Significance of Tissue Expression and Serum Levels of Angiopoietin-like Protein 4 in Breast Cancer Progression: Link to NF-κB /P65 Activity and Pro-Inflammatory Cytokines

  • Shafik, Noha M (Department of Medical Biochemistry, Faculty of Medicine, Tanta University) ;
  • Mohamed, Dareen A (Department of Pathology, Faculty of Medicine, Tanta University) ;
  • Bedder, Asmaa E (Department of Pathology, Faculty of Medicine, Tanta University) ;
  • El-Gendy, Ahmed M (Department of General Surgery, Faculty of Medicine, Tanta University)
  • Published : 2016.01.11


Background: The molecular mechanisms linking breast cancer progression and inflammation still remain obscure. The aim of the present study was to investigate the possible association of angiopoeitin like protein 4 (ANGPTL4) and its regulatory factor, hypoxia inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$), with the inflammatory markers nuclear factor kappa B/p65 (NF-${\kappa}B$/P65) and interleukin-1 beta (IL-$1{\beta}$) in order to evaluate their role in inflammation associated breast cancer progression. Materials and Methods: Angiopoietin-like protein 4 (ANGPTL4) mRNA expressions were evaluated using quantitative real time PCR and its protein expression by immunohistochemistry. DNA binding activity of NF-${\kappa}B$/P65 was evaluated by transcription factor binding immunoassay. Serum levels of ANGPTL4, HIF-$1{\alpha}$ and IL-$1{\beta}$ were immunoassayed. Tumor clinico-pathological features were investigated. Results: ANGPTL4 mRNA expressions and serum levels were significantly higher in high grade breast carcinoma ($1.47{\pm}0.31$ and $184.98{\pm}18.18$, respectively) compared to low grade carcinoma ($1.21{\pm}0.32$ and $171.76{\pm}7.58$, respectively) and controls ($0.70{\pm}0.02$ and $65.34{\pm}6.41$, respectively), (p<0.05). Also, ANGPTL4 high/moderate protein expression was positively correlated with tumor clinico-pathological features. In addition, serum levels of HIF-$1{\alpha}$ and IL-$1{\beta}$ as well as NF-${\kappa}B$/P65 DNA binding activity were significantly higher in high grade breast carcinoma ($148.54{\pm}14.20$, $0.79{\pm}0.03$ and $247.13{\pm}44.35$ respectively) than their values in low grade carcinoma ( $139.14{\pm}5.83$, $0.34{\pm}0.02$ and $184.23{\pm}37.75$, respectively) and controls ($33.95{\pm}3.11$, $0.11{\pm}0.02$ and $7.83{\pm}0.92$, respectively), (p<0.001). Conclusion: ANGPTL4 high serum levels and tissue expressions in advanced grade breast cancer, in addition to its positive correlation with tumor clinico-pathological features and HIF-$1{\alpha}$ could highlight its role as one of the signaling factors involved in breast cancer progression. Moreover, novel correlations were found between ANGPTL4 and the inflammatory markers, IL-$1{\beta}$ and NF-${\kappa}B$/p65, in breast cancer, which may emphasize the utility of these markers as potential tools for understanding interactions for axes of carcinogenesis and inflammation contributed for cancer progression. It is thus hoped that the findings reported here would assist in the development of new breast cancer management strategies that would promote patients' quality of life and ultimately improve clinical outcomes. However, large-scale studies are needed to verify these results.


Breast cancer;angiopoietin-like protein-4 (ANGPTL4);hypoxia inducible factor-1 alpha


  1. Biswas DK, Martin KJ, McAlister C, et al (2003). Apoptosis caused by chemotherapeutic inhibition of nuclear factor-kappaB activation. Cancer Res, 63, 290-5.
  2. Bradford MM (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248-54.
  3. Brunckhorst MK, Xu Y, Lu R, et al (2014). Angiopoietins promote ovarian cancer progression by establishing a procancer microenvironment. Am J Pathol, 184, 2285-96.
  4. Cheng ZX, Wang DW, Liu T, et al (2014). Effects of the HIF-$1{\alpha}$ and NF-${\kappa}B$ loop on epithelial-mesenchymal transition and chemoresistance induced by hypoxia in pancreatic cancer cells. Oncol Rep, 31, 1891-8.
  5. Feingold KR, Shigenaga JK, Cross AS, et al (2012). Angiopoietin like protein 4 expression is decreased in activated macrophages. Biochem Biophys Res Commun, 421, 612-5.
  6. Gaballah HH, Zakaria SS, Ismail SA (2014). Activity and expression pattern of NF-${\kappa}B$ /P65 in peripheral blood from hepatocellular carcinoma patients - link to hypoxia inducible factor -$1{\alpha}$. Asian Pac J Cancer Prev, 15, 6911-7.
  7. Galaup A, Cazes A, Le Jan S, et al (2006). Angiopoietinlike4preventsmetastasis through inhibition of vascularpermeability and tumorcellmotility and invasiveness. Proc Natl Acad Sci U S A, 103, 18721-6.
  8. Gong JP, Liu CA, Wu CX, et al (2002). Nuclear factor ${\kappa}B$ activity in patients with acute severe cholangitis. World J Gastroenterol, 8, 346-9.
  9. Gonzalez-Ramos R, Rocco J, Rojas C, et al (2012). Physiologic activation of nuclear factor kappa-B in the endometrium during the menstrual cycle is altered in endometriosis patients. Fertil Steril, 97, 645-51.
  10. Aggarwal BB, Shishodia S, Sandur SK, et al (2006). Inflammation and cancer: how hot is the link? Biochem Pharmacol, 72, 1605-21.
  11. Anand K, Asthana P, Kumar A, et al (2011). Quercetin mediated reduction of angiogenic markers and chaperones in DLAinduced solid tumours. Asian Pac J Cancer Prev, 12, 2829-35.
  12. Gorlach A, Bonello S (2008). The cross-talk between NF-kappaB and HIF-1: further evidence for a significant liaison. Biochem J, 412, 17-9.
  13. Holliday DL, Speirs V (2011). Choosing the right cell line for breast cancer research. Breast Cancer Res, 13, 215.
  14. Hu J, Jham BC, Ma T, et al (2011). Angiopoietin-like 4: a novel molecular hallmark in oral Kaposi's sarcoma. Oral Oncol, 47, 371-5.
  15. Inoue T, Kohro T, Tanaka T, et al (2014). Cross-enhancement of ANGPTL4 transcription by HIF1 alpha and PPAR beta/delta is the result of the conformational proximity of two response elements. Genome Biol, 15, 63.
  16. Jung YJ, Isaacs JS, Lee S, et al (2003). IL-1beta-mediated upregulation of HIF-1alpha via an NFkappaB/COX-2 pathway identifies HIF-1 as a critical link between inflammation and oncogenesis. FASEB J, 17, 2115-7.
  17. Kafshdooz L, Tabrizi AD, Mohaddes SM, (2014). The polymorphism of hypoxia-inducible factor-1a gene in endometrial cancer. Asian Pac J Cancer Prev, 15, 10393-6.
  18. Katanov C, Lerrer S, Liubomirski Y, et al (2015). Regulation of the inflammatory profile of stromal cells in human breast cancer: prominent roles for TNF-${\alpha}$ and the NF-${\kappa}B$ pathway. Stem Cell Res Ther, 6, 87.
  19. Khong TL, Thairu N, Larsen H, et al (2013). Identification of the angiogenic gene signature induced by EGF and hypoxia in colorectal cancer. BMC Cancer, 13, 518.
  20. Kim SH, Park YY, Kim SW, et al (2011). ANGPTL4induction by prostaglandin E2 under hypoxic conditions promotes colorectal cancer progression. Cancer Res, 71, 7010-20.
  21. Li H, Ge C, Zhao F, et al (2011). HIF-1-activated ANGPTL4 contributes to tumor metastasis via VCAM-1/integrin b1 signaling in human hepatocellular carcinoma. Hepatology, 54, 910-9.
  22. Li Y, Miao LY, Xiao YL, et al (2015). Hypoxia induced high expression of thioredoxin interacting protein (TXNIP) in Non-small cell lung cancer and its prognostic effect. Asian Pac J Cancer Prev, 16, 2953-8.
  23. Ng KT, Xu A, Cheng Q, et al (2014). Clinical relevance and therapeutic potential of angiopoietin-like protein 4 in hepatocellular carcinoma. Mol Cancer, 13, 196.
  24. Padua D, Zhang XH, Wang Q, et al (2008). TGF beta primes breast tumors for lung metastasis seeding through angiopoietin-like 4. Cell, 133, 66-77.
  25. Pandey S, Singh S, Anang V, et al (2015). Pattern Recognition Receptors in Cancer Progression and Metastasis. Cancer Growth Metastasis, 8, 25-34.
  26. Potente M, Gerhardt H, Carmeliet P (2011). Basic and therapeutic aspects of angiogenesis. Cell, 146, 873-87.
  27. Philip M, Rowley DA, Schreiber H (2004). Inflammation as a tumor promoter in cancer induction. Semin Cancer Biol, 14, 433-9.
  28. Rider P, Carmi Y, Guttman O, et al (2011). IL-$1{\alpha}$ and IL-$1{\beta}$ recruit different myeloid cells and promote different stages of sterile inflammation. J Immunol, 187, 4835-43.
  29. Saijo Y, Tanaka M, Miki M, et al (2002). Proinflammatory cytokine IL-1 beta promotes tumor growth of Lewis lung carcinoma by induction of angiogenic factors: in vivo analysis of tumor-stromal interaction. J Immunol, 169, 469-75.
  30. Santulli G (2014). Angiopoietin-like proteins: a comprehensive look. Front Endocrinol (Lausanne), 5, 4.
  31. Snoussi K, Strosberg AD, Bouaouina N, et al (2005). Genetic variation in pro-inflammatory cytokines (interleukin-1beta, interleukin-1alpha and interleukin-6) associated with the aggressive forms, survival, and relapse prediction of breast carcinoma. Eur Cytokine Netw, 16, 253-60.
  32. Taghavi A, Fazeli Z, Vahedi M, et al (2012). Increased trend of breast cancer mortality in Iran. Asian Pac J Cancer Prev, 13, 367-70.
  33. Tan MJ, Teo Z, Sng MK, et al (2012). Emerging roles of angiopoietin-like 4 in human cancer. Mol Cancer Res, 10, 677-88.
  34. Taylor CT, Cummins EP (2009). The role of NF-kappaB in hypoxia-induced gene expression. Ann N Y Acad Sci, 1177, 178-84.
  35. Tewari R, Choudhury SR, Ghosh S, et al (2012). Involvement of $TNF{\alpha}$-induced TLR4-NF-${\kappa}B$ and TLR4-HIF-$1{\alpha}$ feedforward loops in the regulation of inflammatory responses in glioma. J Mol Med (Berl), 90, 67-80.
  36. Unwith S, Zhao H, Hennah L, et al (2015). The potential role of HIF on tumour progression and dissemination. Int J Cancer, 136, 2491-503.
  37. Van Laere SJ, Van der Auwera I, Van den Eynden GG, et al (2006). Nuclear factor-kappaB signature of inflammatory breast cancer by cDNA microarray validated by quantitative real-time reverse transcription PCR, immuno-histochemistry, and nuclear factor-kappaB DNA-binding. Clin Cancer Res, 12, 3249-56.
  38. van Loo G, Beyaert R (2011). Negative regulation of NF-${\kappa}B$ and its involvement in rheumatoid arthritis. Arthritis Res Ther, 31, 221.
  39. Wagner KD, Benchetrit M, Bianchini L, et al (2011). Peroxisome proliferator-activated receptor beta/delta (PPARbeta/delta) is highly expressed in liposarcoma and promotes migration and proliferation. J Pathol, 224, 575-588.
  40. Wang F, Ma J, Wang KS, et al (2015). Blockade of TNF-${\alpha}$-induced NF-${\kappa}B$ signaling pathway and anti-cancer therapeutic response of dihydrotanshinone I. Int Immunopharmacol, 28, 764-72.
  41. Wang L, Kang F, Li J, et al (2013). Overexpression of p65 attenuates celecoxib-induced cell death in MDA-MB-231 human breast cancer cell line. Cancer Cell Int, 13, 14.
  42. Xia JT, Chen LZ, Jian WH, (2014). MicroRNA-362 induces cell proliferation and apoptosis resistance in gastric cancer by activation of NF-${\kappa}B$ signaling. J Transl Med, 12, 33.
  43. Xia T, Cheng H, Zhu Y (2014). Knockdown of hypoxia-inducible factor-1 alpha reduces proliferation, induces apoptosis and attenuates the aggressive phenotype of retinoblastoma WERI-Rb-1 cells under hypoxic conditions. Ann Clin Lab Sci, 44, 134-44.
  44. Yi J, Pan BZ, Xiong L, Song HZ (2013). Clinical significance of angiopoietin-likeprotein4expression in tissue and serum of esophageal squamous cell carcinoma patients. Med Oncol, 30, 680.
  45. Yoshida T, Hashimura M, Mastumoto T, et al (2013). Transcriptional upregulation of HIF-$1{\alpha}$ by NF-${\kappa}B$/p65 and its associations with ${\beta}$-catenin/p300 complexes in endometrial carcinoma cells. Lab Invest, 93, 1184-93.
  46. Yotsumoto F, Tokunaga E, Oki E, et al (2013). Molecular hierarchy of heparin-binding EGF-like growth factorregulated angiogenesis in triple-negative breast cancer. Mol Cancer Res, 11, 506-17.
  47. Zhang H, Wong CC, Wei H, et al (2012). HIF-1-dependent expression of angiopoietin-like 4 and L1CAM mediates vascular metastasis of hypoxic breast cancer cells to the lungs. Oncogene, 31,1757-70.
  48. Zhang ZG, Zhang QN, Wang XH, et al (2013). Hypoxiainducible factor 1 alpha (HIF-$1{\alpha}$) as a prognostic indicator in patients with gastric tumors: a meta-analysis. Asian Pac J Cancer Prev, 14, 4195-8.
  49. Zhu P, Tan MJ, Huang RL, et al (2011). Angiopoietin-like 4 protein elevates the prosurvival intracellular O2(-):$H_2O_2$ratio and confers anoikis resistance to tumors. Cancer Cell, 19, 401-15.

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