HSP90 inhibitor PU-H71 increases radiosensitivity of breast cancer cells metastasized to visceral organs and alters the levels of inflammatory mediators

Abstract

Heat shock protein 90 (HSP90) inhibitors are considered as new radiosensitizing agents. PU-H71, a novel HSP90 inhibitor, is under evaluation for the treatment of advanced cancer. It is however not known whether PU-H71 alters radiosensitivity of metastatic breast cancer. Hence, we here evaluated mechanisms of possible anti-tumoral and radiosensitizing effects of PU-H71 on breast carcinoma cells metastasized to vital organs such as the liver and brain. The effect of PU-H71 on proliferation of breast carcinoma cells was determined using 4T1 cells and its brain (4TBM), liver (4TLM), and heart (4THM) metastatic subsets as well as non-metastatic 67NR cells. Changes in radiation sensitivity were determined by clonogenic assays. Changes in client proteins and levels of angiogenic and inflammatory mediators from these cancer cell cultures and ex vivo cultures were detected. PU-H71 alone inhibited ERK1/2, p38, and Akt activation and reduced N-cadherin and HER2 which further documented the anti-tumoral effects of PU-H71. The combination of PU-H71 and radiotherapy induced cytotoxic effect than PU-H71 alone, and PU-H71 showed a radiosensitizing effect in vitro. On the other hand, PU-H71 and radiation co-treatment increased p38 phosphorylation which is one of the hallmarks of inflammatory response. Accordingly, IL-6 secretion was increased following PU-H71 and radiotherapy co-treatment ex vivo. Levels of angiogenic and inflammatory factors such as MIP-2, SDF-1, and VEGF were increased under in vitro conditions but not under ex vivo conditions. These results demonstrated for the first time that PU-H71 enhances therapeutic effects of radiotherapy especially in highly metastatic breast carcinoma but a possible increase in inflammatory response should also be considered.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Abbreviations

HSP90:

Heat shock protein 90

SDF-1:

Stromal-derived factor-1

MIP-2:

Macrophage inflammatory protein-2

VEGF:

Vascular endothelial growth factor

IL-6:

Interleukin 6

RT:

Radiotherapy

4THM:

4T1 heart metastasis

4TLM:

4T1 liver metastasis

4TBM:

4T1 brain metastasis

References

  1. Adachi S et al (2010) HSP90 inhibitors induce desensitization of EGF receptor via p38 MAPK-mediated phosphorylation at Ser1046/1047 in human pancreatic cancer cells. Oncol Rep 23:1709–1714

    CAS  PubMed  Google Scholar 

  2. Akmansu M, Unsal D, Bora H, Elbeg S (2005) Influence of locoregional radiation treatment on tumor necrosis factor-alpha and interleukin-6 in the serum of patients with head and neck cancer. Cytokine 31:41–45. https://doi.org/10.1016/j.cyto.2005.02.009

    CAS  Article  PubMed  Google Scholar 

  3. Ambati SR et al (2014) Pre-clinical efficacy of PU-H71, a novel HSP90 inhibitor, alone and in combination with bortezomib in Ewing sarcoma. Mol Oncol 8:323–336. https://doi.org/10.1016/j.molonc.2013.12.005

    CAS  Article  PubMed  Google Scholar 

  4. Aslakson CJ, Miller FR (1992) Selective events in the metastatic process defined by analysis of the sequential dissemination of subpopulations of a mouse mammary tumor. Cancer res 52:1399–1405

    CAS  PubMed  Google Scholar 

  5. Bao L, Haque A, Jackson K, Hazari S, Moroz K, Jetly R, Dash S (2011) Increased expression of P-glycoprotein is associated with doxorubicin chemoresistance in the metastatic 4T1 breast cancer model. Am J Pathol 178:838–852. https://doi.org/10.1016/j.ajpath.2010.10.029

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Barbero S et al (2003) Stromal cell-derived factor 1alpha stimulates human glioblastoma cell growth through the activation of both extracellular signal-regulated kinases 1/2 and Akt. Cancer Res 63:1969–1974

    CAS  PubMed  Google Scholar 

  7. Bhagwat N et al (2014) Improved targeting of JAK2 leads to increased therapeutic efficacy in myeloproliferative neoplasms. Blood, 123:2075–2083. https://doi.org/10.1182/blood-2014-01-547760

  8. Butler LM, Ferraldeschi R, Armstrong HK, Centenera MM, Workman P (2015) Maximizing the therapeutic potential of HSP90 inhibitors. Mol cancer res 13:1445–1451. https://doi.org/10.1158/1541-7786.MCR-15-0234

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  9. Caldas-Lopes E et al (2009) Hsp90 inhibitor PU-H71, a multimodal inhibitor of malignancy, induces complete responses in triple-negative breast cancer models. Proc Natl Acad Sci U S A 106:8368–8373. https://doi.org/10.1073/pnas.0903392106

    Article  PubMed  PubMed Central  Google Scholar 

  10. Charafe-Jauffret E et al (2009) Breast cancer cell lines contain functional cancer stem cells with metastatic capacity and a distinct molecular signature. Cancer Res 69:1302–1313. https://doi.org/10.1158/0008-5472.CAN-08-2741

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Chin AR, Wang SE (2014) Cytokines driving breast cancer stemness. Mol Cell Endocrinol 382:598–602. https://doi.org/10.1016/j.mce.2013.03.024

    CAS  Article  PubMed  Google Scholar 

  12. Erin N, Boyer PJ, Bonneau RH, Clawson GA, Welch DR (2004) Capsaicin-mediated denervation of sensory neurons promotes mammary tumor metastasis to lung and heart. Anticancer Res 24:1003–1009

    PubMed  Google Scholar 

  13. Erin N, Kale S, Tanriover G, Koksoy S, Duymus O, Korcum AF (2013) Differential characteristics of heart, liver, and brain metastatic subsets of murine breast carcinoma. Breast Cancer Res Treat 139:677–689. https://doi.org/10.1007/s10549-013-2584-0

    CAS  Article  PubMed  Google Scholar 

  14. Erin N, Nizam E, Tanriover G, Koksoy S (2015a) Autocrine control of MIP-2 secretion from metastatic breast cancer cells is mediated by CXCR2: a mechanism for possible resistance to CXCR2 antagonists. Breast Cancer Res Treat 150:57–69. https://doi.org/10.1007/s10549-015-3297-3

    CAS  Article  PubMed  Google Scholar 

  15. Erin N, Podnos A, Tanriover G, Duymus O, Cote E, Khatri I, Gorczynski RM (2015b) Bidirectional effect of CD200 on breast cancer development and metastasis, with ultimate outcome determined by tumor aggressiveness and a cancer-induced inflammatory response. Oncogene 34:3860–3870. https://doi.org/10.1038/onc.2014.317

    CAS  Article  PubMed  Google Scholar 

  16. Erin N et al (2009) Altered gene expression in breast cancer liver metastases. Int J Cancer 124:1503–1516. https://doi.org/10.1002/ijc.24131

    CAS  Article  PubMed  Google Scholar 

  17. Erin N, Zhao W, Bylander J, Chase G, Clawson G (2006) Capsaicin-induced inactivation of sensory neurons promotes a more aggressive gene expression phenotype in breast cancer cells. Breast Cancer Res Treat 99:351–364. https://doi.org/10.1007/s10549-006-9219-7

    CAS  Article  PubMed  Google Scholar 

  18. Erin N, Bronson SK, Billingsley ML (2003) Calcium-dependent interaction of calcineurin with Bcl-2 in neuronal tissue. Neurosci. 117:541–555

    CAS  Article  Google Scholar 

  19. Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C (2006) Clonogenic assay of cells in vitro. Nat Protoc 1:2315–2319. https://doi.org/10.1038/nprot.2006.339

    CAS  Article  PubMed  Google Scholar 

  20. Gandhi N et al (2013) Novel Hsp90 inhibitor NVP-AUY922 radiosensitizes prostate cancer cells. Cancer biol ther 14:347–356. https://doi.org/10.4161/cbt.23626

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  21. Gao X et al (2015) Anti-VEGF treatment improves neurological function and augments radiation response in NF2 schwannoma model. Proc Natl Acad Sci U S A 112:14676–14681. https://doi.org/10.1073/pnas.1512570112

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Giulino-Roth L et al (2017) Inhibition of Hsp90 suppresses PI3K/AKT/mTOR signaling and has antitumor activity in Burkitt lymphoma. Mol Cancer Ther 16:1779–1790. https://doi.org/10.1158/1535-7163.MCT-16-0848

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  23. Goldstein RL et al (2015) Pharmacoproteomics identifies combinatorial therapy targets for diffuse large B cell lymphoma. J Clin Invest 125:4559–4571. https://doi.org/10.1172/JCI80714

    Article  PubMed  PubMed Central  Google Scholar 

  24. Guo A, Lu P, Lee J, Zhen C, Chiosis G, Wang YL (2017) HSP90 stabilizes B-cell receptor kinases in a multi-client interactome: PU-H71 induces CLL apoptosis in a cytoprotective microenvironment. Oncogene 36:3441–3449. https://doi.org/10.1038/onc.2016.494

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Gupta J, Nebreda AR (2015) Roles of p38alpha mitogen-activated protein kinase in mouse models of inflammatory diseases and cancer. FEBS J 282:1841–1857. https://doi.org/10.1111/febs.13250

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  26. Ha K, Fiskus W, Rao R, Balusu R, Venkannagari S, Nalabothula NR, Bhalla KN (2011) Hsp90 inhibitor-mediated disruption of chaperone association of ATR with hsp90 sensitizes cancer cells to DNA damage. Mol Cancer Ther 10:1194–1206. https://doi.org/10.1158/1535-7163.MCT-11-0094

    CAS  Article  PubMed  Google Scholar 

  27. Hashida S et al (2015) Hsp90 inhibitor NVP-AUY922 enhances the radiation sensitivity of lung cancer cell lines with acquired resistance to EGFR-tyrosine kinase inhibitors. Oncol Rep 33:1499–1504. https://doi.org/10.3892/or.2015.3735

    CAS  Article  PubMed  Google Scholar 

  28. Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D (2011) Global cancer statistics. CA Cancer J Clin 61:69–90. https://doi.org/10.3322/caac.20107

    Article  PubMed  Google Scholar 

  29. Kamal A, Thao L, Sensintaffar J, Zhang L, Boehm MF, Fritz LC, Burrows FJ (2003) A high-affinity conformation of Hsp90 confers tumour selectivity on Hsp90 inhibitors. Nature 425:407–410. https://doi.org/10.1038/nature01913

    CAS  Article  PubMed  Google Scholar 

  30. Knupfer H, Preiss R (2007) Significance of interleukin-6 (IL-6) in breast cancer (review). Breast Cancer Res Treat 102:129–135. https://doi.org/10.1007/s10549-006-9328-3

    CAS  Article  PubMed  Google Scholar 

  31. Kollmar O, Menger MD, Schilling MK (2006) Macrophage inflammatory protein-2 contributes to liver resection-induced acceleration of hepatic metastatic tumor growth. World J Gastroenterol 12:858–867

    CAS  Article  Google Scholar 

  32. Kwon Y et al (2015) MicroRNA-26a/-26b-COX-2-MIP-2 loop regulates allergic inflammation and allergic inflammation-promoted enhanced tumorigenic and metastatic potential of cancer cells. J Biol Chem 290:14245–14266. https://doi.org/10.1074/jbc.M115.645580

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  33. Lee J, Cacalano G, Camerato T, Toy K, Moore MW, Wood WI (1995) Chemokine binding and activities mediated by the mouse IL-8 receptor. J Immunol 155:2158–2164

    CAS  PubMed  Google Scholar 

  34. Lee Y et al (2016) The purine scaffold Hsp90 inhibitor PU-H71 sensitizes cancer cells to heavy ion radiation by inhibiting DNA repair by homologous recombination and non-homologous end joining. Radiother Oncol 121:162–168. https://doi.org/10.1016/j.radonc.2016.08.029

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Li HK, Matsumoto Y, Furusawa Y, Kamada T (2016) PU-H71, a novel Hsp90 inhibitor, as a potential cancer-specific sensitizer to carbon-ion beam therapy. J Radiat Res 57:572–575. https://doi.org/10.1093/jrr/rrw054

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Liu Y et al (2017) STK33 participates to HSP90-supported angiogenic program in hypoxic tumors by regulating HIF-1alpha/VEGF signaling pathway. Oncotarget 8:77474–77488. https://doi.org/10.18632/oncotarget.20535

    Article  PubMed  PubMed Central  Google Scholar 

  37. Lushchak VI (2014) Dissection of the hormetic curve: analysis of components and mechanisms. Dose Response 12:466–479. https://doi.org/10.2203/dose-response.13-051.Lushchak

    Article  PubMed  PubMed Central  Google Scholar 

  38. Mao AW, Jiang TH, Sun XJ, Peng J (2015) Application of chemokine receptor antagonist with stents reduces local inflammation and suppresses cancer growth. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 36:8637–8643. https://doi.org/10.1007/s13277-015-3557-1

    CAS  Article  Google Scholar 

  39. McMahon G (2000) VEGF receptor signaling in tumor angiogenesis. The oncologist 5(Suppl 1):3–10

    CAS  Article  Google Scholar 

  40. Mimnaugh EG, Chavany C, Neckers L (1996) Polyubiquitination and proteasomal degradation of the p185c-erbB-2 receptor protein-tyrosine kinase induced by geldanamycin. The Journal of biological chemistry 271:22796–22801

    CAS  Article  Google Scholar 

  41. Miyamoto Y et al (2001) Interleukin-6 inhibits radiation induced apoptosis in pancreatic cancer cells. Anticancer research 21:2449–2456

    CAS  PubMed  Google Scholar 

  42. Morimoto RI, Kline MP, Bimston DN, Cotto JJ (1997) The heat-shock response: regulation and function of heat-shock proteins and molecular chaperones. Essays Biochem 32:17–29

    CAS  PubMed  Google Scholar 

  43. Moulick K et al (2011) Affinity-based proteomics reveal cancer-specific networks coordinated by Hsp90. Nature chemical biology 7:818–826. https://doi.org/10.1038/nchembio.670

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. Nagaraju GP et al (2013) Antiangiogenic effects of ganetespib in colorectal cancer mediated through inhibition of HIF-1alpha and STAT-3. Angiogenesis 16:903–917. https://doi.org/10.1007/s10456-013-9364-7

    CAS  Article  PubMed  Google Scholar 

  45. Neckers L (2007) Heat shock protein 90: the cancer chaperone. J Biosci 32:517–530

    CAS  Article  Google Scholar 

  46. Pathak S et al (2015) Radiation and SN38 treatments modulate the expression of microRNAs, cytokines and chemokines in colon cancer cells in a p53-directed manner. Oncotarget. https://doi.org/10.18632/oncotarget.5815

  47. Provencio M, Sanchez A (2014) Therapeutic integration of new molecule-targeted therapies with radiotherapy in lung cancer. Translational lung cancer research 3:89–94. https://doi.org/10.3978/j.issn.2218-6751.2014.03.06

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  48. Qu Z, Wang S, Teng R, Yi X (2014) PU-H71 effectively induces degradation of IkappaB kinase beta in the presence of TNF-alpha. Mol Cell Biochem 386:135–142. https://doi.org/10.1007/s11010-013-1852-y

    CAS  Article  PubMed  Google Scholar 

  49. Rae C, Mairs RJ (2017) Evaluation of the radiosensitizing potency of chemotherapeutic agents in prostate cancer cells. Int J Radiat Biol 93:194–203. https://doi.org/10.1080/09553002.2017.1231946

    CAS  Article  PubMed  Google Scholar 

  50. Rezaei M, Friedrich K, Wielockx B, Kuzmanov A, Kettelhake A, Labelle M, Schnittler H, Baretton G, Breier G (2012) Interplay between neural-cadherin and vascular endothelial-cadherin in breast cancer progression. Breast cancer res 14:R154. https://doi.org/10.1186/bcr3367

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Seaton A, Maxwell PJ, Hill A, Gallagher R, Pettigrew J, Wilson RH, Waugh DJ (2009) Inhibition of constitutive and cxc-chemokine-induced NF-kappaB activity potentiates ansamycin-based HSP90-inhibitor cytotoxicity in castrate-resistant prostate cancer cells. Br j cancer 101:1620–1629. https://doi.org/10.1038/sj.bjc.6605356

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. Segawa T, Fujii Y, Tanaka A, Bando S, Okayasu R, Ohnishi K, Kubota N (2014) Radiosensitization of human lung cancer cells by the novel purine-scaffold Hsp90 inhibitor, PU-H71. Int j mol med 33:559–564. https://doi.org/10.3892/ijmm.2013.1594

    CAS  Article  PubMed  Google Scholar 

  53. Shen XY, Wang SH, Liang ML, Wang HB, Xiao L, Wang ZH (2008) The role and mechanism of CXCR4 and its ligand SDF-1 in the development of cervical cancer metastasis Ai zheng = Aizheng = Chinese journal of cancer 27:1044-1049

  54. Singh JK, Simoes BM, Howell SJ, Farnie G, Clarke RB (2013) Recent advances reveal IL-8 signaling as a potential key to targeting breast cancer stem cells. Breast cancer res 15:210. https://doi.org/10.1186/bcr3436

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  55. Terwisscha van Scheltinga AG et al (2014) Visualising dual downregulation of insulin-like growth factor receptor-1 and vascular endothelial growth factor-A by heat shock protein 90 inhibition effect in triple negative breast cancer. Eur j cancer 50:2508–2516. https://doi.org/10.1016/j.ejca.2014.06.008

    CAS  Article  PubMed  Google Scholar 

  56. Valastyan S, Weinberg RA (2011) Tumor metastasis: molecular insights and evolving paradigms. Cell 147:275–292. https://doi.org/10.1016/j.cell.2011.09.024

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  57. Wagner M, Bjerkvig R, Wiig H, Melero-Martin JM, Lin RZ, Klagsbrun M, Dudley AC (2012) Inflamed tumor-associated adipose tissue is a depot for macrophages that stimulate tumor growth and angiogenesis. Angiogenesis 15:481–495. https://doi.org/10.1007/s10456-012-9276-y

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  58. Weigelt B, Peterse JL, van’t Veer LJ (2005) Breast cancer metastasis: markers and models. Nat Rev Cancer 5:591–602. https://doi.org/10.1038/nrc1670

    CAS  Article  PubMed  Google Scholar 

  59. Wu CT, Chen MF, Chen WC, Hsieh CC (2013) The role of IL-6 in the radiation response of prostate cancer. Radiat oncol 8:159. https://doi.org/10.1186/1748-717X-8-159

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  60. Xiang L et al (2014) Ganetespib blocks HIF-1 activity and inhibits tumor growth, vascularization, stem cell maintenance, invasion, and metastasis in orthotopic mouse models of triple-negative breast cancer. J mol med 92:151–164. https://doi.org/10.1007/s00109-013-1102-5

    CAS  Article  PubMed  Google Scholar 

  61. Xu C, Zhao H, Chen H, Yao Q (2015) CXCR4 in breast cancer: oncogenic role and therapeutic targeting. Drug des devel ther 9:4953–4964. https://doi.org/10.2147/DDDT.S84932

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  62. Xu Y, Zhang C, Chen D, Zhao J, Shen Z, Wu Y, Zhu Y (2013) Effect of HSP90 inhibitor in pheochromocytoma PC12 cells: an experimental investigation. Tumour biology : the journal of the International Society for Oncodevelopmental Biology and Medicine 34:4065–4071. https://doi.org/10.1007/s13277-013-0996-4

    CAS  Article  Google Scholar 

  63. Yoshida S et al (2011) Low-dose Hsp90 inhibitors tumor-selectively sensitize bladder cancer cells to chemoradiotherapy. Cell cycle 10:4291–4299. https://doi.org/10.4161/cc.10.24.18616

    CAS  Article  PubMed  Google Scholar 

  64. Zong H et al (2015) A hyperactive signalosome in acute myeloid leukemia drives addiction to a tumor-specific Hsp90 species. Cell Rep 13:2159–2173. https://doi.org/10.1016/j.celrep.2015.10.073

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  65. Zuehlke A, Johnson JL (2010) Hsp90 and co-chaperones twist the functions of diverse client proteins. Biopolymers 93:211–217. https://doi.org/10.1002/bip.21292

    CAS  Article  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgments

This study was supported by Akdeniz University Research Unit, Grant No: 2014.03.0122.005.

Author information

Affiliations

Authors

Contributions

ŞK conducted the experiments; ŞK and NE were involved in planning and analyzing the experiments as well as writing the manuscript; AK and ED are involved in planning and conducting of the radiotherapy experiments.

Corresponding author

Correspondence to Nuray Erin.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material

ESM 1

(PDF 541 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Kale, Ş., Korcum, A.F., Dündar, E. et al. HSP90 inhibitor PU-H71 increases radiosensitivity of breast cancer cells metastasized to visceral organs and alters the levels of inflammatory mediators. Naunyn-Schmiedeberg's Arch Pharmacol 393, 253–262 (2020). https://doi.org/10.1007/s00210-019-01725-z

Download citation

Keywords

  • PU-H71
  • Radiosensitivity
  • HSP90 inhibitor
  • Breast cancer
  • Metastasis