Open Access Peer-reviewed Research Article

HIF and COX-2 expression in triple negative breast cancer cells with hypoxia and 5-fluorouracil

Main Article Content

Noriko Mori corresponding author
Yelena Mironchik
Flonné Wildes
Sherry Y. Wu
Kanami Mori
Balaji Krishnamachary
Zaver M. Bhujwalla


Our purpose was to understand the effects of normoxia or hypoxia on 5-fluorouracil  (5-FU) treatment in triple negative breast cancer (TNBC) cells, and characterize the molecular changes in hypoxia inducible factors (HIFs) and cyclooxygenase-2 (COX-2) following treatment.  Cell viability and protein levels of HIFs and COX-2 were determined after wild type and HIF silenced MDA-MB-231 cells, and wild type SUM-149 cells, were treated with 5-FU under normoxia or hypoxia.  5-FU reduced cell viability to the same levels irrespective of normoxia or hypoxia.  HIF silenced MDA-MB-231 cells showed comparable changes in cell viability, supporting observations that hypoxia and the HIF pathways did not significantly influence cell viability reduction by 5-FU.  Our data suggest that HIF-2aaccumulation may predispose cancer cells to cell death under hypoxia.  SUM-149 cells that have higher COX-2 and HIF-2afollowing 24 h of hypoxia, were more sensitive to 96 h of hypoxia compared to MDA-MB-231 cells, and were more sensitive to 5-FU than MDA-MB-231 cells.  COX-2 levels changed with hypoxia and with 5-FU treatment but patterns were different between the two cell lines.  At 96 h, COX-2 increased in both untreated and 5-FU treated cells under hypoxia in MDA-MB-231 cells.  In SUM-149 cells, only treatment with 5-FU increased COX-2 at 96 h of hypoxia.  Cells that survive hypoxia and 5-FU treatment may exhibit a more aggressive phenotype.  Our results support understanding interactions between HIF and COX-2 with chemotherapeutic agents under normoxia and hypoxia, and investigating the use of COX-2 inhibitors in these settings.

5-FU, COX-2, HIF, Hypoxia, TNBC

Article Details

Supporting Agencies
This work was supported by National institutes of health (NIH) R01CA82337
How to Cite
Mori, N., Mironchik, Y., Wildes, F., Wu, S., Mori, K., Krishnamachary, B., & Bhujwalla, Z. (2020). HIF and COX-2 expression in triple negative breast cancer cells with hypoxia and 5-fluorouracil. Current Cancer Reports, 2(1), 54-63.


  1. Waks AG and Winer EP. Breast Cancer Treatment: A Review. JAMA, 2019, 321(3): 288-300.
  2. Hon JD, Singh B, Sahin A, et al. Breast cancer molecular subtypes: from TNBC to QNBC. American Journal of Cancer Research, 2016, 6(9): 1864-1872.
  3. Hudis CA and Gianni L. Triple-negative breast cancer: an unmet medical need. Oncologist, 2011, 16(Suppl 1): 1-11.
  4. Tan DS, Marchio C, Jones RL, et al. Triple negative breast cancer: molecular profiling and prognostic impact in adjuvant anthracycline-treated patients. Breast Cancer Res Treat, 2008, 111(1): 27-44.
  5. Graham K and Unger E. Overcoming tumor hypoxia as a barrier to radiotherapy, chemotherapy and immunotherapy in cancer treatment. International Journal of Nanomedicine, 2018, 13: 6049-6058.
  6. Schito L and Semenza GL. Hypoxia-Inducible Factors: Master Regulators of Cancer Progression. Trends Cancer, 2016, 2(12): 758-770.
  7. Semenza GL. Targeting HIF-1 for cancer therapy. Nature Reviews Cancer, 2003, 3(10): 721-732.
  8. Bertout JA, Patel SA and Simon MC. The impact of O2 availability on human cancer. Nature Reviews Cancer, 2008, 8(12): 967-975.
  9. O’Reilly EA, Gubbins L, Sharma S, et al. The fate of chemoresistance in triple negative breast cancer (TNBC). BBA Clinic, 2015, 3: 257-275.
  10. Helczynska K, Larsson AM, Holmquist Mengelbier L, et al. Hypoxia-inducible factor-2alpha correlates to distant recurrence and poor outcome in invasive breast cancer. Cancer Research, 2008, 68(22): 9212-9220.
  11. Shah T, Krishnamachary B, Wildes F, et al. HIF isoforms have divergent effects on invasion, metastasis, metabolism and formation of lipid droplets. Oncotarget, 2015, 6(29): 28104-28119.
  12. Keith B, Johnson RS and Simon MC. HIF1alpha and HIF2alpha: sibling rivalry in hypoxic tumour growth and progression. Nature Review Cancer, 2011, 12(1): 9-22.
  13. Bharti SK, Mironchik Y, Wildes F, et al. Metabolic consequences of HIF silencing in a triple negative human breast cancer xenograft. Oncotarget, 2018, 9(20): 15326-15339.
  14. Half E, Tang XM, Gwyn K, et al. Cyclooxygenase-2 expression in human breast cancers and adjacent ductal carcinoma in situ. Cancer Research, 2002, 62(6): 1676-1681.
  15. Ristimaki A, Sivula A, Lundin J, et al. Prognostic significance of elevated cyclooxygenase-2 expression in breast cancer. Cancer Research, 2002, 62(3): 632-635.
  16. Lim W, Park C, Shim MK, et al. Glucocorticoids suppress hypoxia-induced COX-2 and hypoxia inducible factor- 1alpha expression through the induction of glucocorticoidinduced leucine zipper. British Journal Of Pharmacology, 2014, 171(3): 735-745.
  17. Kaidi A, Qualtrough D, Williams AC, et al. Direct transcriptional up-regulation of cyclooxygenase-2 by hypoxiainducible factor (HIF)-1 promotes colorectal tumor cell survival and enhances HIF-1 transcriptional activity during hypoxia. Cancer Research, 2006, 66(13): 6683-6691.
  18. Xue X and Shah YM. Hypoxia-inducible factor-2alpha is essential in activating the COX2/mPGES-1/PGE2 signaling axis in colon cancer. Carcinogenesis, 2013, 34(1): 163-169.
  19. Zhao J, Du F, Shen G, et al. The role of hypoxia-inducible factor-2 in digestive system cancers. Cell Death & Disease, 2015, 6(1): e1600.
  20. Zhao CX, Luo CL and Wu XH. Hypoxia promotes 786- O cells invasiveness and resistance to sorafenib via HIF- 2alpha/COX-2. Medical Oncology, 2015, 32(1): 419.
  21. Stasinopoulos I, O’Brien DR, Wildes F, et al. Silencing of cyclooxygenase-2 inhibits metastasis and delays tumor onset of poorly differentiated metastatic breast cancer cells. Molecular Cancer Research, 2007, 5(5): 435-442.
  22. Stasinopoulos I, Mori N and Bhujwalla ZM. The malignant phenotype of breast cancer cells is reduced by COX-2 silencing. Neoplasia, 2008, 10(11): 1163-1169.
  23. Krishnamachary B, Stasinopoulos I, Kakkad S, et al. Breast cancer cell cyclooxygenase-2 expression alters extracellular matrix structure and function and numbers of cancer associated fibroblasts. Oncotarget, 2017, 8(11): 17981-17994.
  24. Harris RE, Casto BC and Harris ZM. Cyclooxygenase-2 and the inflammogenesis of breast cancer.World Journal of Clinical Oncology, 2014, 5(4): 677-692.
  25. de la Cueva A, Ramirez de Molina A, Alvarez-Ayerza N, et al. Combined 5-FU and ChoKalpha inhibitors as a new alternative therapy of colorectal cancer: evidence in human tumor-derived cell lines and mouse xenografts. PLoS One, 2013, 8(6): e64961.
  26. Hammond WA, Swaika A and Mody K. Pharmacologic resistance in colorectal cancer: a review. Therapeutic Advances in Medical Oncology, 2016, 8(1): 57-84.
  27. Matsuda N, Wang X, Lim B, et al. Safety and Efficacy of Panitumumab Plus Neoadjuvant Chemotherapy in Patients With Primary HER2-Negative Inflammatory Breast Cancer. JAMA Oncology, 2018, 4(9): 1207-1213.
  28. Krishnamachary B, Penet MF, Nimmagadda S, et al. Hypoxia regulates CD44 and its variant isoforms through HIF- 1alpha in triple negative breast cancer. PLoS One, 2012, 7(8): e44078.
  29. Strese S, Fryknas M, Larsson R, et al. Effects of hypoxia on human cancer cell line chemosensitivity. BMC Cancer, 2013, 13(1): 331.
  30. Dubsky P, Sevelda P, Jakesz R, et al. Anemia is a significant prognostic factor in local relapse-free survival of premenopausal primary breast cancer patients receiving adjuvant cyclophosphamide/methotrexate/5- fluorouracil chemotherapy. Clinical Cancer Research, 2008, 14(7): 2082-2087.
  31. Sakata K, Kwok TT, Murphy BJ, et al. Hypoxia-induced drug resistance: comparison to P-glycoprotein-associated drug resistance. British Journal Of Cancer, 1991, 64(5): 809- 814.
  32. Rohwer N, Dame C, Haugstetter A, et al. Hypoxia-inducible factor 1alpha determines gastric cancer chemosensitivity via modulation of p53 and NF-kappaB. PLoS One, 2010, 5(8): e12038.
  33. Ahmadi M, Ahmadihosseini Z, Allison SJ, et al. Hypoxia modulates the activity of a series of clinically approved tyrosine kinase inhibitors. British Journal Of Pharmacology, 2014, 171(1): 224-236.
  34. Westerlund I, Shi Y, Toskas K, et al. Combined epigenetic and differentiation-based treatment inhibits neuroblastoma tumor growth and links HIF2alpha to tumor suppression. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(30): e6137-e6146.
  35. Nakazawa MS, Eisinger-Mathason TS, Sadri N, et al. Epigenetic re-expression of HIF-2alpha suppresses soft tissue sarcoma growth. Nature Communications, 2016, 7(1): 1-13.
  36. Mercer SJ, Di Nicolantonio, F, Knight LA, et al. Rapid upregulation of cyclooxygenase-2 by 5-fluorouracil in human solid tumors. Anticancer Drugs, 2005, 16(5): 495-500.
  37. Bos PD, Zhang XH, Nadal C, et al. Genes that mediate breast cancer metastasis to the brain. Nature, 2009, 459(7249): 1005-1009.
  38. Sagara A, Igarashi K, Otsuka M, et al. Intrinsic Resistance to 5-Fluorouracil in a Brain Metastatic Variant of Human Breast Cancer Cell Line, MDA-MB-231BR. PLoS One, 2016, 11(10): e0164250.
  39. Chow LW, Loo WT, Wai CC, et al. Study of COX-2, Ki67, and p53 expression to predict effectiveness of 5-flurouracil, epirubicin and cyclophosphamide with celecoxib treatment in breast cancer patients. Biomed Pharmacother, 2005, 59 (Suppl 2): S298-301.
  40. Fabi A, Metro G, Papaldo P, et al. Impact of celecoxib on capecitabine tolerability and activity in pretreated metastatic breast cancer: results of a phase II study with biomarker evaluation. Cancer Chemother Pharmacol, 2008, 62(4): 717- 725.