intervention inhibited cell proliferation and increased apoptosis in AML cells, while PBMC cells remained unaffected. In an AML model constructed with C1498 cell injection in C57BL/6J mice, we observed that SETD8 knockdown and treatment with UNC0379 inhibited tumor formation and infiltration. To elucidate the molecular mechanism of SETD8's impact on AML, we conducted transcriptome sequencing on SETD8 knockdown AML cells and found significantly decreased mRNA expression of CXCR4, which was further validated at the protein level. Also, BeatAML and TCGA datasets demonstrated a positive correlation between SETD8 and CXCR4 mRNA expression. These phenomena motivated us to clarify the regulation relationship between SETD8 and CXCR4. CXCR4 is a crucial membrane receptor. After stimulation, it promotes AML cell proliferation and survival. CHIP experiments in SETD8 knockdown cells revealed reduced H4K20me1 enrichment in the CXCR4 promoter region. Subsequent rescue experiments demonstrated that overexpressing CXCR4 or using its corresponding agonist SDF-1alpha in SETD8 knockdown cells moderately restored cell proliferation and reduced apoptosis. In summary, we discovered that SETD8 promotes CXCR4 transcriptional activation through catalyzing the H4K20me1 mark, thereby influencing AML cell proliferation and apoptosis. SETD8 presents a promising therapeutic target through genetic or pharmacological intervention.

Nuclear Tubulin Enhances CXCR4 Transcription and Promotes Chemotaxis Through TCF12 Transcription Factor in human Hematopoietic Stem Cells.

Tubulins are cytoskeleton components in all eukaryotic cells and play crucial roles in various cellular activities by polymerizing into dynamic microtubules. A subpopulation of tubulin has been shown to localize in the nucleus, however, the function of nuclear tubulin remains largely unexplored. Here we report that microtubule depolymerization specifically upregulates surface CXCR4 expression in human hematopoietic stem cells (HSCs). Mechanistically, microtubule depolymerization results in accumulation of tubulin subunits in the nucleus, leading to elevated CXCR4 transcription and increased chemotaxis of human HSCs. Treatment with microtubule stabilizer Epothilone B strongly suppresses the phenotypes induced by microtubule depolymerizing agents in human HSCs. Furthermore, chromatin immunoprecipitation assay reveals an increased binding of nuclear tubulin and TCF12 transcription factor at the CXCR4 promoter region. Depletion of TCF12 significantly suppresses microtubule depolymerization mediated upregulation of CXCR4 surface expression. These results demonstrate a previously unknown function of nuclear tubulin in regulating gene transcription through TCF12. New strategy targeting nuclear tubulin-TCF12-CXCR4 axis may be applicable to enhance HSC transplantation.

Mycobacterium indicus pranii therapy suppresses systemic dissemination of tumor cells in B16F10 murine model of melanoma

Cancer associated morbidity is mostly attributed to the dissemination of tumor cells from their primary niche into the circulation known as “metastasis”. Mycobacterium indicus pranii (MIP) an approved immunotherapeutic agent against lung cancer (NSCLC) has shown potent anti-tumor activity in prior studies. While evaluating anti-tumor activity of MIP in mouse model, MIP treated animals typically exhibited less metastatic lesions in their pulmonary compartment. To study the role of MIP in metastasis closely, B16F10 melanoma cells were implanted subcutaneously in the mice, and the dissemination of tumor cells from the solid tumor was evaluated over a period of time. When B16F10 melanoma cells were treated with MIP in vitro, downregulation of epithelial mesenchymal transition markers was observed in these cells, which in turn suppressed the invasion, migration and adhesion of tumor cells. Notably, MIP therapy was found to be effectively reducing the metastatic burden in murine model of melanoma. Molecular characterization of MIP treated tumor cells substantiated that MIP upregulates the PPARγ expression within the tumor cells, which attenuates the NFκB/p65 levels within the nucleus, resulting in the suppression of Mmp9 expression in tumor cells. Besides that, MIP also downregulated the surface expression of chemokine receptor CXCR4 in murine melanoma cells, where chromatin immunoprecipitation confirmed the impeded recruitment of p50 and c-Rel factors to the Cxcr4 promoter, resulting in its downregulation transcriptionally. Taken together, MIP suppressed the dissemination of tumor cells in vivo, by regulating the expression of MMP9 and CXCR4 on these cells.

FOXA2 promotes esophageal cancer migration and metastasis by activating CXCR4 expression

Esophageal cancer is one of the most common malignant tumors worldwide and half of the patients present tumor metastasis at initial diagnosis. CXCR4 has been reported to be upregulated in esophageal cancer tissues and associated with tumor metastasis. However, the upstream transcriptional regulator of CXCR4 in esophageal cancer is still unclear. In this study, we found that transcription factor FOXA2 directly bound on the CXCR4 promoter region and activated its expression in esophageal cancer cells. The expression of FOXA2 was upregulated in esophageal cancer tissues and positively correlated with CXCR4. Moreover, knockout of FOXA2 significantly inhibited esophageal cancer migration and metastasis, which can be rescued by ectopically expressed CXCR4. Taken together, we reveal a novel transcriptional activator of CXCR4 in esophageal cancer, which might provide new strategies for esophageal cancer treatment.

The Embryonic Key Pluripotent Factor NANOG Mediates Glioblastoma Cell Migration via the SDF1/CXCR4 Pathway.

NANOG is a key transcription factor required for maintaining pluripotency of embryonic stem cells. Elevated NANOG expression levels have been reported in many types of human cancers, including lung, oral, prostate, stomach, breast, and brain. Several studies reported the correlation between NANOG expression and tumor metastasis, revealing itself as a powerful biomarker of poor prognosis. However, how NANOG regulates tumor progression is still not known. We previously showed in medaka fish that Nanog regulates primordial germ cell migration through Cxcr4b, a chemokine receptor known for its ability to promote migration and metastasis in human cancers. Therefore, we investigated the role of human NANOG in CXCR4-mediated cancer cell migration. Of note, we found that NANOG regulatory elements in the CXCR4 promoter are functionally conserved in medaka fish and humans, suggesting an evolutionary conserved regulatory axis. Moreover, CXCR4 expression requires NANOG in human glioblastoma cells. In addition, transwell assays demonstrated that NANOG regulates cancer cell migration through the SDF1/CXCR4 pathway. Altogether, our results uncover NANOG-CXCR4 as a novel pathway controlling cellular migration and support Nanog as a potential therapeutic target in the treatment of Nanog-dependent tumor progression.

The Novel Target of Colorectal Carcinoma: TRIM44 Regulates Cell Migration and Invasion via Activation of CXCR4/NF-κB Signaling.

Tripartite motif containing 44 (TRIM44) has been reported to regulate various biological effects in malignant cancers and matrix Metalloproteinases has been demonstrated to be associated with cancer cell migration and invasion. Nonetheless, the expression and molecular mechanism of TRIM44 in colorectal cancer (CRC) remain rarely known. TRIM44 was overexpressed or knocked down in CRC cells. Subsequently, the effects of TRIM44 on cell migration and invasion as well as underlying molecular mechanisms were detected. Data showed that TRIM44 was highly expressed in CRC cell lines. Downregulation of TRIM44 inhibited the cell viability, migration, and invasion in SW-480 cells. In addition, overexpression of TRIM44 enhanced the expression of NF-κB and CXCR4, and enhanced the binding between NF-κB and CXCR4 promoter region. In summarize, TRIM44 may serve as a potential target for CRC diagnosis and progression.

SOX4 promotes the growth and metastasis of breast cancer

PurposeIncreasing evidence has shown that the transcription factor SOX4 is closely associated with the development and progression of many malignant tumors. However, the effect of SOX4 on breast cancer is unclear. In this study, we purposed to investigate the role of SOX4 in the growth and metastasis in breast cancer and the underlying mechanism. Moreover, the effect of SOX4 on cancer cell resistance to chemotherapeutic agents was also evaluated in vitro and in vivo.MethodsWe used lentivirus technique to ectopically express SOX4 in MDA-MB-231 and SUM149 cells or knockdown SOX4 in BT474 cells, and examined the effect of these changes on various cellular functions. MTT assay was used to determine the cell viability as well as resistance to chemotherapeutic agents. The regulation of SOX4 on epithelial-mesenchymal transition (EMT)-related genes was analyzed using qRT-PCR. The binding of SOX4 to the CXCR7 gene was demonstrated using chromatin immunoprecipitation assay and dual-luciferase reporter activity assay. The effect of SOX4/CXCR7 axis on metastasis was examined using Transwell migration and Matrigel invasion assays. The expression of SOX4/CXCR7 in primary tumors and metastatic foci in lymph nodes was assessed using immunohistochemistry. Cellular morphology was investigated under phase contrast microscope and transmission electron microscopy. Moreover, the effect of SOX4 on tumor growth, metastasis, and resistance to chemotherapy was also studied in vivo by using bioluminescent imaging.ResultsSOX4 increased breast cancer cell viability, migration, and invasion in vitro and enhanced tumor growth and metastasis in vivo. It regulated EMT-related genes and bound to CXCR7 promoter to upregulate CXCR7 transcription. Both SOX4 and CXCR7 were highly expressed in human primary tumors and metastatic foci in lymph nodes. Treatment of breast cancer cells with the CXCR7 inhibitor CCX771 reversed the SOX4 effect on cell migration and invasion. Ectopic expression of SOX4 increased the susceptibility of cells to paclitaxel.ConclusionsSOX4 plays an important role in the growth and metastasis of breast cancer. SOX4/CXCR7 may serve as potential therapeutic targets for the treatment. Paclitaxel may be a good therapeutic option if the expression level of SOX4 is high.

CXCR4 induces cell autophagy and maintains EBV latent infection in EBVaGC.

Rationale: Epstein-Barr virus (EBV) is found in ~7% of gastric carcinoma cases worldwide, and all tumour cells harbour the clonal EBV genome. EBV can regulate pathways and protein expression to induce gastric carcinoma; however, the molecular mechanism underlying EBV-associated gastric carcinoma (EBVaGC) remains elusive.Methods: GEO microarray and molecular experiments were performed to compare CXCR4 expression between EBV-positive and EBV-negative gastric carcinoma (EBVnGC). Transfections with LMP2A plasmid or siRNA were carried out to assess the role of LMP2A in CXCR4 expression. The effects and mechanisms of CXCR4 on cell autophagy were analysed in vitro using molecular biological and cellular approaches. Additionally, we also determined the regulatory role of CXCR4 in latent EBV infection.Results: CXCR4 expression was significantly upregulated in EBVaGC tissues and cell lines. LMP2A could induce AKT phosphorylation to increase NRF1 expression, thereby binding to the CXCR4 promoter to increase its transcriptional level. Moreover, CXCR4 promoted ZEB1 expression to upregulate ATG7 synthesis, which could then activate autophagy. Moreover, CXCR4 increased the number of cells entering the G2/M phase and inhibited cell apoptosis via the autophagy pathway. Finally, CXCR4 knockdown was associated with elevated BZLF1 expression, but this effect was not influenced by autophagy.Conclusions: Our data suggested new roles for CXCR4 in autophagy and EBV replication in EBVaGC, which further promoted cell survival and persistent latent infection. These new findings can lead to further CXCR4-based anticancer therapy.

Epigenetic Alterations in Juvenile Spondyloarthritis Patients: a Preliminary Study of Selected Genes Promoter Methylation and Silencing

Juvenile spondyloarthritis (jSpA) is a complex disease with both genetic and environmental factors contributing to etiology. Multiple studies have shown that epigenetic mechanisms could link the environment and gene expression and thus provide a potential explanation for external contribution in the pathogenesis of numerous diseases, including rheumatic. Previously obtained gene signatures in jSpA patients revealed distinctive expression of important immune-related genes, though the mechanism(s) responsible for those alterations remained unknown. The purpose of this study was to evaluate the methylation levels of the TLR4, CXCR4, NLRP3, and PTPN12 gene promoter, along with the expression of several non-coding microRNAs (miR-150, miR-146a, miR-181a, and miR-223) in jSpA patients. Peripheral blood samples were obtained from 19 patients newly diagnosed with jSpA according to ILAR classification criteria for enthesitis-related arthritis (ErA) and seven gender- and age-matched subjects without any symptoms or signs of inflammatory disease. The expression of specific microRNAs was analyzed using qRT-PCR with predeveloped microRNA assays. DNA promoter region methylation status of selected genes was assessed by methylated DNA immunoprecipitation (MeDIP) analysis. Fold enrichment of immunoprecipitated DNA differed significantly for NLRP3 promoter site, while the expression analysis of selected microRNAs showed no significant difference in fold change between jSpA patients and healthy controls. The results indicated that epigenetic modifications in the initial phase of the disease could be responsible for some of the expression alterations in jSpA patients. Since NLRP3 has a crucial role in inflammasome assembly and inflammasomes have been shown to shape microbiota, it is tempting to assume that dysbiosis in jSpA patients can at least partially be explained by reduced NLRP3 expression due to hypermethylation, stressing for the first time the epigenetic contribution to jSpA pathophysiology.

Hypoxia-induced cancer stemness acquisition is associated with CXCR4 activation by its aberrant promoter demethylation

BackgroundA hypoxic microenvironment leads to an increase in the invasiveness and the metastatic potential of cancer cells within tumors via the epithelial-mesenchymal transition (EMT) and cancer stemness acquisition. However, hypoxia-induced changes in the expression and function of candidate stem cell markers and their possible molecular mechanism is still not understood.MethodsLung cell lines were analyzed in normoxic or hypoxic conditions. For screening among the stem cell markers, a transcriptome analysis using next-generation sequencing was performed. For validation, the EMT and stem cell characteristics were analyzed. To determine whether an epigenetic mechanism was involved, the cell lines were treated with a DNA methyltransferase inhibitor (AZA), and methylation-specific PCR and bisulfite sequencing were performed.ResultsNext-generation sequencing revealed that the CXCR4 expression was significantly higher after the hypoxic condition, which functionally resulted in the EMT and cancer stemness acquisition. The acquisition of the EMT and stemness properties was inhibited by treatment with CXCR4 siRNA. The CXCR4 was activated by either the hypoxic condition or treatment with AZA. The methylation-specific PCR and bisulfite sequencing displayed a decreased CXCR4 promoter methylation in the hypoxic condition.ConclusionsThese results suggest that hypoxia-induced acquisition of cancer stem cell characteristics was associated with CXCR4 activation by its aberrant promoter demethylation.

TCF12 promotes the tumorigenesis and metastasis of hepatocellular carcinoma via upregulation of CXCR4 expression

TCF12, which is known to be involved in the regulation of cell growth and differentiation, has been reported to function as an oncogene or a tumor suppressor gene in the progression of various malignant tumors. However, its function and molecular mechanism in hepatocellular carcinoma (HCC) remain unclear.Methods: Stable ectopic TCF12 expression or knockdown in HCC cell lines was established by lentiviral infection. Then, MTT, colony formation, migration, invasion and HUVECs tube formation assays as well as an orthotopic xenograft model were used to investigate the biologic function of TCF12 in HCC cells in vitro and in vivo. Subsequently, RNA-Seq analysis was utilized to explore the target genes regulated by TCF12. RT-qPCR, western blotting, a dual-luciferase reporter assay, Ch-IP, CHIP-Seq and functional rescue experiments were used to confirm the target gene regulated by TCF12. Finally, RT-qPCR, western blot and immunohistochemical (IHC) staining were performed to detect the expression level of TCF12 and to analyze the correlation of TCF12 with downstream genes as well as the clinical significance of TCF12 in human primary HCC.Results: Our functional studies revealed that stable overexpression of TCF12 in human HCC cells enhanced cell proliferation, migration and invasion in vitro and in vivo, whereas knockdown of TCF12 showed opposing effects. Mechanistically, CXCR4 was a downstream target of TCF12, and TCF12 directly bound to the CXCR4 promoter to regulate its expression. Moreover, CXCR4, with its ligand CXCL12, played a critical role in tumor progression induced by TCF12 via activation of the MAPK/ERK and PI3K/AKT signaling pathways. Clinically, IHC analysis revealed that TCF12 was significantly associated with poor survival of HCC patients and that TCF12 expression was closely correlated with CXCR4 expression in primary HCC tissues.Conclusion: Our findings are the first to indicate that TCF12 could promote the tumorigenesis and progression of HCC mainly by upregulating CXCR4 expression and is a prognostic indicator for patients with HCC.

Thymoquinone Inhibits Bone Metastasis of Breast Cancer Cells Through Abrogation of the CXCR4 Signaling Axis.

Overexpression of chemokine receptor type 4 (CXCR4) has been found to be associated with increased cell proliferation, metastasis and also act as an indicator of poor prognosis in patients with breast cancer. Therefore, new agents that can abrogate CXCR4 expression have potential against breast cancer metastasis. In this study, we examined the potential effect of thymoquinone (TQ), derived from the seeds of Nigella sativa, on the expression and regulation of CXCR4 in breast cancer cells. TQ was found to inhibit the expression of CXCR4 in MDA-MB-231 triple negative breast cancer (TNBC) cells in a dose- and time-dependent manner. It was noted that suppression of CXCR4 by TQ was possibly transcriptionally regulated, as treatment with this drug caused down-regulation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation and suppression of NF-κB binding to the CXCR4 promoter. Pretreatment with a proteasome inhibitor and/or lysosomal stabilization did not affect TQ induced suppression of CXCR4. Down-regulation of CXCR4 was further correlated with the inhibition of CXCL12-mediated migration and invasion of MDA-MB-231 cells. Interestingly, it was observed that the deletion of p65 could reverse the observed anti-invasive/anti-migratory effects of TQ in breast cancer cells. TQ also dose-dependently inhibited MDA-MB-231 tumor growth and tumor vascularity in a chick chorioallantoic membrane assay model. We also observed TQ (2 and 4 mg/kg) treatment significantly suppressed multiple lung, brain, and bone metastases in a dose-dependent manner in a metastasis breast cancer mouse model. Interestingly, H&E and immunohistochemical analysis of bone isolated from TQ treated mice indicated a reduction in number of osteolytic lesions and the expression of metastatic biomarkers. In conclusion, the results indicate that TQ primarily exerts its anti-metastatic effects by down-regulation of NF-κB regulated CXCR4 expression and thus has potential for the treatment of breast cancer.

Feedback control of the CXCR7/CXCL11 chemokine axis by estrogen receptor α in ovarian cancer.

Ovarian cancer (OC) is one of the most intractable diseases, exhibiting tremendous molecular heterogeneity and lacking reliable methods for screening, resulting in late diagnosis and widespread peritoneal dissemination. Menopausal estrogen replacement therapy is a well‐recognized risk factor for OC, but little is known about how estrogen might contribute to this disease at the cellular level. This study identifies chemokine receptor CXCR7/ACKR3 as an estrogen‐responsive gene, whose expression is markedly enhanced by estrogen through direct recruitment of ERα and transcriptional active histone modifications in OC cells. The gene encoding CXCR7 chemokine ligand I‐TAC/CXCL11 was also upregulated by estrogen, resulting in Ser‐118 phosphorylation, activation, and recruitment of estrogen receptor ERα at the CXCR7 promoter locus for positive feedback regulation. Both CXCR7 and CXCL11, but not CXCR3 (also recognized to interact with CXCL11), were found to be significantly increased in stromal sections of microdissected tumors and positively correlated in mesenchymal subtype of OC. Estrogenic induction of mesenchymal markers SNAI1, SNAI2, and CDH2 expression, with a consequent increase in cancer cell migration, was shown to depend on CXCR7, indicating a key role for CXCR7 in mediating estrogen upregulation of mesenchymal markers to induce invasion of OC cells. These findings identify a feed‐forward mechanism that sustains activation of the CXCR7/CXCL11 axis under ERα control to induce the epithelial–mesenchymal transition pathway and metastatic behavior of OC cells. Such interplay underlies the complex gene profile heterogeneity of OC that promotes changes in tumor microenvironment and metastatic acquisition.

Abstract 3559: Galectin-1 upregulates CXCR4 in glioblastoma through upregulation of CXCR4 via NF-κB activation

Abstract Background: Despite aggressive treatment, including surgical resection, radiation and chemotherapy, over 90% of glioblastoma (GB) patients experience tumor recurrence. This may be due to high migratory potential, abnormal angiogenesis, molecular heterogeneity and a strong immunosuppressive environment. An earlier report from our laboratory demonstrated that GB cells express high levels of carbohydrate-binding galectin protein: galectin-1 (Gal-1). However, the role of galectin-1 in GB remains elusive. The aim of this study was to evaluate whether galectin-1 promotes tumor progression through upregulation of CXCR4 via NF-κB in in vitro GB models. Methods: To investigate the molecular mechanisms of Gal-1 on tumor progression, we used RT-PCR and western blots techniques. Gal-1 was stably over-expressed in the U118 and U87MG cell line. Gal-1 (+/-) sub-clones were assayed for attachment, proliferation, migration, and tube formation. Results: Gal-1 protein expression was assessed in human GB cells. We found increased Gal-1 protein expression in human GB cells and tissue. To investigate the effects of Gal-1 silencing on angiogenesis in vitro, we collected conditioned media from Gal-1-knockdown U118 and U87MG cells then applied this to human umbilical vein endothelial cells (HUVEC) culture to analyze endothelial cell proliferation, migration, and tube formation. Proliferation rate, migrating capability, and tube formation decreased significantly when HUVECs were cultured with concentrated conditioned media from Gal-1-knockdown cells. This data suggests that Gal-1 in GB cells may promote tumor angiogenesis both in vitro and co-culture. Results showed that silencing of Gal-1 in U118 and U87MG cells significantly repressed CXCR4 expression at the mRNA level and protein levels. To further explore the role of CXCR4 in GB cells, CXCR4 knockdown resulted in significant inhibition of cell migration and clonogenic ability in vitro. Thus, Gal-1 might upregulate CXCR4 expression to promote tumor progression. A previous laboratory study indicated that NF-κB promotes cancer cell motility by upregulating CXCR4 expression. Luciferase reporter assay was used to determine the involvement of NF-κB in Gal-1-induced CXCR4 expression in U118 and U87MG cells. NF-κB luciferase activity was significantly decreased in Gal-1-knockdown cells. Moreover, decreased CXCR4 promoter activity and protein expression in Gal-1-knockdown cells were observed. This data indicates that Gal-1 might modulate CXCR4 expression via NF-κB activation. Conclusion: Our in vitro cell culture results suggest that upregulation of galectin-1 and CXCR4 might at least in part explain the aggressive infiltrative biology of GB and the Gal-1-CXCR4 axis may serve as a therapeutic target. Further studies in different animal models are warranted. Citation Format: David Cachia, Rachel Malley, William A. Vandergrift, Sunil Patel, Gabriel A. Rabinovich, Arabinda Das. Galectin-1 upregulates CXCR4 in glioblastoma through upregulation of CXCR4 via NF-κB activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3559.

Tetramethylpyrazine in a Murine Alkali-Burn Model Blocks NFκB/NRF-1/CXCR4-Signaling-Induced Corneal Neovascularization.

Tetramethylpyrazine (TMP) is the active ingredient extracted from the Chinese herb Chuanxiong. The purpose of our study was to identify the mechanism of therapeutic TMP suppression of pathologic chemokine receptor 4 (CXCR4) transcription. C57BL/6J mice with alkali-burned corneas were treated with either TMP eye drops (1.5 mg/mL) or PBS. Corneal neovascularization (CNV) was measured and a clinical assessment was made by slit lamp microscopy. Expression of CXCR4 and the transcription factors nuclear respiratory factor-1 (NRF-1), nuclear factor kappa B (NFκB), forkhead box C1, and yin yang 1 were tracked by real-time RT-PCR and immunofluorescence staining of murine corneas. Western blot, real-time PCR, and immunofluorescence evaluated expression of related genes in human umbilical vein endothelial cells (HUVECs) after 200-μmol/L TMP treatment. In addition, plasmid transfection and chromatin immunoprecipitation assays elucidated the relationship among NRF-1, NFκB, and CXCR4. Corneas treated with TMP had smaller areas of neovascularization and scored better in clinical assessments. Injured corneas showed significantly elevated expressions of NRF-1, NFκB, and CXCR4 that were normalized in vivo by TMP treatment. Similarly, in HUVECs in vitro, TMP decreased expression of NRF-1, NFκB, and CXCR4. Overexpression of NFκB or NRF-1 raised the expression of CXCR4 in HUVECs, but not synergistically. Chromatin immunoprecipitation assays detected only NRF-1 bound to the CXCR4 promoter region, suggesting NFκB controls CXCR4 expression by upregulating NRF-1. Together, our data suggest TMP downregulates CXCR4 by repressing NRF-1 expression in CNV, likely indirectly by downregulating NFκB. Our results implicate a novel mechanism wherein TMP inhibits neovascularization via an NFκB/NRF-1/CXCR4 circuit.

Doxorubicin upregulates CXCR4 via miR-200c/ZEB1-dependent mechanism in human cardiac mesenchymal progenitor cells

Doxorubicin (DOXO) treatment is limited by its cardiotoxicity, since it causes cardiac-progenitor-cell depletion. Although the cardioprotective role of the stromal cell-derived factor-1/C-X-C chemokine receptor type 4 (SDF1/CXCR4) axis is well established, its involvement during DOXO-induced cardiotoxicity has never been investigated. We showed that in a mouse model of DOXO-induced cardiomyopathy, CXCR4+ cells were increased in response to DOXO, mainly in human cardiac mesenchymal progenitor cells (CmPC), a subpopulation with regenerative potential. Our in vitro results showed a CXCR4 induction after 24 h of DOXO exposure in CmPC. SDF1 administration protected from DOXO-induced cell death and promoted CmPC migration. CXCR4 promoter analysis revealed zinc finger E-box binding homeobox 1 (ZEB1) binding sites. Upon DOXO treatment, ZEB1 binding decreased and RNA-polymerase-II increased, suggesting a DOXO-mediated transcriptional increase in CXCR4. Indeed, DOXO induced the upregulation of miR-200c, that directly targets ZEB1. SDF1 administration in DOXO-treated mice partially reverted the adverse remodeling, decreasing left ventricular (LV) end diastolic volume, LV ejection fraction and LV anterior wall thickness in diastole, recovering LV end systolic pressure and reducing±dP/dt. Moreover, in vivo administration of SDF1 partially reverted DOXO-induced miR-200c and p53 protein upregulation in mouse hearts. In addition, downmodulation of ZEB1 mRNA and protein by DOXO was significantly increased by SDF1. In keeping, p21 mRNA, that is induced by p53 and inhibited by ZEB1, is induced by DOXO treatment and is decreased by SDF1 administration. This study showed new players of the DOXO-induced cardiotoxicity, that can be exploited to ameliorate DOXO-associated cardiomyopathy.

Glucocorticoid hormone-induced chromatin remodeling enhances human hematopoietic stem cell homing and engraftment.

Efficient hematopoietic stem cell (HSC) homing is important for hematopoietic cell transplantation (HCT), especially when HSC numbers are limited, as in the use of cord blood (CB). In a screen of small-molecule compounds, we identified glucocorticoid (GC) hormone signaling as an activator of CXCR4 expression in human CB HSCs and hematopoietic progenitor cells (HPCs). Short-term GC pretreatment of human CB HSCs and HPCs promoted SDF-1-CXCR4-axis-mediated chemotaxis, homing, and long-term engraftment when these cells were transplanted into primary- and secondary-recipient NSG mice. Mechanistically, activated glucocorticoid receptor binds directly to a glucocorticoid response element in the CXCR4 promoter and recruits the SRC-1-p300 complex to promote H4K5 and H4K16 histone acetylation, facilitating transcription of CXCR4. These results suggest a new and readily available means to enhance the clinical efficacy of CB HCT.

Discordant mRNA and protein expression of CXCR4 under in vitro CoCl2-induced hypoxic conditions

Cobalt chloride (CoCl2) has long been accepted as a suitable in vitro hypoxia-mimetic agent. The gene CXCR4, which encodes a chemokine receptor, plays a key role in hypoxic retinal disease. Here, we investigated the mRNA and protein expression of CXCR4 in WERI-Rb1 retinoblastoma cells and human umbilical vein endothelial cells (HUVECs) under CoCl2-induced hypoxic conditions, by means of real-time PCR and western blot. We found that CoCl2-induced hypoxia profoundly increased CXCR4 expression at the mRNA level, but not at the protein level, at 12, 24, 48 and 72 h in these cells. Interestingly, this result differed from observations of 1% O2 hypoxic conditions. Additionally, luciferase assays demonstrated that CoCl2-induced hypoxia significantly increased transcription at the CXCR4 promoter. In order to compare our in vitro findings with the effects of hypoxia in vivo, an OIR (Oxygen-induced retinopathy) rat model was constructed. However, both CXCR4 mRNA and protein levels in OIR rats were significantly increased compared to controls. Thus taken together, our findings suggest that the relationship between CXCR4 mRNA and protein expression is not strictly linear under in vitro CoCl2-induced hypoxic conditions. through comparative in vitro and in vivo experiments, this study implies that CoCl2 is an imperfect simulation of hypoxia in retinal disease.