11P - Expression of erythropoietin receptor variant forms is associated with lymphatic invasion and metastasis formation in breast cancer

Abstract

Background: Anemia not only impairs the quality of life of patients, but it leads to tumor hypoxia, resistance to therapy and reduces survival. Although recombinant Epo has revolutionized the treatment of anemia, clinical trials suggested a potential adverse effect of Epo on tumor recurrence and patient survival. Previously we identified altered EpoR mRNA transcripts in breast cancer cells lacking ligand binding domain exon 1-3 sequences in vitro. Our hypothesis is that altered expression of these proteins may be associated with unique clinicopathologic features. Methods: The expression level of C-and N-terminal portion of EpoR were measured by quantitative RT-PCR in 157 retrospective cohort of archived breast cancers. In order to identify the variant EpoR mRNA forms we have established a SMART RACE PCR assay using EpoR specific primer sets producing large overlapping sequences. Total RNA was frozen sections of 20 primary breast cancers after the H&E diagnoses.The products of the SMART RACE and nested PCR reactions was subjected to sequence analysis. Results: Out of the 157 samples 95 (61%) showed more than 2-fold change between the expression levels of the C and N-terminal regions of EpoR by quantitative RT-PCR. Significant correlation was found between the expression levels of EpoR mRNA variants and lymphatic invasion of the tumors. Logistic regression model demonstrated the association of metastatic capacity and expression differences of extra and intracellular regions of EpoR in primary breast cancers (P < 0.05, greater than 2-fold change). Using a SMART RACE PCR assay a single EpoR mRNA species at ∼1500 kb, corresponding to the full length wild type EpoR mRNA, and several distinct bands at ∼1300 kb, ∼1000 kb and ∼800 kb, lacking exon 1-4 N-terminal ligand binding domain, were seen in the samples. Conclusions: We showed that different variant forms of EPOR present not only in vitro but also in the individual breast tumor samples. These altered forms of EpoR may be responsible for the observed differences in the Epo responsiveness of EpoR bearing breast cancers. Legal entity responsible for the study: Zsuzsa Rakosy. Funding: OTKA PD 116996, 2015-2018. Disclosure: The author has declared no conflicts of interest.