CXCR4 Protein Research Articles

Progressive Increase of Inflammatory CXCR4 and TNF-Alpha in the Dorsal Root Ganglia and Spinal Cord Maintains Peripheral and Central Sensitization to Diabetic Neuropathic Pain in Rats.

Diabetic neuropathic pain (DNP) is a common and serious complication of diabetic patients. The pathogenesis of DNP is largely unclear. The proinflammation proteins, CXCR4, and TNF-α play critical roles in the development of pain, while their relative roles in the development of DNP and especially its progression is unknown. We proposed that establishment of diabetic pain models in rodents and evaluating the stability of behavioral tests are necessary approaches to better understand the mechanism of DNP. In this study, Von Frey and Hargreaves Apparatus was used to analyze the behavioral changes of mechanical allodynia and heat hyperalgesia in streptozotocin-induced diabetic rats at different phases of diabetes. Moreover, CXCR4 and TNF-α of spinal cord dorsal and dorsal root ganglia (DRG) were detected by western blotting and immunostaining over time. The values of paw withdrawal threshold (PWT) and paw withdrawal latencies (PWL) were reduced as early as 1 week in diabetic rats and persistently maintained at lower levels during the progression of diabetes as compared to control rats that were concomitant with significant increases of both CXCR4 and TNF-α protein expressions in the DRG at 2 weeks and 5 weeks (the end of the experiments) of diabetes. By contrast, CXCR4 and TNF-α in the spinal cord dorsal horn did not significantly increase at 2 weeks of diabetes while both were significantly upregulated at 5 weeks of diabetes. The results indicate that central sensitization of spinal cord dorsal may result from persistent peripheral sensitization and suggest a potential reference for further treatment of DNP.

Unilesional mycosis fungoides is associated with increased expression of microRNA-17~92 and T helper 1 skewing.

The molecular basis of unilesional mycosis fungoides (MF), characterized by a solitary lesion that is clinicopathologically indistinguishable from multifocal patch or plaque MF (early MF), is unknown. To investigate the microRNA profile in unilesional MF distinguishing it from early MF. Biopsy samples of unilesional MF and early MF were evaluated with the Affymetrix microRNA array, with further comparison with inflammatory dermatosis, using quantitative polymerase chain reaction. NanoString technology was applied to analyse the gene expression of T helper (Th)1 immune markers, and immunohistochemistry was used to evaluate CXCR3 and GATA-binding protein 3 (GATA3) markers for Th1 and Th2 cells, respectively. Unilesional MF had a significantly higher level of expression of all members of the microRNA miR-17~92 cluster than early MF. Specifically, unilesional MF had a higher miR-17 level than early MF and inflammatory dermatoses. There was downregulation of the expression of phosphatase and tensin homolog (PTEN) and CREB1, known targets of miR-17~92 members; higher gene expression of interleukin-2 and interferon-γ; and a statistically lower average percentage of GATA3+ dermal cells (6·7% vs. 42·3%), were detected in unilesional MF compared with early MF. High immunoreactivity of CXCR3 was noted in both unilesional and early MF. Unilesional MF exhibits a microRNA profile distinct from that of conventional early MF, with a higher level of miR-17~92 members along with Th1 skewing. These findings suggest a robust reactive T-cell immune response in unilesional MF and might account for the localized nature of this disease.

Bone marrow-derived interstitial cells of cajal are increased by electroacupuncture in the colon of diabetic mice.

Insufficiency of interstitial cells of cajal (ICC) in the muscular plexus of colon has been proved in diabetic mice. Electroacupuncture (EA) at ST36 can accelerate ICC supplement in the colon of diabetic mice, but the source of increased ICC has not been clearly explored. Bone marrow cells possess the potential of migration and differentiation into ICC of the intestine. Our aim is to explore the effects of EA on variations of bone marrow-derived ICC in the colon of diabetic mice as well as its mechanism. Wild C57BL/6 mice were divided into six groups with random assignment method: control group, diabetic mellitus (DM) group, bone marrow transplantation (BMT)+DM group, BMT+DM+sham EA group, BMT+DM+low-frequency EA group, and BMT+DM+high-frequency EA group. Flow cytometric method was adopted to identify the chimera model. The specific location and expression levels of c-Kit+ green fluorescent protein (GFP+) cells in colon were detected by immunofluorescence. Western blot and quantitative polymerase chain reaction were used to assess the expression level of c-Kit, GFP, membrane-band stem cell factor (mSCF), p-ERK, p-c-Jun, ETV1, stromal cell-derived factor 1 (SDF-1), CXCR4, transforming growth factor β1 (TGF-β1), and smad3. c-Kit+ GFP+ cells in the muscular plexus of colon were apparently increased in the EA groups; the protein and mRNA expression level of CXCR4, SDF-1, TGF-β1, smad3, c-kit, mSCF, p-ERK, p-c-Jun, and ETV1 were elevated in the EA groups. Electroacupuncture can effectively increase bone marrow-derived ICC in the colon by SDF-1/CXCR4, TGF-β1/Smad3, and mSCF/Kit-p-ERK/p-c-Jun-ETV1 signal pathway.

Assessment of Short-Term Engraftment Potential of Ex Vivo Expanded Hematopoietic Stem Cells Using Normal Fetal Mouse in Utero Transplantation Model.

Objective:Ex vivo expansion is a promising strategy to overcome the low number of human umbilical cord blood hematopoietic stem cells (hUCB-HSCs). Although based on the obtained results in unnatural physiological condition of irradiated genetically immune-deficient mouse models, there has always been concern that the expanded cells have less engraftment potential. The purpose of this study was to investigate effect of common ex vivo expansion method on engraftment potential of hUCB-mononuclear cells (MNCs), using normal fetal mouse, as a model with more similarity to human physiological conditions.Materials and Methods:In this experimental study, briefly, isolated hUCB-MNCs were cultured in common expansion medium containing stem cell factor, Flt3 ligand and thrombopoietin. The unexpanded and expanded cells were transplanted to the fetal mice on gestational days of 11.5-13.5. After administration of human hematopoiesis growth factors (hHGFs), presence of human CD45+ cells, in the peripheral blood of recipients, was assessed at various time points after transplantation.Results:The expanded MNCs showed 32-fold increase in the expression of CD34+38- phenotype and about 3-fold higher clonogenic potential as compared to the uncultured cells. Four weeks after transplantation, 73% (19/26) of expanded-cell recipients and 35% (7/20) of unexpanded-cell recipients were found to be successfully engrafted with human CD45+ cells. The engraftment level of expanded MNCs was significantly (1.8-fold) higher than unexpanded cells. After hHGFs administration, the level was increased to 3.2, 3.8 and 2.6-fold at respectively 8, 12, and 16 weeks of post transplantation. The increased expression of CXCR4 protein in expanded MNCs is a likely explanation for the present findings.Conclusion:The presented data showed that expanded MNCs compared to unexpended cells are capable of more rapid and higher short-term engraftment in normal fetal mouse. It could also be suggested that in utero transplantation (IUT) of normal fetal mice could be an appropriate substitute for NOD/SCID mice in xenotransplantation studies.

Significant changes in circular RNA in the mouse cerebral cortex around an injury site after traumatic brain injury

Background and objectiveCircular RNA (circRNA) is an important type of non-coding RNA that has not been widely researched in traumatic brain injury (TBI). The present study aimd to detect the altered circRNA expression around an injury site in the mouse cerebral cortex after TBI and explore its potential functions. MethodC57BL/6 mice were used to construct a controlled cortical impact (CCI) model to simulate TBI. At 24 h post-TBI, the cortex around the injury site was collected, and the total RNA was extracted to perform RNA sequencing (RNA-seq). The differentially expressed circRNAs were determined according to the following criteria: |log2(fold change)| > 1, P < .05 and FDR < 0.05. Among them, circRNA chr8_87,859,283–87,904,548 was preliminarily explored to determine its function. ResultsA total of 8036 altered circRNAs were discovered, and among them, 16 were significantly changed (5 up-regulated and 11 down-regulated). The circRNA chr8_87,859,283–87,904,548 significantly increased by approximately 4 times in the cerebral cortex around the injury site after TBI and promoted neuro-inflammation through increasing the CXCR2 protein by sponging mmu-let-7a-5p. As a result, the increased circRNA chr8_87,859,283–87,904,548 blocked the restoration of neurological function after TBI. ConclusionMany circRNAs are significantly up-regulated or down-regulated in the traumatic cerebral penumbra cortex after TBI. Among them, the circRNA chr8_87,859,283–87,904,548 potentially plays a pro-inflammatory role, which may have a deleterious effect on neurological restoration after TBI..

The Pan-PIM Kinase Inhibitor LGB321 Strongly Increases Ibrutinib Effects in CLL, By Blocking Anti-Apoptotic Pathways and Microenvironmental Interactions

Despite remarkable anti-tumor activity of ibrutinib in CLL, some patients develop resistance due to acquired mutations. By adding drugs which target survival pathways and the CLL protective microenvironmental interactions, we aim to increase ibrutinib efficacy and to prevent ibrutinib resistance. The three PIM kinases are involved in important disease mechanisms in CLL, with PIM1 regulating CXCR4 surface expression impacting the interaction with the protective microenvironment, and PIM2/3 affecting the apoptotic machinery by regulating BAD.Here, we investigated the effect of the Pan-PIM kinase inhibitor LGB321 in CLL in vitro and in vivo and its cooperative effect with ibrutinib. LGB321 was highly effective in inducing apoptosis in primary CLL cells, independent of prognostic factors or microenvironmental interactions, and reduced pBAD and total BAD protein levels in CLL. In vitro combination of ibrutinib and LGB321 demonstrated strong additive effects with doubled apoptosis rates compared to single treatment. Further, LGB321 treatment blocked the CXCR4/CXCL12 axis by dephosphorylating the CXCR4 receptor on Ser339, by reducing total CXCR4 protein levels, and by blocking the externalization of the CXCR4 receptor. Concordantly, LGB321 blocked CXCR4 functions regarding migration towards a CXCL12 gradient and homing of LGB321-pretreated primary CLL cells towards the bone marrow of NOG mice.In vivo experiments confirmed the efficacy of LGB321 in 5 different CLL xenograft studies. Transplantation of primary CLL cells into NOG mice and treatment with LGB321 for 2 weeks strongly reduced WBC counts, spleen size and spleen infiltration with human CLL cells in all 5 CLL cases, and blocked BAD as well as CXCR4 phosphorylation also in vivo.Xenograft studies combining ibrutinib with LGB321 demonstrated much faster reduction of WBC counts than ibrutinib single treatment and strongly reduced disease burden in the double treated mice, confirming the cooperative effects of those two inhibitors also in vivo.Our results demonstrate that LGB321 might be an effective treatment option for CLL patients by impairing PIM2/3 mediated CLL-cell survival, and by blocking the PIM1/CXCR4-mediated interaction of CLL cells with the protective microenvironment. Furthermore, LGB321 enhances ibrutinib treatment effects and might help to increase its efficacy and to avoid the development of ibrutinib resistance. DisclosuresNo relevant conflicts of interest to declare.

Growth Arrest-Specific 2 Promotes the Growth of T-Cell Acute Lymphoblastic Leukemia Cells Via Its Regulation of CXCR4

Growth arrest-specific 2 (GAS2) has multiple functions including the regulation of cell morphology, cell cycle, apoptosis and calpain activity.GAS2 has a dual function in cancer cells, however its expression and underlying mechanism in human T-cell acute lymphoblastic leukemia (T-ALL) remain unclear.In the present study, qRT-PCR analysis showed that GAS2 has significantly higher expression (155.5-fold, P=0.0048) in CD3+ cells from T-ALL patients (n=25) than healthy donors (n=13). GAS2 was present in Jurkat cells, while absent in MOLT-4 or HPB-ALL cells. A tiny CpG island of GAS2 was almost fully methylated in both MOLT-4 (100%) and HPB-ALL cells (80%), while 40% methylation in Jurkat cells; suggesting that DNA methylation played a subtle role in regulating GAS2 expression.Two independent shRNA sequences were delivered into Jurkat cells with lentiviral vector. GAS2 silencing inhibited the growth and colony-forming cell (CFC) production significantly. Conversely, GAS2 overexpression enhanced the growth and CFC production of both MOLT-4 and HPB-ALL cells. In addition, GAS2 overexpression promoted HPB-ALL cell induced leukemia in a xenoengraftment model (5 mice in each control group). In GAS2 expressed group, the disease latency was shortened, the splenomegaly was more severe than control group (0.35±0.04g vs. 0.27±0.05g), and more leukemic cells were present in bone marrow (85±3% vs. 45±7%).To obtain the molecular insights of how GAS2 acts, RNA-seq data comparing GAS2 silenced Jurkat cells with control cells were generated. Several Notch signaling molecules were inhibited, including NOTCH1, HES1 and HES4. Despite the differential expression of these transcripts was validated in Jurkat cells, GAS2 overexpression did not elevated the expression of these transcripts in MOLT-4 or HPB-ALL cells, suggesting GAS2 did not have a consistent impact on Notch signaling. However, we found that GAS2 silencing reduced CXCR4 protein expression in Jurkat cells and GAS2 overexpression enhanced CXCR4 protein expression in MOLT-4 cells, while CXCR4 transcript was not altered upon GAS2 manipulation. Consequently, GAS2 silencing significantly reduced migration ability of Jurkat cells and GAS2 overexpression enhanced migration ability of MOLT-4 cells. Overexpression of CXCR4 “rescued” the inhibited CFC production and migration upon GAS2 silencing. A truncated GAS2 (Δ171-313) coined as GAS2DN (dominant negative form of GAS2) has been known to inhibit normal function of GAS2. Herein, we showed that GAS2DN inhibited the growth of Jurkat cells and the expression of CXCR4. To delineate the role of calpain1 and calpain2 in GAS2 function, shRNA sequences against calpain1 and calpain2 was delivered into GAS2DN expressed Jurkat cells respectively, the results showed that calpain2 but not calpain1 silencing was able to enhance the cell growth and CXCR4 expression.Taken together, the present study has demonstrated that GAS2 is aberrantly expressed in human T-ALL cells, which promotes the growth of T-ALL cells partially via its post-transcriptional regulation of CXCR4 depending on calpain2 activity. These data provide new insights of the pathogenesis of T-ALL and possibly new clues to improve the management of the disease. DisclosuresNo relevant conflicts of interest to declare.

Clinical Implications of MYD88 and CXCR4 in Patients with Waldenström's Macroglobulinemia

BackgroundWaldenström's macroglobulinemia (WM) is a rare B-cell neoplasm defined as lymphoplasmacytic lymphoma with bone marrow infiltration and monoclonal IgM in the serum. More than 90% of WM patients carry a point mutation L265P in the MYD88 gene and concurrently, almost one third of MYD88L265P-positive patients harbor frameshift (WHIM-FS) or non-sense (WHIM-NS) mutation in gene CXCR4. The mutations in CXCR4 result in premature stop codons and in shortening of CXCR4 protein product. Incomplete C-terminal domain of CXCR4 chemokine receptor is known to hyperactivate CXCR4-mediated signalization. The presence and type of mutation in genes MYD88 and CXCR4 appears to be significant in diagnostics and prognostic stratification of WM patients and it also influences the clinical manifestation of the disease.AimsTo analyze mutational status of MYD88 and CXCR4 genes in patients with WM, to compare our results with laboratory parameters and to evaluate the prognostic stratification of the patients according to MYD88 and CXCR4 mutational status.MethodsAnalyzed DNA was isolated from mononuclear fraction of bone marrow or peripheral blood cells at the time of diagnosis. Mutational status of analyzed genes was determined using allele-specific PCR (in the case of MYD88) and using direct Sanger sequencing (in the case of CXCR4). All found mutations were confirmed by specific cleavage with restriction endonucleases at defined conditions.ResultsWe analyzed 23 patients with WM. All patients were MYD88L265P-positive (100 %), and 7 of them (30,4%) were also CXCR4 mutants (1 patient harbored WHIM-FS mutation and 6 harbored WHIM-NS mutation). CXCR4 mutations were associated with more aggressive disease: higher ISSWM score (low 0/intermediate 1/ high 6 risk), anemia (7/7), hyperviscosity syndrome (2/7) at time of diagnosis. CXCR4WHIM-WT patients were often asymptomatic (5/16) with lower ISSWM score (low 5/intermediate 5/ high 6 risk) but with common adenopathy (11/16). CXCR4 mutations were also associated with worse treatment response (2 CXCR4WHIM-MUT patients were refractory to initial therapy and needed 2nd line treatment, 3 patients had only partial response to first-line therapy, one patient died after 2nd cycle due to abdominal septic complication and only one patient reached VGPR). Progression-free survival in treated patients was 32 vs. 8 months in CXCR4 mutants.Conclusion/summaryThe mutational analysis of MYD88 and CXCR4 genes is essential for the diagnostics and prognostic stratification of patients with WM and it allows a deeper understanding of the molecular pathogenesis of the diseases. CXCR4 mutations were found in nearly one third of WM patients. In accordance with published data, we confirmed that CXCR4 mutations are associated with more aggressive disease presentation and thus affect treatment outcome. On the contrary CXCR4WHIM-WT patients have more indolent course of the disease.This work was supported by grant IGA-LF-2018-004 and MH CR - RVO (FNOL, 00098892). DisclosuresNo relevant conflicts of interest to declare.

MiR-142-3p as tumor suppressor miRNA in the regulation of tumorigenicity, invasion and migration of human breast cancer by targeting Bach-1 expression.

Breast cancer is the most common type of cancer among women, and despite improved treatments, it remains a major challenge. However, improved mechanistic insight may lead to novel therapeutic strategies. miR-142-3p belongs to the miR-142 family and is involved in pathogenesis and metastasis of various types of malignancies by targeting several important messenger RNAs (mRNAs) including Bach-1. This is especially true for breast cancer, where Bach-1 is involved in the metastatic spread by deregulation of metastasis-associated genes. In this study, we collected 24 breast cancer tissues with 24 adjusted normal tissues to measure the expression levels of miR-142-3p and Bach-1 mRNA using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) and IHC. miR-142-3p targeting of Bach-1 expression in MCF-7 and MDA-MB-468 breast cancer cells was evaluated using bioinformatics, qRT-PCR and western blot analyses. The cellular proliferation, invasion, and migration were assessed by MTT, transwell matrigel and wound healing assay and the EMT-associated proteins C-X-C chemokine receptor type 4 (CXCR-4), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor receptor (VEGFR) were analyzed by western blot analysis. Also, the expression levels of tumor suppressors including miR-330, miR-145, and miR-34a were estimated by qRT-PCR. Analysis of paired specimens of primary malignant and normal tissues showed that miR-142-3p was downregulated, while Bach-1 mRNA and protein both were overexpressed in the breast cancer tumors. This inverse relationship was confirmed by cell line experiments demonstrating that miR-142-3p expression reduced Bach-1 mRNA levels. Furthermore, replacement of miR-142-3p could inhibit the proliferation, invasion, and migration in breast cancer potentially by targeting of Bach-1 mRNA and subsequent inhibition of CXCR4, MMP9, and VEGFR protein expressions. In addition, induction of miR-142-3p could upregulate tumor suppressor miRNAs, including miR-330, miR-145, and miR34a. For the first time, our results revealed that miR-142-3p could target Bach-1in breast cancer cells leading to the reduction of EMT-related proteins and reduced cell proliferation, invasion, and migration. The results also demonstrated that miR-142-3p could regulate important tumor suppressor miRNAs in breast cancer cells. In conclusion, our results suggest that miR-142-3p could be a good candidate for the targeted therapy of breast cancer, especially for the invasive type.

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.

Disparate roles of CXCR3A and CXCR3B in regulating progressive properties of colorectal cancer cells.

Human C-X-C Motif Chemokine Receptor 3A (CXCR3A) and CXCR3B are two splice variants of CXCR3 that is involved in a variety of progressive processes of cancer cells, including proliferation, migration, invasion, and tumorigenicity. However, the molecular mechanisms of CXCR3 in colorectal cancer (CRC) remain incomplete understood. In the present study, a significantly up-regulated CXCR3 protein was firstly observed in CRC tissues and cell lines in comparison with the paired non-tumor tissues and normal intestinal epithelial cells, which was positively associated with CRC TNM stages. In contrast, CXCR3B was down-regulated in CRC tumor tissues compared with that in the corresponding paired paracancerous tissues, and negatively correlated with the TNM stages of cancer. Of interest, the overexpression of CXCR3A enhanced the progressive capacity of cell proliferation, migration, invasion in CRC LOVO and HCT116 cells in vitro, and the tumorigenicity in nude mice in vivo. Conversely, the overexpression of CXCR3B exhibited an opposite phenotype of CXCR3A, with an ability to inhibit the progressive properties in CRC cell lines in vitro and tumorigenesis in vivo. In addition, immunoblotting analysis further demonstrated that an increased expression of CXCR3A inhibited the expression of CXCR3B in CRC cells and NCM460 normal colon epithelial cells; vice verse, an overexpression of CXCR3B suppressed the expression of CXCR3A in these cells. These data imply that an interaction between the CXCR3A and CXCR3B may play an important regulatory role in tumorigenicity of CRC, which warrants for further investigation.

An omega-3 polyunsaturated fatty acid derivative, 18-HEPE, protects against CXCR4-associated melanoma metastasis.

Melanoma has a high propensity to metastasize and exhibits a poor response to classical therapies. Dysregulation of the chemokine receptor gene CXCR4 is associated with melanoma progression, and although n-3 polyunsaturated fatty acids (PUFAs) are known to be beneficial for melanoma prevention, the underlying mechanism of this effect is unclear. Here, we used the n-3 fatty acid desaturase (Fat-1) transgenic mouse model of endogenous n-3 PUFA synthesis to investigate the influence of elevated n-3 PUFA levels in a mouse model of metastatic melanoma. We found that relative to wild-type (WT) mice, Fat-1 mice exhibited fewer pulmonary metastatic colonies and improved inflammatory indices, including reduced serum tumor necrosis factor alpha (TNF-α) levels and pulmonary myeloperoxidase activity. Differential PUFA metabolites in serum were considered a key factor to alter cancer cell travelling to lung, and we found that n-6 PUFAs such as arachidonic acid induced CXCR4 protein expression although n-3 PUFAs such as eicosapentaenoic acid (EPA) decreased CXCR4 levels. In addition, serum levels of the bioactive EPA metabolite, 18-HEPE, were elevated in Fat-1 mice relative to WT mice, and 18-HEPE suppressed CXCR4 expression in B16-F0 cells. Moreover, relative to controls, numbers of pulmonary metastatic colonies were reduced in WT mice receiving intravenous injections either of 18-HEPE or 18-HEPE-pretreated melanoma cells. Our results indicate that 18-HEPE is a potential anticancer metabolite that mediates, at least in part, the preventive effect of n-3 PUFA on melanoma metastasis.

CXCR2 is a novel cancer stem-like cell marker for triple-negative breast cancer

BackgroundBreast cancer is the leading cause of mortality from cancer in women worldwide, and cancer stem-like cell (CSC) is responsible for failure treatment of breast cancer. It plays an important role in resistant disease and metastasis. CD44/CD24 and ALDH are well-accepted protein markers of breast CSC, and it was reported that distinct subtypes of breast CSC were identified by the 2 markers. It is possible that there are various kinds of breast CSC which could be identified by different markers, and CSC markers utilized at present are not enough to fully understand breast CSC. Finding out more novel CSC markers is necessary. CXCR2 is involved in breast cancer metastasis, treatment resistance, and recurrence and has positive cross-talk with known breast CSC protein markers. It can be concluded that CXCR2 is related to breast CSC, and further study is in need.ResultsIn this study, we assessed expression of CXCR2 with immunohistochemistry in breast cancer tissues from 37 patients and discovered that level of CXCR2 was significantly lower in triple-negative breast cancer (TNBC) compared with non-TNBC. CXCR2 expression decreased in estrogen receptor-negative or HER2-negative breast cancer, but not progesterone receptor-negative counterparts. By immunofluorescence, we observed high coexpression rate of CXCR2 and CSC-related proteins, including NANOG and SOX2. To prove our speculation that CXCR2 was a novel CSC marker for TNBC, we used 4T1 cell, which is a TNBC cell line, to analyze CXCR2-positive subpopulations and observed that CXCR2-positive 4T1 cells showed characteristics of CSC, including resistance to cisplatinum, radiation, and hypoxia, low proportion (around 1%), much more tumor xenografts, tumor spherule formation, and higher levels of CSC-related mRNA compared with CXCR2-negative cells.ConclusionCXCR2 is an acceptable and newly discovered CSC marker for only TNBC.

Spermatogonial stem cell potential of CXCR4-positive cells from prepubertal bull testes

Spermatogonial stem cells (SSC) have the potential to restore spermatogenesis when transplanted into testes depleted of germ cells. Due to this property, SSC could be used in breeding programs and in transgenic animal research. Particularly in cattle, SSC are not as well characterized as in mice or humans. In mice, C-X-C Motif Chemokine Receptor 4 positive (CXCR4+) testicular cells have high SSC potential. It, therefore, was hypothesized that CXCR4 is a marker of undifferentiated spermatogonia in cattle. Using samples from pre-pubertal calves, the CXCR4 protein was detected by immunohistochemistry in a few cells of the seminiferous tubules. Testicular cells were isolated, frozen-thawed and submitted to magnetic-activated cell sorting using anti-CXCR4 antibody. Quantitative RT-PCR analysis revealed that CXCR4+ cells had THY1, OCT4 and ZBTB16 (or PLZF) mRNA in these cells. Flow cytometry results indicated that the proportion of THY1+ cells is enriched in CXCR4+ populations. Colonization potential of CXCR4+ cells was assessed after xenotransplantation into testes of nude mice treated with busulfan. Transplantation of CXCR4+ cells yielded an increase of 5.4-fold when compared to CXCR4- cells. These results indicate that CXCR4 could be used as a marker to enrich and sort cells of bulls with putative spermatogonial stem cell potential.

Bufalin inhibits cell proliferation and migration of hepatocellular carcinoma cells via APOBEC3F induced intestinal immune network for IgA production signaling pathway

ObjectiveThis study aimed to evaluate functions of APOBEC3F gene in biological process of hepatocellular carcinoma (HCC) and anti-tumor mechanisms of bufalin. MethodsEffect of APOBEC3F and bufalin on cell proliferation and migration abilities were evaluated by CCK-8, wounding healing tests and transwell assays in SK-Hep1 and Bel-7404 cells. Bioinformatic analysis were also used to compare APOBEC3F expression levels, detect coexpressed genes and enrichment of pathways. ResultsAPOBEC3F was overexpressed in tumor tissues compared to adjacent tissues in HCC patients. And, APOBEC3F promotes cell proliferation and migration in SK-Hep1 and Bel-7404 cells. Bufalin inhibits cell proliferation and migration and reduces APOBEC3F expression. GO and KEGG enrichment of APOBEC3F-coexpressed genes revealed that APOBEC3F might active intestinal immune network for IgA production signaling pathway, leading to malignant biological behaviors of HCC cells. Additionally, siAPOBEC3F could decrease pIgR, CCR9, CCR10 and CXCR4 protein levels. And, bufalin inhibits the pIgR, CCR9, CCR10 and CXCR4 protein expressions. ConclusionsBufalin inhibits cell proliferation and migration of HCC cells via APOBEC3F induced intestinal immune network for IgA production signaling pathway.

Targeting Chemokine Receptor CXCR4 and Translocator Protein for Characterization of High-Risk Plaque in Carotid Stenosis Ex Vivo.

Background and Purpose- This pilot study aims to demonstrate the feasibility of targeting molecular characteristics of high-risk atherosclerotic plaque in symptomatic and asymptomatic carotid stenosis (CS), that is, upregulation of the translocator protein (TSPO) and the chemokine receptor type 4 (CXCR4), by means of molecular imaging. Methods- In a translational setting, specimens of carotid plaques of patients with symptomatic and asymptomatic CS obtained by carotid endarterectomy were analyzed for the presence of TSPO and CXCR4 by autoradiography, using the positron emission tomography tracers 18F-GE180 and 68Ga-Pentixafor and evaluated by histopathology. In addition, 68Ga-Pentixafor positron emission tomography/computed tomography was performed in a patient with high-grade CS. Results- Distinct patterns of upregulation of TSPO (18F-GE180 uptake) and CXCR4 (68Ga-Pentixafor uptake) were identified in carotid plaque by autoradiography. The spatial distribution was associated with specific histological hallmarks that are established features of high-risk plaque: TSPO upregulation correlated with activated macrophages infiltration, whereas CXCR4 upregulation also corresponded to areas of intraplaque hemorrhage. 68Ga-Pentixafor uptake was significantly higher in plaques of symptomatic compared with asymptomatic CS. Clinical positron emission tomography revealed marked 68Ga-Pentixafor uptake in carotid plaque of a patient with high-grade CS. Conclusions- Clinical imaging of molecular signatures of high-risk atherosclerotic plaque is feasible and may become a promising diagnostic tool for comprehensive characterization of carotid disease. This methodology provides a platform for future studies targeting carotid plaque.

Correlation of CXCR4/CXCR7 signaling pathway with pre-eclampsia.

The possible role of C-X-C chemokine receptor type 4 (CXCR4)/CXCR7 signaling pathway in pre-eclampsia (PE) was investigated to study the expression of CXCR4/CXCR7 in serum of PE patients and its correlation and relationship with PE. Twenty patients with mild PE and 40 cases with severe PE enrolled into the PE group and 60 cases of the normal pregnancy group during the same period were selected as the objects of study. The changes in expression of serum CXCR4 messenger ribonucleic acid (mRNA) and CXCR7 mRNA were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and CXCR4/CXCR7 protein concentration in serum was determined by enzyme-linked immunosorbent assay (ELISA). The pregnancy termination time of gravid was earlier in the severe PE group than that in the normal pregnancy group (P<0.05); the mean arterial pressure (MAP), systolic pressure, diastolic pressure were higher in patients with mild and severe PE than those in the normal pregnancy group (P<0.05). The results of RT-qPCR showed that the mRNA expression of serum CXCR4 and CXCR7 in PE patients were distinctly higher than those in the normal pregnancy group (P<0.05). The expression level of CXCR4 mRNA was positively correlated with that of CXCR7 mRNA (r=0.567, P=0.02). The results of ELISA displayed that the content of CXCR4/CXCR7 in serum of patients with PE was remarkably higher than that in the normal pregnancy group (P<0.05); the expression of serum CXCR4/CXCR7 in patients with severe PE was higher than in those with mild PE (P<0.05). The expression level of serum CXCR4 protein was positively correlated with that of CXCR7 protein (r=0.563, P=0.01). The expression level of CXCR4/CXCR7 may be closely related to the formation of PE.

The role of lncRNA XIST/miR-211 axis in modulating the proliferation and apoptosis of osteoarthritis chondrocytes through CXCR4 and MAPK signaling

Long noncoding RNAs (lncRNAs) participate in multiple diverse diseases, including osteoarthritis (OA). Here, we explored the role of lncRNA XIST in OA and identified the potential molecular mechanisms. The expression of XIST in cartilage samples in patients with OA was significantly upregulated. XIST knockdown remarkably suppressed IL-1β-suppressed OA chondrocyte proliferation while promoted IL-1β-induced cell apoptosis. By employing online tools, miRNAs related to CXCR4, a major contributor to chondrocyte apoptosis, and XIST were selected. miR-211 expression could be significantly inhibited by IL-1β stimulation, and miR-211 negatively regulated XIST expression and CXCR4 protein levels. Through direct binding, XIST served as a ceRNA for miR-211 to counteract miR-211-mediated CXCR4 repression, thereby modulating chondrocyte proliferation and apoptosis through downstream MAPK signaling. In OA tissues, miR-211 expression was significantly downregulated while CXCR4 mRNA expression was upregulated. miR-211 was negatively correlated with XIST and CXCR4, respectively, while XIST and CXCR4 was positively correlated in tissue samples. In conclusion, the study revealed that lncRNA XIST can promote the proliferation of OA chondrocytes and promote apoptosis through the miR-211/CXCR4 axis. Thus, lncRNA XIST might be considered as a potential therapeutic target for OA treatment.

Alternative splicing of helicase-like transcription factor (Hltf): Intron retention-dependent activation of immune tolerance at the feto-maternal interface.

Hltf is regulated by intron retention, and global Hltf-deletion causes perinatal lethality from hypoglycemia. In heart, full-length Hltf is a transcriptional regulator of Hif-1α that controls transport systems. Thus, we tested the hypothesis that Hltf deletion from placenta caused or exacerbated neonatal hypoglycemia via Hif-1α regulation of nutrient transporters. RNA-seq data analyses identified significant changes in transcript expression and alternative splicing (AS) in E18.5 placentome. iPathwayGuide was used for gene ontology (GO) analysis of biological processes, molecular functions and cellular components. Elim pruning algorithm identified hierarchical relationships. The methylome was interrogated by Methyl-MiniSeq Epiquest analysis. GO analysis identified gene enrichment within biological processes. Protein expression was visualized with immunohistochemistry. Although two Hltf mRNA isoforms are quantifiable in most murine tissues, only the truncated Hltf isoform is expressed in placenta. The responsible intron retention event occurs in the absence of DNA methylation. iPathwayGuide analysis identified 157 target genes of 11,538 total genes with measured expression. These were obtained using a threshold of 0.05 for statistical significance (p-value) and a long fold change of expression with absolute value of at least 0.6. Hltf deletion altered transcription of trophoblast lineage-specific genes, and increased transcription of the Cxcr7 (p = 0.004) gene whose protein product is a co-receptor for human and simian immunodeficiency viruses. Concomitant increased Cxcr7 protein was identified with immunolabeling. Hltf deletion had no effect on transcription or site-specific methylation patterns of Hif-1α, the major glucose transporters, or System A amino acid transporters. There was no measureable evidence of uteroplacental dysfunction or fetal compromise. iPathGuide analysis revealed Hltf suppresses cytolysis (10/21 genes; p-value 1.900e-12; p-value correction: Elim pruning; GO:019835) including the perforin-granzyme pathway in uterine natural killer cells. Our findings 1) prove the truncated Hltf protein isoform is a transcription factor, 2) establish a functional link between AS of Hltf and immunosuppression at the feto-maternal interface, 3) correlate intron retention with the absence of DNA methylation, and 4) underscore the importance of differential splicing analysis to identify Hltf’s functional diversity.

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.