Overexpression Of Insulin-like Growth Factor-binding Protein-3 Research Articles

IGFBP3 induces PD-L1 expression to promote glioblastoma immune evasion

BackgroundGlioblastoma (GBM) characterized by immune escape is the most malignant primary brain tumors, which has strong immunosuppressive effect. Programmed death ligand-1 (PD-L1) is a recognized immunosuppressive member on the surface of tumor cells, and plays a crucial role in immune evasion of tumors. Actually, little is known about the regulation of PD-L1 expression in GBM. Insulin-like growth factor binding protein 3 (IGFBP3) is upregulated in GBM and is related to poor patient prognosis. However, it remains unclear whether IGFBP3 plays a role in the regulation of PD-L1 expression in GBM.MethodsThe role of IGFBP3 in the glioma immune microenvironment was investigated using the CIBERSORT algorithm. The correlation between IGFBP3 and PD-L1 expression was analyzed using TCGA and CGGA databases. QRT-PCR, immunoblotting and RNA-seq were used to examine the regulatory effect of IGFBP3 on PD-L1 expression. Co-culture assay, cell counting kit (CCK-8), qRT-PCR, ELISA and flow cytometry were performed to explore the function of IGFBP3 in inducing immunosuppression. The biological role of IGFBP3 was verified using immunohistochemical, immunofluorescence and mice orthotopic tumor model.ResultsIn this study, we analyzed immune cells infiltration in gliomas and found that IGFBP3 may be associated with an immunosuppressive microenvironment. Then, by analyzing TCGA and CGGA databases, our results showed that IGFBP3 and PD-L1 expression were positively correlated in GBM patients, but not in LGG patients. In vitro experiments conducted on different GBM cell lines revealed that the overexpression of IGFBP3 led to an increase in PD-L1 expression, which was reversible upon knockdown IGFBP3. Mechanistically, IGFBP3 activated the JAK2/STAT3 signaling pathway, leading to an increase in PD-L1 expression. Additionally, co-culture experiments results showed IGFBP3 overexpression induced upregulation of PD-L1 expression promoted apoptosis in Jurkat cells, and this effect was blocked by IGFBP3 antibody and PDL-1 inhibitors. Importantly, in vivo experiments targeting IGFBP3 suppressed tumor growth and significantly prolonged the survival of mice.ConclusionsThis research demonstrated IGFBP3 is a novel regulator for PD-L1 expression in GBM, and identified a new mechanism by which IGFBP3 regulates immune evasion through PD-L1, suggesting that IGFBP3 may be a potential novel target for GBM therapy.

Secreted insulin-like growth factor binding protein 5 functions as a tumor suppressor and chemosensitizer through inhibiting insulin-like growth factor 1 receptor/protein kinase B pathway in acute myeloid leukemia

BackgroundIn addition to being secreted into the intercellular spaces by exocytosis, insulin-like growth factor binding protein 5 (IGFBP5) may also remain in the cytosol or be transported to the nucleus. Depending on the different cellular context and subcellular distribution, IGFBP5 can act as a tumor suppressor or promoter through insulin-like growth factor -dependent or -independent mechanisms. Yet, little is known about the impacts of IGFBP5 on acute myeloid leukemia (AML) and its underlying mechanism. MethodsHere we investigated the roles of IGFBP5 in human AML by using recombinant human IGFBP5 (rhIGFBP5) protein and U937 and THP1 cell lines which stably and ectopically expressed IGFBP5 or mutant IGFBP5 (mtIGFBP5) with the lack of secretory signal peptide. Cell counting kit-8 and flow cytometry assay were conducted to assess the cell viability, cell apoptosis and cell cycle distribution. Cytotoxicity assay was used to detect the chemosensitivity. Leukemia xenograft model and hematoxylin-eosin staining were performed to evaluate AML progression and extramedullary infiltration in vivo. ResultsIn silico analysis demonstrated a positive association between IGFBP5 expression and overall survival of the AML patients. Both IGFBP5 overexpression and extrinsic rhIGFBP5 suppressed the growth of THP1 and U937 cells by inducing cell apoptosis and arresting G1/S transition and promoted the chemosensitivity of U937 and THP1 cells to daunorubicin and cytarabine. However, overexpression of mtIGFBP5 failed to demonstrate these properties. An in vivo xenograft mouse model of U937 cells also indicated that overexpression of IGFBP5 rather than mtIGFBP5 alleviated AML progression and extramedullary infiltration. Mechanistically, these biological consequences depended on the inactivation of insulin-like growth factor 1 receptor -mediated phosphatidylinositol-3-kinase/protein kinase B pathway. ConclusionsOur findings revealed secreted rather than intracellular IGFBP5 as a tumor-suppressor and chemosensitizer in AML. Upregulation of serum IGFBP5 by overexpression or addition of extrinsic rhIGFBP5 may serve as a suitable therapeutic approach for AML.

Porcine IGFBP3 promotes porcine circovirus type 2 replication via PERK/eIF2α mediated DNA damage

The infection of porcine circovirus type 2 (PCV2) triggers activation of the protein kinase RNA–like endoplasmic reticulum kinase (PERK) pathway and leads to DNA damage. Insulin-like growth factor-binding protein 3 (IGFBP3) may interact with the endoplasmic reticulum (ER). It remains unclear whether IGFBP3 regulates DNA damage via ER stress to mediate PCV2 replication. In this study, we observed an upregulation of porcine IGFBP3 expression during PCV2 infection, and overexpression of IGFBP3 enhanced the expression of PCV2 Cap protein, PCV2 DNA copy number, and viral titers in PK-15 B6 cells and 3D4/21 cells. Additionally, overexpression of IGFBP3 induced an increase in the DNA damage marker γH2AX by activating the PERK/eIF2α pathway without concomitant activation of ATF4, IRE1α, and ATF6α/GRP78 pathways in PK-15 B6 cells and 3D4/21 cells. Knockdown of IGFBP3 had a reverse effect on PCV2 replication in PK-15 B6 cells and 3D4/21 cells. Furthermore, treatment with etoposide enhanced PCV2 replication while KU57788 decreased it. GSK2606414 and salubrinal limited both DNA damage and viral replication. Therefore, our findings suggest that porcine IGFBP3 promotes PCV2 replication through the PERK/eIF2α pathway-mediated induction of DNA damage in PK-15 B6 cells and 3D4/21 cells. Our study provides a basis for exploring novel antiviral strategies via the extensive understanding of the relationships between host cellular proteins and viral replication.

Chordin Like-1 Regulates Osteoblast and Adipocyte Differentiation Through Stabilizing Insulin-Like Growth Factor Binding Protein 3.

Chordin like-1 (CHRDL1) is an antagonist of bone morphogenetic proteins (BMPs) that acts through binding BMPs and blocking their interaction with BMP receptors. CHRDL1 plays a role in osteoblast differentiation but controversial effects were reported. On the other hand, the role of CHRDL1 in adipogenesis is unknown. In the present study, we investigated the function of CHRDL1 in regulating differentiation of osteoblasts and adipocytes and elucidated the underlying mechanism. CHRDL1 expression was downregulated during osteogenesis while it was upregulated during adipogenesis in primary cultured and established mesenchymal progenitor cell lines. Functional experiments revealed that CHRDL1 suppressed osteoblast differentiation and promoted adipocyte differentiation. Mechanistic explorations revealed that CHRDL1 is directly bound to insulin-like growth factor binding protein 3 (IGFBP3) and attenuated the degradation of the latter. Furthermore, CHRDL1 and IGFBP3 suppressed the activity of insulin receptor substrate 1 (IRS1)/AKT serine/threonine kinase (AKT)/mechanistic target of rapamycin kinase complex 1 (mTORC1) signaling in progenitor cells undergoing osteogenic differentiation. By contrast, they activated AKT/mTORC1 signaling independently of IRS1 during adipogenic differentiation. CHRDL1 enhanced the interaction of nuclear IGFBP3 and retinoid X receptor α (RXRα) during adipogenesis, and inhibition of RXR inactivated AKT and attenuated the stimulation of adipogenic differentiation by CHRDL1. Overexpression of IGFBP3 relieved the perturbation of osteogenic and adipogenic differentiation of progenitor cells induced by CHRDL1 silencing. Finally, CHRDL1 and IGFBP3 were upregulated in the trabecular bone of aged mice. Our study provides evidence that CHRDL1 reciprocally regulates osteoblast and adipocyte differentiation through stabilizing IGFBP3 and differentially modulating AKT/mTORC1 signaling.

Testosterone promotes human foreskin fibroblast growth through miR-143-3p targeting IGFBP-3

Testosterone is an important male hormone, which could improve the maintenance and recovery of gonadal function in males as well as the repair of human hypospadias and cell fibrosis. Our study focused on investigating the regulatory effect of testosterone in human foreskin fibroblasts (HFF-1) and regulatory mechanisms involved. In this study, HFF-1 cells were treated with testosterone, and cell viability and migration were assessed by cell counting kit-8 (CCK8) and Transwell assays. The expression levels of androgen receptor (AR), miR-143-3p and insulin-like growth factor binding protein-3 (IGFBP-3) were measured by quantitative real-time PCR (qRT-PCR), Western blotting, and immunofluorescence. In addition, a potential binding site for miR-143-3p on IGFBP-3 was predicted and its direct binding was further confirmed by a dual luciferase reporter assay. These results showed that testosterone increased the viability and migration of HFF-1 cells. Testosterone could down-regulate miR-143-3p and up-regulate IGFBP-3 and AR. Overexpression of miR-143-3p hindered HFF-1 cell viability and negatively regulated IGFBP-3, whereas inhibition of IGFBP-3 impeded cell viability and migration. Furthermore, miR-143-3p was found to directly bind to IGFBP-3. Overexpression of IGFBP-3 countered the regulation of HFF-1 cells by miR-143-3p mimics. In conclusion, this study showed that testosterone promoted the proliferation and migration of HFF-1 cells and AR signaling, at least via the miR-143-3p/IGFBP-3 axis. This discovery presents a novel insight for testosterone application in male disorders like hypospadias.

Co-amplified with PDGFRA, IGFBP7 is a prognostic biomarker correlated with the immune infiltrations of glioma.

A subgroup of glioma carry genetic 4q12 amplification including platelet derived growth factor receptor α (PDGFRA) and insulin like growth factor binding protein 7 (IGFBP7). However, the prognosis of PDGFRA and IGFBP7 in glioma is unclear. The prognosis of PDGFRA and IGFBP7 was determined using cox regression and Kaplan-Meier survival analysis. Pathways associated with IGFBP7 were analyzed through gene set enrichment analysis (GSEA). Immune profiling of glioma was determined using "ESTIMATE" and "TIMER" database. PDGFRA amplification or expression was not correlated with the outcomes of glioblastoma (GBM). IGFBP7 but not PDGFRA was over-expressed in GBM. IGFBP7 over-expression was correlated with the unfavorable outcomes of GBM. In lower grade glioma (LGG), PDGFRA over-expression was not correlated with the unfavorable prognosis of LGG, while, IGFBP7 was a prognostic biomarker of LGG. LGG patients with IGFBP7 lower expressions had prolonged clinical overall survival. Combination of IDH mutation, LGG grade and IGFBP7 achieved even better prognostic effects in LGG. Moreover, IGFBP7 was over-expressed in glioma patients with wild type IDH or with high grades. IGFBP7 over-expression was correlated with the unfavorable outcomes of glioma. Furthermore, IGFBP7 was hypo-methylated in GBM or LGG patients without IDH mutations. IGFBP7 hyper-methylation was correlated with the lower overall survival of GBM or LGG. LGG patients with wild type IDH and with IGFBP7 hypo-methylation demonstrated even worse prognosis. IGFBP7 was associated with multiple immune-related signaling pathways in GBM or LGG. The stromal score, immune score and the infiltrations of immune cells were also correlated with IGFBP7 and the prognosis of LGG. IGFBP7 but not PDGFRA served an ideal prognostic marker and therapeutic target of glioma.

Long non-coding RNA IGFBP7-AS1 accelerates the odontogenic differentiation of stem cells from human exfoliated deciduous teeth by regulating IGFBP7 expression

Stem cells from human exfoliated deciduous teeth (SHED) are attractive seed cells for dental tissue engineering. We identified the effect of the long noncoding RNA insulin-like growth factor-binding protein 7 antisense RNA 1 (lncRNA IGFBP7-AS1) in vivo and its underlying mechanism during SHED odontogenic differentiation. IGFBP7-AS1 and insulin-like growth factor-binding protein 7 (IGFBP7) were overexpressed using lentiviruses. IGFBP7 expression was knocked down with small interfering RNA. The effect of IGFBP7-AS1 in vivo was confirmed by animal experiments. The effect of IGFBP7 on SHED odontogenic differentiation was assessed with alkaline phosphatase staining, alizarin red S staining, quantitative reverse transcription-PCR, and western blotting. The relationship between IGFBP7-AS1 and IGFBP7 was confirmed by quantitative reverse transcription–PCR and western blotting. IGFBP7-AS1 promoted SHED odontogenesis in vivo, and regulated the expression of the coding gene IGFBP7 positively. Inhibiting IGFBP7 led to suppress SHED odontogenic differentiation while IGFBP7 overexpression had the opposite effect. IGFBP7-AS1 enhanced the stability of IGFBP7. IGFBP7-AS1 promoted SHED odontogenic differentiation in vivo. The underlying mechanism may involve the enhancement of IGFBP7 stability. This may provide novel potential targets for dental tissue engineering.

Abstract P2082: Igfbp-3 Activates Pro-survival Pathways To Mitigate Anthracycline-induced Cardiomyocyte Injury

Rationale: Anthracyclines such as doxorubicin are among the most effective chemotherapeutic agents; however, their use leads to frequent cardiovascular complications, including heart failure, due to the activation of multiple cardiomyocyte injury pathways. Insulin-like growth factor binding protein-3 (IGFBP3) is strongly upregulated in cardiomyocytes after administration of doxorubicin. However, its role in doxorubicin-induced cardiomyocyte apoptosis and survival remains controversial. Methods and Results: We demonstrate that IGFBP3 is strongly induced by doxorubicin in neonatal ventricular myocytes (NRVMs) 12 hours after doxorubicin treatment by RT-PCR (4.2 fold vs. control, n=3, P<0.05) and Western blot. Adenoviral overexpression of IGFBP3 in NRVMs leads to a significant reduction of phosphorylated H2A histone (γH2AX) after doxorubicin treatment, a hallmark of DNA double-strand breaks (DSBs), as well as topoisomerase IIβ, a key mediator of doxorubicin induced DNA DSBs. Conversely, silencing of IGFBP3 with siRNA leads to a significant increase of γH2AX expression in NRVMs. Adenoviral shRNA silencing of IGFBP3 in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) further demonstrates a 44% and 52% decrease in contraction magnitude and velocity in response to doxorubicin compared with scramble shRNA (scramble shRNA, n=5, shIGFBP3, n=3, P<0.05). Immunofluorescent imaging and cell fractionation assays conducted in NRVMs indicate that doxorubicin initiates IGFBP3 nuclear translocation, suggesting an active role in the cardiomyocyte response to doxorubicin-induced cell injury. Next generation sequencing of hiPSC-CMs treated with doxorubicin reveals that overexpression of IGFBP3 leads to upregulation of cardiomyocyte survival pathways, including TNF receptors without cytoplasmic death domain (TNFRSF10C and D), BCL2 family gene BCL2L1, the p53 target ACER2, the p53 co-factor FBXW7, the β-catenin mediator TBX3 and the ataxia telangiectasia mutated (ATM) activator ZNF423. Conclusion: Our findings indicate that IGFBP3 is strongly induced in cardiomyocytes by doxorubicin treatment. Overexpression of IGFBP3 attenuates doxorubicin induced DNA DSBs and promotes pro-survival signaling pathways.

MiR-133a-5p Inhibits Glioma Cell Proliferation by Regulating IGFBP3.

Objective This research aims to investigate the expression of miR-133a-5p in glioma tissues and its impact on glioma cell proliferation. Methods Fluorescence-quantitative PCR was used to detect the expression of miR-133a-5p in 25 cases of glioma and adjuncent tissues. CCK-8 and colony formation analyses were used to evaluate the impact of transfection with miR-133a-5p inhibitors or mimics on glioma cell growth and colony formation. The IGFBP3 (insulin-like growth factor-binding protein-3) and miR-133a-5p binding sites were predicted using Starbase, and the miR-133a-5p binding capacity with 3'UTR of IGFBP3 gene was determined using a luciferase gene reporter system. Following transfection with miR-133a-5p mimics or inhibitors, the IGFBP3 protein expression in glioma cells was determined by western blotting. The colony formation assay was applied to evaluate the influence of IGFBP3 overexpression on the miR-133a-5p in glioma cell proliferation. For assessment of the IGFBP3 expression in glioma tissues and prognosis, TCGA database was employed. Results The expression of miR-133a-5p was considerably reduced in glioma tissue compared to adjuncent control tissue. In addition, miR-133a-5p expression decreased with increasing glioma malignancy. Glioma cell growth and colony formation were reduced after miR-133a-5p mimics were transfected, while transfection of miR-133a-5p inhibitors had a reverse impact. The expression of IGFBP3 was affected by miR-133a-5p by binding to its 3'UTR region. Additional study demonstrated that the overall survival (OS) of subjects with increased IGFBP3 expression was considerably lower compared to patients with decreased IGFBP3 expression. The IGFBP3 overexpression effectively counteracts the glioma cell proliferation-inhibiting impact of miR-133a-5p. Conclusion miR-133a-5p acts as a glioma tumor suppressor gene. It reduces glioma cell proliferation by modulating IGFBP3 and could be a target for glioma therapy.

Diallyl Trisulfide Suppresses High-Glucose-Induced Cardiomyocyte Apoptosis by Targeting Reactive Oxygen Species-Mediated Hypoxia-Inducible Factor-1α/Insulin-like Growth Factor Binding Protein 3 Activation.

It has been reported that 80% of diabetic patients die due to cardiovascular diseases. We previously demonstrated that activated hypoxia-inducible factor-1α (HIF-1 α)/insulin-like growth factor binding protein-3 (IGFBP-3) signaling by reactive oxygen species (ROS)-regulated prolyl hydroxylase domain-containing protein (PHD) is involved in high-glucose (HG)-induced cardiac apoptosis. Diallyl trisulfide (DATS), a garlic component, shows the strongest inhibitory effect on diabetic cardiomyopathy. In this study, we investigated whether HIF-1α/IGFBP-3 signaling governs the antiapoptotic effect by DATS on HG-exposed cardiomyocytes. It was observed that significantly increased levels of cell apoptosis and decreased Akt phosphorylation were reversed by DATS in HG-exposed cardiac cells. H2O2 and PHD small interfering RNA treatments increased HIF-1α and IGFBP-3 protein levels, which were decreased by DATS treatment. Overexpression of HIF-1α and IGFBP-3 increased HG-induced cell apoptosis, which was suppressed by DATS. The coimmunoprecipitation assay results showed that DATS not only increased the IGF-1 level and reduced IGFBP-3 level but also suppressed their extracellular association for cardiac cells exposed to HG. Experiments using neonatal cardiomyocytes and hearts showed similar results. These findings indicate that the effect of ROS-regulated PHD on the activation of HIF-1α/IGFBP-3 signaling governs the antiapoptotic effect by DATS on HG-exposed cardiomyocytes.

IGFBP7 overexpression promotes acquired resistance to AZD9291 in non-small cell lung cancer

AZD9291 (osimertinib) is the third-generation EGFR-TKI treat for EGFR mutated NSCLC patients. Despite its encouraging efficacy in clinical, acquired resistance is still inevitable. The mechanism of drug resistance needs to be further explored. In a previous study, we established an AZD9291-resistant cell strain named HCC827/AZDR. We found that insulin-like growth factor binding protein 7 (IGFBP7) expression was markedly increased in HCC827/AZDR cells and AZD9291-resistant patients by RNA sequencing and immunohistochemical analysis, respectively. Reduced IGFBP7 in HCC827/AZDR cells by si-RNA interference recovered the sensitivity to AZD9291 partially and increased AZD9291-induced cell apoptosis. Enhancing IGFBP7 expression in EGFR-mutated non-small cell lung cancer (NSCLC) cells using lentiviruses infection reduced their sensitivity to AZD9291. This study is the first to discover that high IGFBP7 expression could occur following treatment with AZD9291. This might be one of the mechanisms underlying AZD9291 resistance and a potential therapeutic target following AZD9291 resistance.

Insulin-Like Growth Factor Binding Protein-3 Exerts Its Anti-Metastatic Effect in Aerodigestive Tract Cancers by Disrupting the Protein Stability of Vimentin.

Simple SummaryLocal invasion and distal metastasis are the main causes of cancer-related death and the poor prognosis of patients with aerodigestive tract cancers. Therefore, understanding the biology of invasion and metastasis is important for the development of effective therapeutic strategies. The present study shows that insulin-like growth factor binding protein-3 (IGFBP-3) inhibits the migration and invasion of non-small cell lung cancer (NSCLC) and head and neck squamous cell carcinoma (HNSCC) cells in vitro and the development of metastasized tumors in vivo. Mechanistic studies suggest vimentin as a cellular target for the antimetastatic effect of IGFBP-3. These results contribute to a better understanding on the regulation of metastasis of cancer cells, providing the rationale to utilize IGFBP-3 as an effective therapeutic strategy targeting migration and metastasis of aerodigestive tract cancers.The proapoptotic, antiangiogenic, and antimetastatic activities of insulin-like growth factor binding protein-3 (IGFBP-3) through IGF-dependent or -independent mechanisms have been suggested in various types of human cancers. However, a mechanistic explanation of and downstream targets involved in the antimetastatic effect of IGFBP-3 is still lacking. In this study, by applying various in vitro and in vivo models, we show that IGFBP-3 suppresses migration and invasion of human head and neck squamous carcinoma (HNSCC) and non-small cell lung cancer (NSCLC) cells. Silencing IGFBP-3 expression elevated the migration and invasion of NSCLC and HNSCC cells in vitro and their local invasion and metastasis in vivo, whereas overexpression of IGFBP-3 decreased such prometastatic changes. Local invasion of 4-nitroquinoline-1-oxide (4-NQO)-induced HNSCC tumors was consistently significantly potentiated in Igfbp3 knockout mice compared with that in wild-type mice. Mechanistically, IGFBP-3 disrupted the protein stability of vimentin via direct binding and promoting its association with the E3 ligase FBXL14, causing proteasomal degradation. The C-terminal domain of IGFBP-3 and the head domain of vimentin are essential for their interaction. These results provide a molecular framework for IGFBP-3′s IGF-independent antimetastatic and antitumor activities.

Insulin-Like Growth Factor Binding Protein 5: an Important Regulator of Early Osteogenic Differentiation of hMSCs

Insulin-like growth factor binding protein 5 (IGFBP5) is broadly bioactive, but its role in osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) remains to be clarified. Here, we demonstrated that IGFBP5 expression was markedly increased during the early osteogenic differentiation of hMSCs. We then over-expressed and knocked down this gene in hMSCs and evaluated the impact of manipulation of IGFBP5 expression on osteogenic differentiation based upon functional assays, ALP staining, and expression of osteogenic markers. Together, these analyses revealed that IGFBP5 over-expression enhanced early osteogenic differentiation, as evidenced by increased ALP staining and osteogenic marker induction, whereas knocking down this gene impaired the osteogenic process. Over-expression of IGFBP5 also markedly bolstered the extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation level, while IGFBP5 knockdown suppressed this signalling activity. We additionally compared the impact of simultaneous IGFBP5 overexpression and ERK1/2 inhibitor treatment to the effect of IGFBP5 over-expression alone in these hMSCs, revealing that small molecule-mediated EKR1/2 inhibition was sufficient to impair osteogenic differentiation in the context of elevated IGFBP5 levels. These findings indicated that IGFBP5 drives the early osteogenic differentiation of hMSCs via the ERK1/2 signalling pathway. Our results offer value as a foundation for future efforts to study and treat serious bone-related diseases including osteoporosis.

Overexpression of IGFBP5 Enhances Radiosensitivity Through PI3K-AKT Pathway in Prostate Cancer.

BackgroundRadiotherapy is the main treatment for localized prostate cancer. The therapeutic effects of radiotherapy are highly dependent on radiosensitivity of target tumors. Here, we investigated the impact of insulin-like growth factor-binding protein 5 (IGFBP5) on irradiation therapy in prostate cancer.MethodsIGFBP5 gene was overexpressed in human prostate cancer cell lines, PC3 and DU145, with transfection of lentivirus expression vector. Radiosensitivity of the cell lines was assessed with colony formation, cell cycle and cell proliferation assays. The expression of proteins associated with the PI3K-AKT pathway was determined by Western blotting. The effect of IGFBP5 knockdown on PI3K-AKT pathway was tested using PI3K inhibitor.ResultsHigher expression of IGFBP5 improved the efficacy of radiotherapy for prostate cancer patients. The effects of IGFBP5 were linked to the PI3K-AKT signaling pathway. Overexpression of IGFBP5 enhanced radiosensitivity and induced G2/M phase arrest in prostate cancer cells. In contrast, it decreased PI3K, p-AKT expression and cell viability. These effects were reversed by IGFBP5 knockdown.ConclusionOur results reveal that IGFBP5 regulates radiosensitivity in prostate cancer via the PI3K-AKT pathway. It is, therefore, a potential biomarker of tumors that influences the therapeutic effect of radiotherapy.

IGFBP5 promotes angiogenic and neurogenic differentiation potential of dental pulp stem cells.

Dental stem cells for dental pulp regeneration have become a new strategy for pulpitis treatment. Angiogenesis and neurogenesis play a vital role in the pulp-dentin complex regeneration, and appropriate growth factors will promote the process of angiogenesis and neurogenesis. Insulin-like growth factor-binding protein 5 (IGFBP5) is involved in the regulation of tooth growth and development. A previous study showed that IGFBP5 enhanced osteo/odontogenic differentiation of dental stem cells. Our research intends to reveal the function of IGFBP5 in the angiogenic and neurogenic differentiation of human dental stem cells. Human dental pulp stem cells (DPSCs) were used in the present study. Lentiviral IGFBP5 shRNA was used to silence the IGFBP5. Retroviruses expressing Wild-type IGFBP5 were used to over-express IGFBP5. Angiogenic and neurogenic differentiation were carried out by in vitro study. Real-time RT-PCR and western blot results showed that over-expression of IGFBP5 upregulated the expressions of angiogenic markers, including VEGF, PDGFA and ANG-1, and neurogenic markers, including NCAM, TH, Nestin, βIII-tubulin, and TH, in DPSCs. Moreover, microscope observation confirmed that over-expression of IGFBP5 enhanced neurosphere formation in DPSCs in size and amount. Immunofluorescence staining results showed that over-expression of IGFBP5 also prompted the percentage of Nestin and βIII-tubulin positive neurospheres in DPSCs. While depletion of IGFBP5 downregulated the expressions of VEGF, PDGFA, ANG-1, NCAM, TH, Nestin, βIII-tubulin, and TH, it decreased the neurosphere formation and percentage of Nestin and βIII-tubulin positive neurospheres in DPSCs. In conclusion, our results revealed that IGFBP5 promoted angiogenic and neurogenic differentiation potential of DPSCs in vitro and provided the possible potential target for enhancing directed differentiation of dental stem cells and dental pulp-dentin functional regeneration.

Downregulated IGFBP7 facilitates liver metastasis by modulating epithelial‑mesenchymal transition in colon cancer.

Distant metastasis is a major cause of cancer-associated mortality in patients with colon cancer. Insulin-like growth factor binding protein 7 (IGFBP7) has been identified as a crucial inhibitor of human cancer. However, the role of IGFBP7 in the pathogenesis of metastatic colon cancer has not been investigated. In the present study, the expression of IGFBP7 in 81 pairs of colon cancer tissues and adjacent normal tissues were investigated using immunohistochemistry. Furthermore, 24 pairs of primary colon cancer and matched liver metastasis tissues were analyzed. LοVο cells with IGFBP7-knockdown and HT-29 cells with IGFBP7-overexpression were employed. The expression levels of E-cadherin, N-cadherin and Vimentin were quantified and compared. Significant alterations in the expression of IGFBP7 between late stage (III + IV) colon cancer and adjacent normal colonic mucosa were observed. (P=0.031). The association between IGFBP7 and epithelial-mesenchymal transition (EMT) markers were validated in primary colon cancer and matched liver metastasis tissues. The invasive front of liver metastatic colon tissues revealed reduced IGFBP7 expression. Additionally, knockdown of IGFBP7 in LοVο cells resulted in decreased E-cadherin, and increased N-cadherin and Vimentin expression compared with the control group. Overexpression of IGFBP7 in HT-29 cells induced an upregulation of E-cadherin; however, the N-cadherin and Vimentin levels were decreased. In conclusion, the results of the present study suggested that IGFBP7 may prevent colon cancer metastasis by inhibiting EMT, and serves as a potential diagnostic marker and therapeutic target for patients with colon cancer.

CircPIP5K1A serves as a competitive endogenous RNA contributing to ovarian cancer progression via regulation of miR-661/IGFBP5 signaling.

Increasing evidence demonstrates the crucial regulatory functions of circular RNAs in different cancer types. The major aim of the current study was to establish functions of circPIP5K1A during ovarian cancer. Our results showed an increased expression of circPIP5K1A in both ovarian cancers and cell lines, which was associated with poor prognosis. In functional analyses, downregulation of circPIP5K1A suppressed ovarian cancer cell migration, proliferation, and invasion in vitro. The miR-661 was indicated as a target of circPIP5K1A and insulin-like growth factor-binding protein 5 (IGFBP5) as a target of miR-661. circPIP5K1A silencing triggered downregulation of IGFBP5 through inducing an increase in miR-66 levels, as determined by the luciferase reporter assay. Data from cell counting kit-8, colony formation, wound healing, and Transwell assays showed that overexpression of IGFBP5 effectively reversed the circPIP5K1A depletion effects. The results collectively indicated that circPIP5K1A contributed to ovarian cancer progression via targeting the miR-661/IGFBP5 axis, supporting its utility as a candidate target for therapy of the disease.

IGFBP7 inhibits cell proliferation by suppressing AKT activity and cell cycle progression in thyroid carcinoma

BackgroundThyroid cancer is the most common malignant endocrine tumor and is classified into papillary thyroid cancer (PTC), follicular thyroid cancer (FTC) and anaplastic thyroid cancer (ATC), which have substantially different characteristics. Insulin-like growth factor binding protein 7 (IGFBP7) has recently been recognized as a tumor suppressor in many cancer types. However, the expression pattern of IGFBP7 and its biological function in various types of thyroid carcinoma remain poorly understood.ResultsWe found that the protein levels of IGFBP7 in FTC and ATC tissues were significantly lower or even absent compared with those in normal thyroid, benign thyroid adenoma and classical PTC tissues. Moreover, overexpression of IGFBP7 in two undifferentiated ATC cell lines, ARO and FRO, and one differentiated FTC cell line, WRO, significantly inhibited cell proliferation in vitro. In vivo experiments revealed that ectopic IGFBP7 expression markedly suppressed growth of tumor xenografts derived from these thyroid cancer cell lines, while IGFBP7 silencing accelerated tumor growth. At the mechanistic level, overexpression of IGFBP7 dramatically suppressed phosphorylation-mediated activation and kinase activity of AKT, causing an upregulation of cyclin-dependent kinase (CDK) inhibitors p27Kip1 and p21Cip1 and induction of G1/S cell cycle arrest, while silencing IGFBP7 exerted the opposite effects.ConclusionsIGFBP7 expression is decreased or even absent in FTC and ATC. Acting as a cell cycle repressor, IGFBP7 plays an important tumor-suppressive role in human thyroid cancer, especially in FTC and ATC subtypes and may represent a promising biomarker and therapeutic target for human thyroid cancer treatment.

IGFBP7 contributes to epithelial-mesenchymal transition of HPAEpiC cells in response to radiation.

Radiation-induced lung injury (RILI) frequently occurs in patients with thoracic malignancies. In response to radiation, alveolar epithelial cells (AEC) undergo epithelial-mesenchymal transition (EMT) and contribute to the pathogenesis of RILI. Insulin-like growth factor binding protein 7 (IGFBP7) is reported as a downstream mediator of transforming growth factor-β1 (TGF-β1) pathway, which plays a crucial role in radiation-induced EMT. In the present study, the levels of IGFBP7 and TGF-β1 were simultaneously increased in experimental RILI models and radiation-treated AEC (human pulmonary alveolar epithelial cells [HPAEpic]). The expression of IGFBP7 in radiation-treated HPAEpic cells was obviously inhibited by the specific inhibitor of TGF-β receptor antagonist SB431542 and TGF-β1 neutralizing antibody, and time-dependently enhanced by TGF-β1 treatment. Moreover, IGFBP7 knockdown significantly attenuated the effects of radiation on morphology change, cell migration, expression of EMT-related markers (E-cadherin, α-SMA, and Vimentin), and phosphorylation of extracellular-signal-regulated kinase (ERK). The effects of IGFBP7 overexpression on the expression of EMT-related markers were partially reversed by the ERK inhibitor PD98059. In conclusion, IGFBP7, was enhanced by TGF-β1, may be involved in radiation-induced EMT of AEC via the ERK signaling pathway, thus contributing to the pathogenesis of RILI.

Insulin-like Growth Factor Binding Protein 7 Activates the Retinoid Acid Differentiation Pathway in Acute Myeloid Leukemia Cells

Acute myeloid leukemia (AML) is characterized by the accumulation of malignant blasts with impaired transcriptional differentiation programs. Despite important advances in AML therapy, the five-year overall survival rate of AML patients remains a disappointing 30-40%. This poor prognosis is mainly caused by survival of chemotherapy resistant leukemic cells, named leukemic stem cells (LSC), re-initiating relapse. However, for AML patients with PML-RARA positive acute promyelocytic leukemia (APL), treatment with all trans retinoic acid (ATRA) results in cure rates of >90%. Upon ATRA treatment, APL cells can restore transcription leading to granulocytic differentiation, and in combination with arsenic trioxide APL cells go into apoptosis. While the success of ATRA treatment has been demonstrated for APL patients, so far it has not proved effective for non-APL AML patients.Previously, we demonstrated that insulin-like growth factor binding protein 7 (IGFBP7), a negative regulator of IGF1 receptor (IGF1R) activity, induces apoptosis of AML cells and sensitizes AML cells to chemotherapy-induced cell death. Since it has been shown that IGF1R inhibitors can eliminate therapy-resistant cells by modifying their chromatin state, we hypothesized that IGFBP7 may also have the capacity to modify an epigenetic state and unlock the ATRA-driven differentiation response. To this end, we generated APL cell lines with down- or upregulated IGFBP7 levels and demonstrated that knockdown of IGFBP7 in NB4 cells blocked ATRA-induced differentiation, whereas overexpression of IGFBP7 led to an 8-fold increase in differentiation in the presence of low concentrations of ATRA, together suggesting a role for IGFBP7 in ATRA-induced differentiation in APL cells.Strategies to increase efficacy of ATRA-based therapy might also improve treatment outcomes for non-APL AML, and therefore we investigated the potential of IGFBP7 to induce susceptibility for ATRA-driven differentiation in this group. ATRA and IGFBP7 treatment of non-APL AML cell lines and primary AML cells derived from patients at diagnosis demonstrated an enhanced efficiency of the combination therapy to induce differentiation of myeloid CD45dimCD33+ AML cells (2.5-fold increase in CD11b-expression) and/or to reduce viability of AML CD45dim cells as compared to single treatments (from 30-39% to 70% reduction upon IGFBP7, ATRA or combination therapy, respectively), in 50% of tested primary AML samples, while this combination therapy did not influence normal hematopoietic cell survival. Remarkably, ATRA-IGFBP7 treatment diminished the in vivo engraftment potential of primary AML cells as compared to treatment with either drug alone in NSG mice (from 28% and 52% to 17% engraftment upon ATRA, IGFBP7 or combination therapy, respectively). Re-transplantation of human AML derived from first transplanted mice into secondary recipients demonstrated that the ATRA-IGFBP7 combination treatment also eliminated LSC more effectively (1.4-fold reduction). Together these data suggest that IGFBP7 enhances sensitivity of AML (stem) cells to ATRA, and is able to induce a transcriptional program sensitizing AML cells for ATRA-induced differentiation and cell death.To identify factors responsible for IGFBP7-induced ATRA sensitivity, we performed gene expression profiling of primary AML samples treated with IGFBP7, and identified growth factor independent protein 1 (GFI1) as one of the top down-regulated genes upon IGFBP7 stimulation. As overexpression of GFI1 in non-APL AML patient samples resulted in a >2.5-fold reduction in IGFBP7-induced susceptibility to ATRA-driven differentiation, low GFI1 expression is suggested to be associated with susceptibility to ATRA in AML cells.In conclusion, our results indicate that treatment of AML patient with a combination of ATRA and IGFBP7 might be successful in preventing relapse and improving AML patient survival, which has to be confirmed in further clinical studies. DisclosuresOssenkoppele:Karyopharm: Consultancy, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Jazz: Consultancy, Honoraria; Genentech: Consultancy, Honoraria; Roche: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Genmab: Research Funding; Celgene: Honoraria, Research Funding; Johnson & Johnson: Consultancy, Honoraria, Research Funding.