Id1 enhances human ovarian cancer endothelial progenitor cell angiogenesis via PI3K/Akt and NF-κB/MMP-2 signaling pathways

Su, Yajuan; Gao, Lingjuan; Teng, Lichen; Wang, Ying; Cui, Jialin; Peng, Shiyun; Fu, Songbin

doi:10.1186/1479-5876-11-132

Id1 enhances human ovarian cancer endothelial progenitor cell angiogenesis via PI3K/Akt and NF-κB/MMP-2 signaling pathways

Research
Open access
Published: 29 May 2013

Volume 11, article number 132, (2013)
Cite this article

Download PDF

You have full access to this open access article

Journal of Translational Medicine Aims and scope Submit manuscript

Id1 enhances human ovarian cancer endothelial progenitor cell angiogenesis via PI3K/Akt and NF-κB/MMP-2 signaling pathways

Download PDF

Yajuan Su¹,
Lingjuan Gao²,
Lichen Teng¹,
Ying Wang¹,
Jialin Cui¹,
Shiyun Peng¹ &
…
Songbin Fu³

4992 Accesses
Explore all metrics

Abstract

Background

Endothelial progenitor cells (EPCs) contribute to tumor angiogenesis and growth. We previously reported that over-expression of an inhibitor of DNA binding/differentiation 1 (Id1) in EPCs can enhance EPC proliferation, migration, and adhesion. In this study, we investigated the role of Id1 in EPC angiogenesis in patients with ovarian cancer and the underlying signaling pathway.

Methods

Circulating EPCs from 22 patients with ovarian cancer and 15 healthy control subjects were cultured. Id1 and matrix metalloproteinase-2 (MMP-2) expression were analyzed by real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blot. EPC angiogenesis was detected by tube formation assays. Double-stranded DNA containing the interference sequences was synthesized according to the structure of a pGCSIL-GFP viral vector and then inserted into a linearized vector. Positive clones were identified as lentiviral vectors that expressed human Id1 short hairpin RNA (shRNA).

Results

Id1 and MMP-2 expression were increased in EPCs freshly isolated from ovarian cancer patients compared to those obtained from healthy subjects. shRNA-mediated Id1 down-regulation substantially reduced EPC angiogenesis and MMP-2 expression. Importantly, transfection of EPCs with Id1 in vitro induced phosphorylation of Akt (p-Akt) via phosphoinositide 3-kinase and increased the expression of MMP-2 via NF-κB. Blockage of both pathways by specific inhibitors (LY294002 and PDTC, respectively) abrogated Id1-enhanced EPC angiogenesis.

Conclusions

Id1 can enhance EPC angiogenesis in ovarian cancer, which is mainly mediated by the PI3K/Akt and NF-κB/MMP-2 signaling pathways. Id1 and its downstream effectors are potential targets for treatment of ovarian cancer because of their contribution to angiogenesis.

Conditioned media from human ovarian cancer endothelial progenitor cells induces ovarian cancer cell migration by activating epithelial-to-mesenchymal transition

Article 23 October 2015

Linc-OIP5 in the breast cancer cells regulates angiogenesis of human umbilical vein endothelial cells through YAP1/Notch/NRP1 signaling circuit at a tumor microenvironment

Article Open access 11 February 2020

EphA1 activation promotes the homing of endothelial progenitor cells to hepatocellular carcinoma for tumor neovascularization through the SDF-1/CXCR4 signaling pathway

Article Open access 11 April 2016

Background

Tumor angiogenesis is recognized as a critical step in tumor progression through which an initially small, localized or non-invasive tumor gradually develops into a large, invasive, metastatic one. Previous studies have shown that bone marrow (BM)-derived EPCs participate in tumor angiogenesis, which accelerates tumor growth [1–3]. Furthermore, EPCs control the angiogenic switch in mouse lung metastasis [4]. Currently, the reasons for ovarian cancer EPC angiogenesis are poorly understood.

Inhibitors of differentiation 1 (Id1) belong to the helix loop helix (HLH) transcription factors family. Maw et al. [5] showed that the level of Id1 expression was positively related to the degree of malignancy in ovarian cancer. A study by Lyden et al. [6] confirmed that Id1 and Id3 played an important role in the vascular endothelial growth factor (VEGF) signal pathway, which is related to angiogenesis. In Id1 knock-out mice, it appeared that tumor growth was significantly inhibited due to an angiogenesis defect. BM-derived EPCs participated in the formation of new blood vessels [7], suggesting that EPCs have a close relationship with Id1. A recent report showed that tumor could induce high expression of Id1 in EPCs derived from BM but not in other cells, suggesting that Id1 might be a key factor for EPCs. A defect of Id1 in BM could lead to decreased numbers of EPCs in peripheral blood, block tumor angiogenesis, and further suppress tumor development [8]. Thus, Id1 may mediate angiogenesis of EPCs however, the mechanism is still poorly understood.

In a previous study, we used real-time RT-PCR to examine mRNA expression of Id1 in EPCs of 25 patients with ovarian cancer [9]. Western blot analysis revealed a higher Id1 expression in human ovarian cancer EPCs than in cells from 20 healthy controls. Compared to healthy controls, ovarian cancer patients showed increased migration and adhesion of EPCs. Statistical analyses revealed that ovarian cancer enhanced proliferation, migration, and adhesion of EPCs [9, Full size image

Id1 up-regulates MMP-2 via NF-κB in EPCs

MMP-2 and MMP-9 are MMPs that are relevant to angiogenic processes. We examined MMP-2 and MMP-9 expression levels of EPCs. Basal expression levels of MMP-2 and MMP-9 mRNA and protein were significantly increased in EPCs (Figure 4A-D). After the Id1-LV and Id1-RNAi-LV construct was transfected into EPCs, we analyzed EPC MMP-2 and MMP-9 expression levels. Id1-LV and Id1-RNAi-LV, respectively, markedly increased and reduced EPC mRNA expression of MMP-2, but not MMP-9. Compared to non-transfected control cells, expression of MMP-2 was significantly decreased by Id1 knock-down, as shown in Figure 4A-C.

Because MMP-2 and Id1 were correlated with each other in EPCs, we postulated that Id1 might control the expression of MMP-2 in EPCs via NF-κB. To test this hypothesis, EPCs were transfected with Id1, co-transfected with NF-κB and β-galactosidase reporters, and harvested for evaluation of NF-κB promoter activity by luciferase assays and of MMP-2 by western blot. Id1 significantly increased NF-κB promoter activity, whereas PDTC abrogated Id1-induced NF-κB promoter activity (Figure 4D). Simultaneously, Id1 significantly increased the expression of MMP −2, and Id1-induced MMP −2 expression was abrogated by PDTC as shown by western lot (Figure 4B-C). This suggests that Id1 increases the expression of MMP-2 via NF-κB.

Discussion

Angiogenesis is an important mechanism for tumorigenesis. Emerging evidence indicates that BM-derived EPCs participate in the tumor vascular network in different ways. They favor the formation of primitive tumor endothelium, control tumor growth, and promote the establishment of the pre-metastatic niche [12, 13]. Moreover, the contribution of BM-derived EPCs to tumor neovascularization has been reported in mice and humans [14, 15]. In our previous study, we found that EPCs from patients with ovarian cancer transfected with Id1-RNAi-LV displayed less proliferation, migration, and adhesion abilities compared to non-transfected control cells [9]. The proliferation, migration, and adhesion properties of ovarian cancer EPCs are attributable to the high expression of Id1, integrin α4 and p-Akt. Id1 contributes to this angiogenesis via the PI3K/Akt and integrin-α4 signaling pathways.

The molecular mechanism involved in EPC-induced tumor angiogenesis is poorly understood. VEGF and placental growth factor (PlGF) have been shown to contribute to EPC mobilization and homing into tumors [16]. Several reports have implicated cytokines, chemokines, hypoxia-inducible 1, integrin, and MMP-9 in regulating tumor angiogenesis. Recent studies indicate that Id1 plays a role in BM-derived hematopoietic progenitor cell mobilization [17–20]. In the present study, we demonstrated that over-expression of Id1 alone can induce angiogenic processes of EPCs in ovarian cancer. Moreover, knock-down of Id1 in EPCs almost completely abolished the EPC angiogenic processes in ovarian cancer. These findings indicate a crucial role for Id1 in ovarian cancer EPCs. Id1-induced EPC angiogenesis is partially blocked by the NF-κB inhibitor (PDTC) or the PI3K inhibitor (LY294002). Activation of NF-κB by angiogenesis factors in normal cells usually increases the expression of VEGF, but not MMP-2. Interestingly, activation of NF-κB by Id1 led to the high expression of MMP-2, instead of VEGF, in EPCs from patients with ovarian cancer in the present study. This may explain why Id1 transfectants are tumorigenic.

Both Id1 and NF-κB are over-expressed in EPCs from patients with ovarian cancer, which contributes to EPC angiogenesis. NF-κB regulates MMP-2 [21], whereas Id1 strengthens this regulation via an increase of NF-κB promoter activity, which contributes to an increase of NF-κB constitutively. However, we could not exclude the possibility that Id1 reduces the tumor volume by inhibition of angiogenesis. Id1 has recently been recognized as a clinical outcome predictor in esophageal squamous carcinoma [22]. We believe that focusing on the entire Id1/NF-κB/MMP-2 signaling pathway or downstream key molecules specific for EPC angiogenesis is more relevant to clinical prognosis than an upstream molecule that has extensive effects on multiple signaling pathways. Id1 is mainly expressed in cancer cells, but is occasionally seen in epithelial basal cells and proliferating fibroblasts surrounding the tumor cells. The function of Id1 may also be offset by other HLH transcription factors, such as E-box proteins, which are involved in cellular differentiation acting against Id1 [23]. In ovarian cancer, we have observed that some Id1-positive specimens are associated with well-differentiated cancer cells. This suggests that Id1 alone does not determine the cellular fate (proliferation or differentiation). It seems that the interaction between Id1 and its antagonists (HLH transcription factors) determines the cell fate. If this is true, Id1-predominant ovarian cancer EPCs may not necessarily be poorly differentiated but surely committed to cellular angiogenesis.

Conclusion

In summary, these data support the rationale of pharmacologic inhibition of the Id1/NF-κB/MMP-2 or Id1/PI3K/Akt pathways for ovarian cancer therapy and suggest that inhibition of Id1 or its downstream molecule MMP-2 removes the protection of ovarian cancer EPC from angiogenesis. Therefore, these EPC properties may be of significant clinical utility for ovarian cancer radiochemosensitization to improve long-term patient outcomes.

Abbreviations

EPCs:: Endothelial progenitor cells
Id1:: inhibitor of DNA binding/differentiation1
MMP-2:: matrix metalloproteinase-2
PDTC:: Pyrrolidine dithiocarbamate
SiRNA:: Small interference RNA
CDNA:: Complementary DNA
SD:: Standard deviation
Real-time PCR:: Real-time quantitative polymerase chain reaction.

References

Kopp HG, Ramos CA, Rafii S: Contribution of endothelial progenitors and proangiogenic hematopoietic cells to vascularization of tumor and ischemic tissue. Curr Opin Hematol. 2006, 13: 175-181. 10.1097/01.moh.0000219664.26528.da.
Article PubMed Central CAS PubMed Google Scholar
Asahara T, Takahashi T, Masuda H: VEGF contributes to postnatal neovascularization by mobilizing bone marrow derived endothelial progenitor cells. Embo J. 1999, 18: 3964-3972. 10.1093/emboj/18.14.3964.
Article PubMed Central CAS PubMed Google Scholar
Urbich C, Dimmeler S: Endothelial progenitor cells: characterization and role in vascular biology. Circ Res. 2004, 95: 343-353. 10.1161/01.RES.0000137877.89448.78.
Article CAS PubMed Google Scholar
Gao D, Nolan DJ, Mellick AS: EPCs control the angiogenic switch in mouse lung metastasis. Science. 2008, 319: 195-198. 10.1126/science.1150224.
Article CAS PubMed Google Scholar
Maw MK, Fujimoto J, Tamaya T: Overexpression of inhibitor of DNA-binding (ID)-1 protein related to angiogenesis in tumor advancement of ovarian cancers. BMC Cancer. 2009, 9: 430-10.1186/1471-2407-9-430.
Article PubMed Central PubMed Google Scholar
Lyden D, Young AZ, Zagzag D: Id1 and Id3 are required for neurogenesis, angiogenesis and vascularization of tumour xenografts. Nature. 2007, 104: 1260-1265.
Google Scholar
Jankovic V, Ciarrocchi A, Boccuni P: Id1 restrains myeloid commitment, maintaining the self-renewal capacity of hematopoietic stem cells. Proc Natl Acad Sci U S A. 2007, 104: 1260-1265. 10.1073/pnas.0607894104.
Article PubMed Central CAS PubMed Google Scholar
Shaked Y, Ciarrocchi A, Franco M, Lee CR, Man S, Cheung AM, Hicklin DJ, Chaplin D, Foster FS, Benezra R, Kerbel RS: Therapy-induced acute recruitment of circulating endothelial progenitor cells to tumors. Science. 2006, 313 (5794): 1785-1787. 10.1126/science.1127592.
Article CAS PubMed Google Scholar
Su YJ, Zheng L, Wang Q, Bao J, Zh C, Ailan L: The PI3K/ Akt pathway upregulates Id1 and integrin α4 to enhance recruitment of human ovarian cancer endothelial progenitor cells. BMC Cancer. 2010, 10: 459-10.1186/1471-2407-10-459.
Article PubMed Central PubMed Google Scholar
Su Y, Zheng L, Wang Q, Li W, Cai Z, **ong S, Bao J: Quantity and clinical relevance of circulating endothelial progenitor cells in human ovarian cancer. J Exp Clin Cancer Res. 2010, 29 (1): 27-10.1186/1756-9966-29-27.
Article PubMed Central PubMed Google Scholar
Tsuchiya K, Kim Y, Ondrey FG, Lin J: Characterization of a temper-ature-sensitive mouse middle ear epithelial cell line. Acta Otolaryngol. 2005, 125: 823-829. 10.1080/00016480510031533.
Article CAS PubMed Google Scholar
Gao DC, Nolan D, McDonnell K, Vahdat L, Benezra R: Bone marrow-derived endothelial progenitor cells contribute to the angiogenic switch in tumor growth and metastatic progression. Biochim Biophys Acta. 2009, 1796: 33-40.
PubMed Central CAS PubMed Google Scholar
Nolan DJ, Ciarrocchi A, Mellick AS, Jaggi JS, Bambino K: Bone marrow-derived endothelial progenitor cells are a major determinant of nascent tumor neovascularization. Genes Dev. 2007, 21: 1546-1558. 10.1101/gad.436307.
Article PubMed Central CAS PubMed Google Scholar
Peters BA, Diaz LA, Polyak K: Contribution of bone marrow-derived endothelial cells to human tumor vasculature. Nat Med. 2005, 11: 261-262. 10.1038/nm1200.
Article CAS PubMed Google Scholar
Duda DG, Cohen KS, Kozin SV: Evidence for incorporation of bone marrow-derived endothelial cells into perfused blood vessels in tumors. Blood. 2006, 107: 2774-2776. 10.1182/blood-2005-08-3210.
Article PubMed Central CAS PubMed Google Scholar
Mieno S, Boodhwani M, Robich MP, Clements RT, Sodha NR, Sellke FW: Effects of diabetes mellitus on VEGF-induced proliferation response in bone marrow derived endothelial progenitor cells. J Card Surg. 2010, 25 (5): 618-625. 10.1111/j.1540-8191.2010.01086.x.
Article PubMed Central PubMed Google Scholar
Spring H, Schuler T, Arnold B, Hammerling GJ, Ganss R: Chemo-kines direct endothelia l progenitors int o tumor neovessels. Proc Natl Acad Sci USA. 2005, 102: 18111-18116. 10.1073/pnas.0507158102.
Article PubMed Central CAS PubMed Google Scholar
Du R, Lu KV, Petritsch C, Liu P, Ganss R, Passegue E, Song H, Vandenberg S, Johnson RS, Werb Z: HIF1 α induces the recruitment of bone marrow–derived vascular modulatory cells to regulate tumor angiogenesis and invasion. Cancer Cell. 2008, 13: 206-220. 10.1016/j.ccr.2008.01.034.
Article PubMed Central CAS PubMed Google Scholar
Li B, Sharpe EE, Maupin AB, Teleron AA, Pyle AL, Carmeliet P, Young PP: VEGF and PlGF promote adult vasculogenesis by enhancing EPC re-cruitment and vessel formation at the site of tumor neovascularization. FASEB J. 2006, 20: 1495-1497. 10.1096/fj.05-5137fje.
Article CAS PubMed Google Scholar
Yang L, Huang J, Ren X, Gorska AE, Chytil A, Aakre M, Carbone DP, Matrisian LM, Richmond A, Lin PC: Abrogation of TGFβ signal-ing in mammary carcinomas recruits Gr-1+CD11b+myeloid cells that promote metastasis. Cancer Cell. 2008, 3: 23-35.
Article Google Scholar
Dutta S, Wang FQ, Wu HS, Mukherjee TJ, Fishman DA: The NF-κB pathway mediates lysophosphatidic acid (LPA)-induced VEGF signaling and cell invasion in epithelial ovarian cancer (EOC). Gynecol Oncol. 2011, 23 (1): 129-137.
Article Google Scholar
Lin J, Guan Z, Wang C, Feng L, Zheng Y, Caicedo E, Bearth E, Peng JR, Gaffney P, Ondrey FG: Inhibitor of differentiation 1 contributes to head and neck squamous cell carcinoma survival via the NF-kappaB/survivin and phosphoinositide 3-kinase/Akt signaling pathways. Clin Cancer Res. 2010, 1;16 (1): 77-87.
Article Google Scholar
Mern DS, Hoppe-Seyler K, Hoppe-Seyler F, Hasskarl J, Burwinkel B: Targeting Id1 and Id3 by a specific peptide aptamer induces E-box promoter activity, cell cycle arrest, and apoptosis in breast cancer cells. Breast Cancer Res Treat. 2010, 124 (3): 623-633. 10.1007/s10549-010-0810-6.
Article CAS PubMed Google Scholar

Download references

Acknowledgements

The study was supported by grants from the National Natural Science Foundation of China (no. 81202039), the Natural Science Foundation of Heilongjiang Province, China (no. QC2012C037), Harbin Medical University the affiliated tumor hospital Science Foundation of China (no. JJ2010-10). We have no conflict of interest to declare.

Author information

Authors and Affiliations

Department of clinical laboratory, the affiliated tumor hospital, Harbin Medical University, Harbin, China
Yajuan Su, Lichen Teng, Ying Wang, Jialin Cui & Shiyun Peng
Department of clinical laboratory, Nan**g Maternity and Child Health Care hospital, Nan**g Medical University, Nan**g, China
Lingjuan Gao
Laboratory of Medical Genetics, Harbin Medical University, Harbin, 150081, China
Songbin Fu

Authors

Yajuan Su
View author publications
You can also search for this author in PubMed Google Scholar
Lingjuan Gao
View author publications
You can also search for this author in PubMed Google Scholar
Lichen Teng
View author publications
You can also search for this author in PubMed Google Scholar
Ying Wang
View author publications
You can also search for this author in PubMed Google Scholar
Jialin Cui
View author publications
You can also search for this author in PubMed Google Scholar
Shiyun Peng
View author publications
You can also search for this author in PubMed Google Scholar
Songbin Fu
View author publications
You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Shiyun Peng or Songbin Fu.

Additional information

Competing interests

The authors declare they have no competing interests.

Authors’ contributions

SBF and SYP conceived of the study and drafted the manuscript. YJS and LJG participated in its design and helped to draft the manuscript. LCT carried out the molecular biological studies and performed the statistical analysis. YW collected the patient information. JLC helped to revise the manuscript and performed the statistical analysis. All authors read and approved the final manuscript.

Yajuan Su, Lingjuan Gao contributed equally to this work.

Authors’ original submitted files for images

Below are the links to the authors’ original submitted files for images.

Authors’ original file for figure 1

Authors’ original file for figure 2

Authors’ original file for figure 3

Authors’ original file for figure 4

Rights and permissions

Open Access This article is published under license to BioMed Central Ltd. This is an Open Access article is distributed under the terms of the Creative Commons Attribution License ( https://creativecommons.org/licenses/by/2.0 ), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Reprints and permissions

About this article

Cite this article

Su, Y., Gao, L., Teng, L. et al. Id1 enhances human ovarian cancer endothelial progenitor cell angiogenesis via PI3K/Akt and NF-κB/MMP-2 signaling pathways. J Transl Med 11, 132 (2013). https://doi.org/10.1186/1479-5876-11-132

Download citation

Received: 11 March 2013
Accepted: 22 May 2013
Published: 29 May 2013
DOI: https://doi.org/10.1186/1479-5876-11-132

Id1 enhances human ovarian cancer endothelial progenitor cell angiogenesis via PI3K/Akt and NF-κB/MMP-2 signaling pathways