Expression of the Epidermal Growth Factor Receptors and Ligands in Paired Samples of Normal Breast Tissue, Primary Breast Carcinomas and Lymph Node Metastases

Anja Brügmann, V. Jensen, J. P. Garne, E. Nexo, B. S. Sorensen

Department of Breast Surgery, Aalborg Hospital, Aalborg, Denmark.
Department of Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark.
Institute of Pathology, Aalborg Hospital, Aalborg, Denmark.
Institute of Pathology, Aarhus University Hospital, Aarhus, Denmark.

DOI: 10.4236/abcr.2014.32005                

Abstract

Purpose: In breast cancer, the EGF receptors host an increasing number of therapeutic targets and the interactive mechanisms of actions of the receptors and their ligands justify investigation of the EGF family as an entity. Experimental design: Paired tissue samples of normal breast tissue and primary breast carcinomas were examined in a prospective study of 163 patients. A third sample was obtained from the paired ipsilateral metastatic lymph node from 58 of these patients. The mRNA expression of four EGF receptors (HER1 - HER4) and 11 activating ligands was quantified with real-time RT-PCR. Results: Expression of HER2, HER3, and HER4 mRNA was upregulated in primary carcinomas compared to normal breast tissue while HER1 was downregulated. The mRNA expression of HER3 and HER4 differed between primary breast carcinomas and lymph node metastases whereas there was no difference in the expression of HER1 and HER2. The combination of low HER3 and low HER4 expression in the primary carcinoma was significantly more frequent in lymph node-negative patients as compared to lymph node positive patients. Distinct correlation patterns of the receptors and their corresponding activating ligands appeared in both normal breast tissue and in carcinomas, notably for the HER3 and HER4 receptors and their 3 specific ligands: HB-EGF, NRG2, and NRG4. Conclusion: HER2, HER3, and HER4 showed increased mRNA expression in carcinomas and were positively correlated to each other and to specific activating ligands. Furthermore, low HER3 and HER4 expression in the carcinomas correlated to the absence of lymph node metastases.

Keywords

Breast Cancer, Lymph Node Metastases, EGF Receptors, EGF Ligands, RT-PCR

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Zahnow, C.A. (2006) ErbB Receptors and Their Ligands in the Breast. Expert Reviews in Molecular Medicine, 23, 121.
[2]	Kalous, O., Conklin, D., Desai, A.J., O’Brien, N.A., Ginther, C., Anderson, L., et al. (2012) Dacomitinib (PF00299804), an Irreversible Pan-HER Inhibitor, Inhibits Proliferation of HER2-Amplified Breast Cancer Cell Lines Resistant to Trastuzumab and Lapatinib. Molecular Cancer Therapeutics, 9, 1978-1987. http://dx.doi.org/10.1158/1535-7163.MCT-11-0730
[3]	McGuire, W.L. (1987) Prognostic Factors for Recurrence and Survival in Human Breast Cancer. Breast Cancer Research and Treatment, 1, 5-9. http://dx.doi.org/10.1007/BF01806129
[4]	Carter, C.L., Allen, C. and Henson, D.E. (1989) Relation of Tumor Size, Lymph Node Status, and Survival in 24, 740 Breast Cancer Cases. Cancer, 1, 181-187. http://dx.doi.org/10.1002/1097-0142 (19890101)63:1<181::AID-CNCR2820630129>3.0.CO;2-H
[5]	Li, J., Gromov, P., Gromova, I., Moreira, J.M., Timmermans-Wielenga, V., Rank, F., et al. (2008) Omics-Based Profiling of Carcinoma of the Breast and Matched Regional Lymph Node Metastasis. Proteomics, 23-24, 5038-5052. http://dx.doi.org/10.1002/pmic.200800303
[6]	Santinelli, A., Pisa, E., Stramazzotti, D. and Fabris, G. (2008) HER-2 Status Discrepancy between Primary Breast Cancer And Metastatic Sites. Impact on target therapy. International Journal of Cancer, 5, 999-1004.
[7]	Vecchi, M., Confalonieri, S., Nuciforo, P., Vigano, M.A., Capra, M., Bianchi, M., et al. (2008) Breast Cancer Metastases Are Molecularly Distinct from their Primary Tumors. Oncogene, 15, 2148-2158. http://dx.doi.org/10.1038/sj.onc.1210858
[8]	Feng, Y., Sun, B., Li, X., Zhang, L., Niu, Y., Xiao, C., et al. (2007) Differentially Expressed Genes between Primary Cancer and Paired Lymph Node Metastases Predict Clinical Outcome of Node-Positive Breast Cancer Patients. Breast Cancer Research and Treatment, 3, 319-329. http://dx.doi.org/10.1007/s10549-006-9385-7
[9]	Pandit, T.S., Kennette, W., Mackenzie, L., Zhang, G., Al-Katib, W., Andrews, J., et al. (2009) Lymphatic Metastasis of Breast Cancer Cells Is Associated with Differential Gene Expression Profiles That Predict Cancer Stem Cell-Like Properties and the Ability to Survive, Establish and Grow in a Foreign Environment. International Journal of Oncology, 2, 297-308.
[10]	Fuchs, I.B., Siemer, I., Buhler, H., Schmider, A., Henrich, W., Lichtenegger, W., et al. (2006) Epidermal Growth Factor Receptor Changes during Breast Cancer Metastasis. Anticancer Research, 6B, 4397-4401.
[11]	Cardoso, F., Di, L.A., Larsimont, D., Gancberg, D., Rouas, G., Dolci, S., et al. (2001) Evaluation of HER2, p53, bcl-2, Topoisomerase II-Alpha, Heat Shock Proteins 27 and 70 in Primary Breast Cancer and Metastatic Ipsilateral Axillary Lymph Nodes. Annals of Oncology, 5, 615-620. http://dx.doi.org/10.1023/A:1011182524684
[12]	Stern, D.F. (2003) ErbBs in Mammary Development. Experimental Cell Research, 1, 89-98. http://dx.doi.org/10.1016/S0014-4827(02)00103-9
[13]	Paez, J.G., Janne, P.A., Lee, J.C., Tracy, S., Greulich, H., Gabriel, S., et al. (2004) EGFR Mutations in Lung Cancer, Correlation with Clinical Response to Gefitinib Therapy. Science, 5676, 1497-1500. http://dx.doi.org/10.1126/science.1099314
[14]	Bang, Y.J, Van, C.E., Feyereislova, A., Chung, H.C., Shen, L., Sawaki, A., et al. (2010) Trastuzumab in Combination with Chemotherapy versus Chemotherapy Alone for Treatment of HER2-Positive Advanced Gastric or Gastro-Oesophageal Junction Cancer (ToGA): a Phase 3, Open-Label, Randomised Controlled Trial. Lancet, 9742, 687-697. http://dx.doi.org/10.1016/S0140-6736(10) 61121-X
[15]	Siena, S., Sartore-Bianchi, A., Di, N.F., Balfour, J. and Bardelli, A. (2009) Biomarkers Predicting Clinical Outcome of Epidermal Growth Factor Receptor-Targeted Therapy in Metastatic Colorectal Cancer. Journal of the National Cancer Institute, 19, 1308-1324. http://dx.doi.org/10.1093/jnci/djp280
[16]	Riese, D.J. and Stern, D.F. (1998) Specificity within the EGF Family/ErbB Receptor Family Signaling Network. Bioessays, 1, 41-48. http://dx.doi.org/10.1002/(SICI)1521-1878(199801)20:1<41::AID-BIES7>3.0.CO;2-V
[17]	Citri, A. and Yarden, Y. (2006) EGF-ERBB Signalling: Towards the Systems Level. Nature Reviews Molecular Cell Biology, 7, 505-516. http://dx.doi.org/10.1038/nrm1962
[18]	Wilson, K.J., Gilmore, J.L., Foley, J., Lemmon, M.A. and Riese, D.J. (2009) Functional Selectivity of EGF Family Peptide Growth Factors: Implications for Cancer. Pharmacology & Therapeutics, 1, 1-8. http://dx.doi.org/10.1016/j.pharmthera.2008.11.008
[19]	Sassen, A., Rochon, J., Wild, P., Hartmann, A., Hofstaedter, F., Schwarz, S., et al. (2008) Cytogenetic Analysis of HER1/EGFR, HER2, HER3 and HER4 in 278 Breast Cancer Patients. Breast Cancer Research, 1, R2. http://dx.doi.org/10.1186/bcr1843
[20]	McIntyre, E., Blackburn, E., Brown, P.J., Johnson, C.G. and Gullick, W.J. (2010) The Complete Family of Epidermal Growth Factor Receptors and Their Ligands Are Co-Ordinately Expressed in Breast Cancer. Breast Cancer Research and Treatment, 1, 105-110. http://dx.doi.org/10.1007/s10549-009-0536-5
[21]	Carlsson, J., Nordgren, H, Sjostrom, J., Wester, K., Villman, K. and Bengtsson, N.O., et al. (2004) HER2 Expression in Breast Cancer Primary Tumours and Corresponding Metastases. Original Data and Literature Review. British Journal of Cancer, 12, 2344-2348.
[22]	Gancberg, D., Di, L.A., Cardoso, F., Rouas, G., Pedrocchi, M. and Paesmans, M., et al. (2002) Comparison of HER-2 Status between Primary Breast Cancer and Corresponding Distant Metastatic Sites. Annals of Oncology, 7, 1036-1043. http://dx.doi.org/10.1093/annonc/mdf252
[23]	Gong, Y., Booser, D.J. and Sneige, N. (2005) Comparison of HER-2 Status Determined by Fluorescence in Situ Hybridization in Primary and Metastatic Breast Carcinoma. Cancer, 9, 1763-1769. http://dx.doi.org/10.1002/cncr.20987
[24]	Tapia, C., Savic, S., Wagner, U., Schonegg, R., Novotny, H. and Grilli, B., et al. (2007) HER2 Gene Status in Primary Breast Cancers and Matched Distant Metastases. Breast Cancer Research, 3, 31. http://dx.doi.org/10.1186/bcr1676
[25]	Zidan, J., Dashkovsky, I., Stayerman, C., Basher, W., Cozacov, C. and Hadary, A. (2005) Comparison of HER-2 Overexpression in Primary Breast Cancer and Metastatic Sites and Its Effect on Biological Targeting Therapy of Metastatic Disease. British Journal of Cancer, 5, 552-556. http://dx.doi.org/10.1038/sj.bjc.6602738
[26]	Santiago, M.P., Vazquez-Boquete, A., Fernandez, B., Masa, C., Antunez, J.R. and Fraga, M., et al. (2009) Whether to Determine HER2 Status for Breast Cancer in the Primary Tumour or in the Metastasis. Histology and Histopathology, 6, 675-682.
[27]	Ohashi, Y., Creek, K.E., Pirisi, L., Kalus, R. and Young, S.R. (2004) RNA Degradation in Human Breast Tissue after Surgical Removal: A Time-Course Study. Experimental and Molecular Pathology, 2, 98-103. http://dx.doi.org/10.1016/j.yexmp.2004.05.005
[28]	Chomczynski, P. and Sacchi, N. (1987) Single-Step Method of RNA Isolation by Acid Guanidinium Thiocyanate-Phenol-Chloroform Extraction. Analytical Biochemistry, 1, 156-159. http://dx.doi.org/10.1016/0003-2697(87)90021-2
[29]	Andersen, C.L., Jensen, J.L. and Orntoft, T.F. (2004) Normalization of Real-Time Quantitative Reverse Transcription-PCR Data: A Model-Based Variance Estimation Approach to Identify Genes Suited for Normalization, Applied to Bladder and Colon Cancer Data Sets. Cancer Research, 15, 5245-5250. http://dx.doi.org/10.1158/0008-5472.CAN-04-0496
[30]	Holbro, T., Beerli, R.R., Maurer, F., Koziczak, M., Barbas III, C.F. and Hynes, N.E. (2003) The ErbB2/ErbB3 Heterodimer Functions as an Oncogenic Unit: ErbB2 Requires ErbB3 to Drive Breast Tumor Cell Proliferation. Proceedings of National Academy of Science of the United States of America, 15, 8933-8938. http://dx.doi.org/10.1073/pnas.1537685100
[31]	Gasparini, G., Gullick, W.J., Maluta, S., Dalla, P.P., Caffo, O. and Leonardi, E., et al. (1994) c-erbB-3 and c-erbB-2 Protein Expression in Node-Negative Breast Carcinoma—An Immunocytochemical Study. European Journal of Cancer, 1, 16-22. http://dx.doi.org/10.1016/S0959-8049(05)80010-3
[32]	Travis, A., Pinder, S.E., Robertson, J.F., Bell, J.A., Wencyk, P. and Gullick, W.J., et al. (1996) C-erbB-3 in Human Breast Carcinoma: Expression and Relation to Prognosis and Established Prognostic Indicators. British Journal of Cancer, 2, 229-233. http://dx.doi.org/10.1038/bjc.1996.342
[33]	Witton, C.J., Reeves, J.R., Going, J.J., Cooke, T.G. and Bartlett, J.M. (2003) Expression of the HER1-4 Family of Receptor Tyrosine Kinases in Breast Cancer. The Journal of Pathology, 3, 290-297. http://dx.doi.org/10.1002/path.1370
[34]	Amin, D.N., Campbell, M.R. and Moasser, M.M. (2010) The Role of HER3, the Unpretentious Member of the HER Family, in Cancer Biology and Cancer Therapeutics. Seminars in Cell & Developmental Biology, 9, 944-950. http://dx.doi.org/10.1016/j.semcdb.2010.08.007
[35]	Garrett, J.T., Olivares, M.G., Rinehart, C., Granja-Ingram, N.D., Sanchez, V. and Chakrabarty, A., et al. (2011) Transcriptional and Posttranslational Up-Regulation of HER3 (ErbB3) Compensates for Inhibition of the HER2 Tyrosine Kinase. Proceedings of National Academy of Science of the United States of America, 12, 5021-5026. http://dx.doi.org/10.1073/pnas.1016140108
[36]	Garrett, J.T., Sutton, C.R., Kuba, M.G., Cook, R.S. and Arteaga, C.L. (2013) Dual Blockade of HER2 in HER2-Overexpressing Tumor Cells Does Not Completely Eliminate HER3 Function. Clinical Cancer Research, 3, 610-619. http://dx.doi.org/10.1158/1078-0432.CCR-12-2024
[37]	Vaught, D.B., Stanford, J.C., Young, C., Hicks, D.J., Wheeler, F. and Rinehart, C., et al. (2012) HER3 is Required for HER2-Induced Preneoplastic Changes to the Breast Epithelium and Tumor Formation. Cancer Research, 10, 2672-2682. http://dx.doi.org/10.1158/0008-5472.CAN-11-3594
[38]	Citri, A., Skaria, K.B. and Yarden, Y. (2003) The Deaf and the Dumb: The Biology of ErbB-2 and ErbB-3. Experimental Cell Research, 1, 54-65. http://dx.doi.org/10.1016/S0014-4827(02)00101-5
[39]	Earp III, H.S., Calvo, B.F. and Sartor, C.I. (2003) The EGF Receptor Family—Multiple Roles in Proliferation, Differentiation, and Neoplasia with an Emphasis on HER4. Transactions of the American Clinical and Climatological Association, 315-333.
[40]	Jones, F.E. (2008) HER4 Intracellular Domain (4ICD) Activity in the Developing Mammary Gland and Breast Cancer. Journal of Mammary Gland Biology and Neoplasia, 2, 247-258. http://dx.doi.org/10.1007/s10911-008-9076-6
[41]	Muraoka-Cook, R.S., Sandahl, M.A., Strunk, K.E., Miraglia, L.C., Husted, C. and Hunter, D.M., et al. (2009) ErbB4 Splice Variants Cyt1 and Cyt2 Differ by 16 Amino Acids and Exert Opposing Effects on the Mammary Epithelium in Vivo. Molecular and Cellular Biology, 18, 4935-4948.
[42]	Sartor, C.I., Zhou, H., Kozlowska, E., Guttridge, K., Kawata, E. and Caskey, L., et al. (2001) Her4 Mediates ligand-Dependent Antiproliferative and Differentiation Responses in Human Breast Cancer Cells. Molecular and Cellular Biology, 13, 4265-4675.
[43]	Thor, A.D., Edgerton, S.M. and Jones, F.E. (2009) Subcellular Localization of the HER4 Intracellular Domain, 4ICD, Identifies Distinct Prognostic Outcomes for Breast Cancer Patients. American Journal of Pathology, 5, 1802-1809.
[44]	Das, P.M., Thor, A.D., Edgerton, S.M., Barry, S.K., Chen, D.F. and Jones, F.E. (2010) Reactivation of Epigenetically Silenced HER4/ERBB4 Results in Apoptosis of Breast Tumor Cells. Oncogene, 37, 5214-5219.
[45]	Foley, J., Nickerson, N.K., Nam, S., Allen, K.T., Gilmore, J.L. and Nephew, K.P., et al. (2010) EGFR Signaling in Breast Cancer: Bad to the Bone. Seminars in Cell & Developmental Biology, 9, 951-960.
[46]	Revillion, F., Lhotellier, V., Hornez, L., Bonneterre, J. and Peyrat, J.P. (2008) ErbB/HER Ligands in Human Breast Cancer, and Relationships with Their Receptors, the Bio-Pathological Features and Prognosis. Annals of Oncology, 1, 73-80.
[47]	Dong, J., Opresko, L.K., Chrisler, W., Orr, G., Quesenberry, R.D. and Lauffenburger, D.A., et al. (2005) The Membrane-Anchoring Domain of Epidermal Growth Factor Receptor Ligands Dictates Their Ability to Operate in Juxtacrine Mode. Molecular Biology of the Cell, 6, 2984-2998.