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Involvement of Osteopontin in the Matrix-Degrading and Proangiogenic Changes Mediated by Nicotine in Pancreatic Cancer Cells

  • 2010 SSAT Plenary Presentation
  • Published:
Journal of Gastrointestinal Surgery Aims and scope

Abstract

Background

Substantial evidence indicates that exposure to cigarette smoke is associated with an elevated risk of pancreatic ductal adenocarcinoma (PDA). However, the mechanisms underlying the effects of nicotine on the development or progression of PDA remain to be investigated. Previously, we showed that nicotine promotes the expression of osteopontin c (OPNc), an isoform of OPN protein that confers on cancer cells a migratory phenotype. In this study, we explored the potential prometastatic role of nicotine in PDA through studying its effect on the expression of matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) and evaluated the role of OPN in mediating these effects.

Materials and Methods

MMP-9 and VEGF mRNA and protein were analyzed in PDA cells treated with or without nicotine (3–300 nM). Transient transfection and luciferase-labeled promoter studies evaluated the effects of OPNc and OPN protein on the transcription and translation of MMP-9 and VEGF. Real-time PCR and immunohistochemistry were used to analyze the mRNA expression levels and localization of OPN, MMP-9, and VEGF proteins in matched invasive human PDA and surrounding nonmalignant tissues.

Results and Discussion

Nicotine significantly enhanced the expression of MMP-9 and VEGF mRNA and protein in PDA cells. Blocking OPN with siRNA or OPN antibody prevented the nicotine-mediated increase of both MMP-9 and VEGF. Transient transfection of OPNc gene in PDA cells or their treatment with recombinant OPN protein significantly (p < 0.05) increased MMP-9 and VEGF mRNA expression levels and induced their promoter activities. In invasive PDA lesions, MMP-9 mRNA levels were significantly (p < 0.005) higher in smokers vs. nonsmokers. VEGF protein co-localized with MMP-9 and OPN in the malignant ducts and correlated well with their higher levels in invasive PDA lesions.

Conclusions

Our data show for the first time that cigarette smoking and nicotine may contribute to PDA metastasis through inducing MMP-9 and VEGF and suggest that OPN plays a central role in mediating these effects. The presence of OPN as a downstream effector of nicotine that is capable of mediating its prometastatic effects in PDA cells is novel and could provide a unique therapeutic target to control pancreatic cancer aggressiveness, especially in the cigarette-smoking population.

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References

  1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Murray T, Thun MJ. Cancer Statistics, 2008. CA Cancer J Clin 2008;52(2):71–96.

    Article  Google Scholar 

  2. Wang XL, Wang J. Smoking-gene interaction and disease development: relevance to pancreatic cancer and atherosclerosis. World Journal of Surgery 2005;29:344–53.

    Article  CAS  PubMed  Google Scholar 

  3. Lowenfels AB, Maisonneuve P. Epidemiology and risk factors for pancreatic cancer. Best Prac Res Clin Gastroenterology 2006;20:197–209.

    Article  Google Scholar 

  4. US Department of Health and Human Services. The health consequences of using smokeless tobacco: a report of the advisory committee to the Surgeon General [NIH Publication no. 86-2874]. Bethesda, MD: USDHHS; April 1996.

  5. Farrow DC, Davis S. Risk of pancreatic cancer in relation to medical history and use of tobacco, alcohol and coffee. Intl J Cancer 1990;45:816–20.

    Article  CAS  Google Scholar 

  6. Silverman DT, Dunn JA, Hoover RN, Schiffman M, Lillemoe KD, Schoenberg JB et al. Cigarette smoking and pancreas cancer: a case control study based on direct interviews. J Natl Cancer Inst 1994;86:1510–6.

    Article  CAS  PubMed  Google Scholar 

  7. Chambers AF, Matrisian LM. Changing views of the role of matrix metalloproteinases in metastasis. J Natl Cancer Inst 1997;89(17):1260–70.

    Article  CAS  PubMed  Google Scholar 

  8. Coussens LM, Werb Z. Matrix metalloproteinases and the development of cancer. Chem Biol 1996;3:895–904.

    Article  CAS  PubMed  Google Scholar 

  9. Kelly T, Yan Y, Osborne RL, Athota AB, Rozypal TL, Colclasure JC, Chu WS. Proteolysis of extracellular matrix by invadopodia facilitates human breast cancer cell invasion and is mediated by matrix metalloproteinases. Clin Exp Metastasis 1998;16:501–12.

    Article  CAS  PubMed  Google Scholar 

  10. Nikkola J, Vihinen P, Vlaykova T, Hahka-Kemppinen M, Kahari VM, Pyrhonen S. High expression levels of collagenase-1 and stromelysin-1 correlate with shorter disease-free survival in human metastatic melanoma. Int J Cancer 2002;97:432–8.

    Article  CAS  PubMed  Google Scholar 

  11. Kahari VM, Saarialho-Kere U. Matrix metalloproteinases and their inhibitors in tumor growth and invasion. Ann Med 1999;31:34–45.

    Article  CAS  PubMed  Google Scholar 

  12. Kleiner DE, Stetler-Stevenson WG. Matrix metalloproteinases and metastasis. Cancer Chemother Pharmacol 1999;43:42–51.

    Article  Google Scholar 

  13. Liotta LA, Tryggvason K, Garbisa S, Hart I, Foltz CM, Shafie S. Metastatic potential correlates with enzymatic degradation of basement membrane collagen. Nature 1980;284:67–8.

    Article  CAS  PubMed  Google Scholar 

  14. Mira E, Lacalle RA, Buesa JM, de Buitrago GG, Jiménez-Baranda S, Gómez-Moutón C, Martínez-A C, Mañes S. Secreted MMP9 promotes angiogenesis more efficiently than constitutive active MMP9 bound to the tumor cell surface. J Cell Sci. 2004;117(Pt 9):1847–57.

    Article  CAS  PubMed  Google Scholar 

  15. Folkman J. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat Med 1995;1:27–31.

    Article  CAS  PubMed  Google Scholar 

  16. Kerbel RS. Tumor angiogenesis. The New England Journal of Medicine 2008;358(19):2039–49.

    Article  CAS  PubMed  Google Scholar 

  17. Takahashi Y, Cleary KR, Mai M, Kitadai Y, Bucana CD, Ellis LM. Significance of vessel count and vascular endothelial growth factor and its receptor (KDR) in intestinal-type gastric cancer. Clin Cancer Res 1996;2:1679–84.

    CAS  PubMed  Google Scholar 

  18. Takahashi Y, Kitadai Y, Bucana CD, Cleary ER, Ellis LM. Expression of vascular endothelial growth factor and its receptor, KDR, correlates with vascularity, metastasis, and proliferation of human colon cancer. Cancer Res1995;55:3964–68.

    CAS  PubMed  Google Scholar 

  19. Chipitsyna G, Gong Q, Anandanadesan R, Alnajar A, Batra SK, Denhardt ST, et al. Induction of osteopontin (OPN) expression by nicotine and cigarette smoke in the pancreas and pancreatic ductal adenocarcinoma cells. Int J Cancer 2009;125:276–85.

    Article  CAS  PubMed  Google Scholar 

  20. Sullivan J, Blair L, Alnajar A, Aziz T, Ng CY, et al. Expression of a prometastatic splice variant of osteopontin, OPNc, in human pancreatic ductal adenocarcinoma. Surgery 2009;146:232–40.

    Article  PubMed  Google Scholar 

  21. Takafuji V, Forgues M, Unsworth E, Goldsmith P, Wang XW. An osteopontin fragment is essential for tumor cell invasion in hepatocellular carcinoma. Oncogene 2007;26(44):6361–71.

    Article  CAS  PubMed  Google Scholar 

  22. Liu J, Zhan M, Hannay JA, Das P, Bolshakov SV, Kotilingam D, Yu D, Lazar AF, Pollock RE, Lev D. Wild-type p53 inhibits nuclear factor-kappaB-induced matrix metalloproteinase-9 promoter activation: implications for soft tissue sarcoma growth and metastasis. Mol Cancer Res. 2006 Nov;4(11):803–10.

    Article  CAS  PubMed  Google Scholar 

  23. Sánchez-López E, López AF, Esteban V, Yagüe S, Egido J, Ruiz-Ortega M, Alvarez-Arroyo MV. Angiotensin II regulates vascular endothelial growth factor via hypoxia-inducible factor-1alpha induction and redox mechanisms in the kidney. Antioxid Redox Signal. 2005;7(9-10):1275–84.

    Article  PubMed  Google Scholar 

  24. Liu H, Chen A, Guo F, Yuan L. A short-hairpin RNA targeting osteopontin downregulates MMP-2 and MMP-9 expressions in prostate cancer PC-3 cells. CancerLett. 2010;295:27–37

    CAS  Google Scholar 

  25. Dai J, Peng L, Fan K, Wang H, Wei R, Ji G, Cai J, Lu B, Li B, Zhang D, Kang Y, Tan M, Qian W, Guo Y. Osteopontin induces angiogenesis through activation of PI3K/AKT and ERK1/2 in endothelial cells. Oncogene. 2009; 28(38):3412–22.

    Article  CAS  PubMed  Google Scholar 

  26. Chakraborty G, Jain S, Kundu GC. Osteopontin promotes vascular endothelial growth factor-dependent breast tumor growth and angiogenesis via autocrine and paracrine mechanisms. Cancer Res. 2008 Jan 1;68(1):152–61.

    Article  CAS  PubMed  Google Scholar 

  27. Tian M, Cui YZ, Song GH, Zong MJ, Zhou XY, Chen Y, Han JX. Proteomic analysis identifies MMP-9, DJ-1 and A1BG as overexpressed proteins in pancreatic juice from pancreatic ductal adenocarcinoma patients. BMC Cancer. 2008;8:241

    Article  PubMed  Google Scholar 

  28. Mroczko B, Lukaszewicz-Zajac M, Wereszczynska-Siemiatkowska U, Groblewska M, Gryko M, Kedra B, Jurkowska G, Szmitkowski M. Clinical significance of the measurements of serum matrix metalloproteinase-9 and its inhibitor (tissue inhibitor of metalloproteinase-1) in patients with pancreatic cancer: metalloproteinase-9 as an independent prognostic factor. Pancreas. 2009;38(6):613–8.

    Article  CAS  PubMed  Google Scholar 

  29. Toomey DP, Manahan E, McKeown C, Rogers A, McMillan H, Geary M, Conlon KC, Murphy JF. Vascular endothelial growth factor and not cyclooxygenase 2 promotes endothelial cell viability in the pancreatic tumor microenvironment. Pancreas. 2010;39:595–603

    Article  CAS  PubMed  Google Scholar 

  30. Sandblom G, Granroth S, Rasmussen IC. TPS, CA 19-9, VEGF-A, and CEA as diagnostic and prognostic factors in patients with mass lesions in the pancreatic head. Ups J Med Sci. 2008;113(1):57–64.

    Article  PubMed  Google Scholar 

  31. Hukkanen, J., Jacob, P. 3 rd and Benowitz, N.L. Metabolism and disposition kinetics of nicotine. Pharmacol Rev 2005;57:79–115.

    Article  CAS  PubMed  Google Scholar 

  32. Mirza M, Shaughnessy E, Hurley JK, Vanpatten KA, Pestano GA, He B, et al. Osteopontin-c is a selective marker of breast cancer. Int J Cancer 2008;122:889–97.

    Article  CAS  PubMed  Google Scholar 

  33. Byrne AM, Bouchier-Hayes DJ, Harmey JH. Angiogenic and cell survival functions of vascular endothelial growth factor (VEGF). J Cell Mol Med. 2005;9(4):777–94.

    Article  CAS  PubMed  Google Scholar 

  34. Denhardt DT, Noda M, O’Regan AW, Pavlin D, Berman JS. Osteopontin as a means to cope with environmental insults: regulation of inflammation, tissue remodeling, and cell survival. J Clin Invest 2001;107:1055–61.

    Article  CAS  PubMed  Google Scholar 

  35. Rocca G, Gaia E, Iuliano R. Increased incidence of cancer in chronic pancreatitis. J Clin Gastroenterol. 1987;9:175–9.

    Article  CAS  PubMed  Google Scholar 

  36. Maisonnneuve P, Lowenfels AB. Chronic pancreatitis and pancreatic cancer. Dig Dis. 2002;20:32–7

    Article  Google Scholar 

Download references

Acknowledgment

This work was supported by NIH grant 1R21 CA133753-02.

Author information

Authors and Affiliations

  1. Department of Surgery, Jefferson Pancreatic, Biliary and Related Cancer Center, Thomas Jefferson University, 1015 Walnut Street, Philadelphia, PA, 19107, USA

    Melissa Lazar, Jennifer Sullivan, Galina Chipitsyna, Qiaoke Gong, Chee Y. Ng, Ahmed F. Salem, Tamer Aziz, Agnes Witkiewicz, David T. Denhardt, Charles J. Yeo & Hwyda A. Arafat

Authors
  1. Melissa Lazar
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  2. Jennifer Sullivan
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  3. Galina Chipitsyna
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  4. Qiaoke Gong
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  7. Tamer Aziz
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Corresponding author

Correspondence to Hwyda A. Arafat.

Additional information

Discussant

DR. MARY MALUCCIO (Indianapolis, IN): You make a very convincing argument for the link between nicotine, osteopontin, MMP9, and VEGF in pancreatic cancer. I think your blocking studies are quite elegant and certainly prove your point. This only adds to the long list of reasons that people should stop smoking.

Unfortunately, even with a much stronger correlation between smoking and the more prevalent lung cancer, most people do not stop smoking until the die is cast. And there are millions upon millions of people that smoke and only 40 some odd thousand cases per year of pancreatic cancer.

So surveying smokers is not a realistic option. Thus, I struggle a bit to envision how these data influence the diagnosis or treatment of pancreatic cancer.

Your data do not suggest that prevention models would work since you did not show that this pathway influences the development of cancer. A preclinical model, whereby you alter the development of cancer, like in the KRAS mutant mouse model or if you can alter the biologic behavior of an established pancreatic cancer by blocking osteopontin would make a much more convincing argument that this is a good target.

The major obstacle in pancreatic cancer continues to be that there is no truly high-risk patient population that we can survey in hopes of altering the detection and behavior of an inevitable pancreatic cancer. And therefore, our ability to intervene early is limited.

There are a couple of questions that I have for your group.

I believe that there are 10 to 20, if not more, pancreatic cancer cell lines through the ATCC, whereas you chose to study two. My question is, how did you decide to use these particular cell lines? And are there cell lines that are more or less likely to respond to nicotine-induced changes?

Secondly, your paper shows very nice images of the human tissue with avid staining of osteopontin in pancreatic cancers from smokers vs. nonsmokers. And that if osteopontin levels are high, then the remaining molecules are also high.

In your experience, if you were to take 100 pancreatic cancer samples, regardless of smoking status, how many would you expect to show significant staining for osteopontin that would suggest that targeting this protein would be of therapeutic benefit?

Closing Discussant

DR. MELISSA LAZAR: To address your first question of how we chose these two cells lines, these are two cell lines that express sort of low levels of basal OPN, which is so that is why we chose them. So when we did overexpress OPNc, that made a difference. We have also used these two cell lines previously in some of our previous other studies with nicotine.

And then, if you did take 100 samples and you stained the tissue for OPN, all of the samples would stain for OPN. When we do stain just normal pancreatic tissue, there are some low levels of OPN staining in the ducts, but there are no levels of MMP9 or VEGF.

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Lazar, M., Sullivan, J., Chipitsyna, G. et al. Involvement of Osteopontin in the Matrix-Degrading and Proangiogenic Changes Mediated by Nicotine in Pancreatic Cancer Cells. J Gastrointest Surg 14, 1566–1577 (2010). https://doi.org/10.1007/s11605-010-1338-0

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