Novel View on Unwanted Side Effects of Radio-Chemotherapy on Bone Marrow (BM) Microenvironment - Radio-Chemotherapy Upregulates BM-Level of Bioactive Lipids, Sphingosine-1- Phosphate (S1P) and Ceramide-1-Phosphate (C1P), That Chemoattract Metastasizing Cancer Cells

Rhabdomyosracoma (RMS), the most common soft-tissue sarcoma of adolescents and children, frequently infiltrates the BM to such a degree that it often mimics acute lymphoblastic leukemia. The prognosis is poor, particularly for the more aggressive and metastatic alveolar RMS (ARMS) compared to embryonal RMS (ERMS). In our previous work, we demonstrated a pivotal role for two signaling axes, a-chemokine stromal-derived factor-1 (SDF-1)–CXCR4 (a seven-transmembrane-spanning G protein-coupled receptor) and hepatocyte growth factor/scatter factor (HGF/SF)–c-met, in metastasis of pediatric sarcomas to bone marrow (BM) (Blood 2002;100:2597-2606,Cancer Research 2003; 63:7926–7935, IJC 2010;127: 2554–2568). Recently, however, we observed that the bioactive lipids sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) are much more potent chemotractants for human rhabdomyosarcoma (RMS) than SDF-1 or HGF/SF. Importantly, we observed that S1P and C1P levels are highly increased in BM after radio-chemotherapy. Hypothesis: Based on these observations, we hypothesized that S1P and C1P direct chemotaxis of RMS cells to BM. This could be particularly important in patients treated with radio-chemotherapy, where upregulation of S1P and C1P levels in BM may facilitate the spread to the bones of tumor cells that survived initial treatment. Material and Methods: Several complementary in vitro and in vivo approaches were employed to demonstrate a novel role of bioactive lipids in BM metastasis of RMS cells. The expression of S1P seven-transmembrane-spanning G protein-coupled receptors, chemotaxis, adhesion, proliferation, and cell signaling studies in response to S1P and C1P were performed on 8 human ARMS and 3 human ERMS cell lines. The secretion of S1P and C1P in BM and by RMS cells was measured by mass spectrometry (MS). The S1P1 receptor was downregulated by employing an shRNA strategy and S1P1-KO cells were evaluated for their ability to grow tumors in immunodeficient mice. Finally, to address the role of the S1 P–S 1P1 axis in the unwanted spread of sarcoma cells after radio-chemotherapy, we compared seeding of S1P1-KO and control RMS cells in irradiated immunodeficient mice. Results: S1P and C1P are much more potent chemoattractants than SDF-1 or HGF/SF, particularly if employed at “physiological” tissue concentrations. S1P1–5 receptors are expressed on RMS cells and stimulation by S1P induced chemotaxis, adhesion of these cells, and phosphorylation of MAPPp42/44 and AKT. However, while receptor/s for C1P have not yet been identified, C1P also exerted similar effects on human RMS cells. Finally, S1P1-KO cells grew smaller tumors in immunodefcient mice and had impaired seeding efficiency in irradiated animals compared to control RMS cells transduced with empty vector. In parallel experiments, we also observed that both bioactive lipids increase stromalization of the RMS by i) chemoattracting and activating cancer-associated fibroblasts (CAF) and ii) promoting tumor angiogenesis. Conclusions: Both systemic and local radio-chemotherapy leads to upregulation of bioactive lipids in damaged tissues and side effect of such treatment is induction of unwanted prometastatic microenvironment in different organs. By employing an RMS model, we confirmed S1P and identified C1P as novel under-appreciated factor directing metastasis of cancer cells. Since S1P and C1P become upregulated in BM after radio-chemotherapy, both bioactive lipids are involved in the unwanted spread to the bones of RMS cells that survived initial treatment. The role of S1P and C1P in metastasis of other pediatric sarcomas and other types of solid tumors and dissemination of leukemias/lymphomas is currently being investigated in our laboratory, similarly as different strategies to inhibit pro-metastatic effects of S1P and C1P.

No relevant conflicts of interest to declare.