Pulmonary Stromal Cells Expressing CC-Chemokine Receptor 5 Promote Metastasis Via Erythroid Differentiation Regulator 1.

Abstract 3601 Poster Board III-538 BACKGROUND The protein erythroid differentiation regulator 1 (ERDR-1) is a stromal-derived factor that was previously found to induce hemoglobin synthesis in erythroleukmia cells and proliferation in hematopoietic progenitors (Dormer et al. Cytokine . 2004). However, previous work had not suggested a role for ERDR-1 in either local or distant tumor growth. We have previously shown that stromal cells, and in particular fibrocytes, which are CD45+ monocyte-derived stromal cell precursors, promote metastasis and this effect is partly dependent on expression of CC-chemokine receptor 5 (CCR5). Here, we sought to determine a mechanism for this finding. METHODS We used Affymetrix Mouse Arrrays to identify genes that were differentially expressed in lung homogenates from wild type (WT) and CCR5 −/− mice 6, 24 and 48 hours after the intravenous (i.v.) injection of B16-F10 tumor cells. After identification of ERDR-1 as a candidate gene, we evaluated WT and CCR5 −/− primary stromal cells for expression of ERDR-1. We also meausred ERDR-1 expression in mouse embryonic fibroblasts (MEFs), and knocked-down the gene in these cells using 2 different shRNA sequences specific for ERDR-1. A third line of MEFs expressing a scrambled (“mock”) shRNA sequence was generated as a control for non-specific effects. To model the effect of stromal ERDR-1 on metastasis, we transferred ERDR-1 knockdown or mock knockdown MEFs into CCR5 −/− or WT mice 48 hours prior to injection of B16-F10 cells. Lung nodules were counted 14 days after tumor injection. We also evaluated the effect of ERDR-1 knockdown on in vitro proliferation and survival. RESULTS ERDR-1 was overexpressed in the lungs of wild type (WT) compared to CCR5 −/− mice by 3.7, 2.7, and 3.5 fold at 6, 24, and 48 hours after tumor injection, respectively. Semi quantitative end-point PCR confirmed this observation in all mice tested (n = 30). Real Time RT-PCR revealed that the in vitro expression of ERDR-1 was higher in primary stromal cells from WT compared to CCR5 −/− mice by 5.2 fold (3.3 to 11.2, p −/− mice receiving ERDR-1 knockdown MEF lines prior to tumor injection we found a 37% and 50% decrease in the number of metastases compared to mice receiving the mock knockdown MEFs ( p = 0.06 and p −/− or WT stromal cells prior to tumor cells. These results indicated that endogenous ERDR-1 expression in WT but not CCR5 −/− mice was sufficient to compensate for lower ERDR-1 expression in transferred stromal cells. Interestingly, we also found that knockdown of ERDR-1 in MEFs led to enhanced apoptosis in vitro (1.7 and 2.8 –fold increase in Annexin-V+ cells) compared to mock knockdown MEFs, suggesting that ERDR-1 acts as a stromal cell survival factor. CONCLUSIONS The relative inability of primary stromal cells from CCR5 −/− mice to promote metastatic tumor growth in the lung was found to correlate with decreased expression of ERDR-1. Knocking down ERDR-1 substantially decreased the number of lung metastasis induced by WT MEFs. Thus, the production of ERDR-1 by stromal cells appears to be important for the CCR5-dependent promotion of metastatic tumor growth. Disclosures: No relevant conflicts of interest to declare.