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Sphingosine Kinase 2 Regulates Non-Cell Autonomous Tumor Suppression Functions of p53 in the Tumor Microenvironment of Metastatic Breast Cancer
Cynthia Weigel1,2*, Melissa A. Maczis1,2*, Elisa N.D. Palladino1,2, Christopher D. Green1,2, Michael Maceyka1,2, Chunqing Guo2,3, Xiang-Yang Wang2,3, Mikhail G. Dozmorov2,4, Sheldon Milstien1,2, and Sarah Spiegel1,21 Department of Biochemistry and Molecular Biology, VCU School of Medicine, Richmond, VA, USA
2 Massey Cancer Center, VCU School of Medicine, Richmond, VA, USA
3 Department of Human and Molecular Genetics, VCU School of Medicine, Richmond, VA, USA
4 Departments of Biostatistics and Pathology, VCU School of Medicine, Richmond, VA, USA
* contributed equally
Background: Breast cancer is the second most common cancer in women. Although research has aimed to prevent and treat metastatic breast cancer effectively, cancer-related deaths due to metastasis have not changed much over the last decades. Therefore, the development of targeted therapies is urgently needed. Mutual interactions between cancer cells and the tumor microenvironment, composed of extracellular matrix (ECM) and various cell types, including immune cells, endothelial cells, and fibroblasts, are essential for cancer progression, metastasis, and prognosis. The bioactive sphingolipid metabolite sphingosine-1-phosphate (S1P) regulates fundamental biological processes that are important for breast cancer progression and metastasis, including cell growth, survival, motility, invasion, and immune cell trafficking. S1P is produced by the phosphorylation of sphingosine by two closely related sphingosine kinases (SphK), SphK1 and SphK2. SphK1 expression is elevated in breast cancer patients and correlates with poor prognosis and resistance to chemotherapy. In mice, SphK1 in tumors increased circulating S1P levels, primed distant premetastatic niches, and correlated with breast cancer growth and pulmonary metastasis. However, it has been suggested that the communication between the host organism and cancer cells is changed by systemic SphK1/S1P signaling to regulate lung colonization and metastasis.
Aims: As nothing is yet known about the role of SphK2 in crosstalk between the tumor microenvironment and cancer cells, we examined the effect of the deletion of endogenous SphK2 on syngeneic breast tumor growth and lung metastasis.
Methods: To study tumor progression and lung metastasis, E0771.LMB, MMTV-Wnt1, and E0771.luc murine breast cancer cells were bilaterally implanted in mammary fat pads of SphK2 knock-out mice, wild-type littermates, or immunodeficient Rag2-/-Il2rg-/- mice. To determine metastatic pulmonary colonization, mice were injected intravenously with E0771 cells. In some experiments, mice were injected intraperitoneally with the SphK2 inhibitor SLM6031434 or vehicle. Tumor growth and the formation of lung metastatic nodules were determined. To assess the involvement of immune cells, tumor samples and lung tissue were evaluated by flow cytometry. Furthermore, primary murine cells from SphK2 knock-out and wild-type mice, mouse embryonic fibroblasts, and primary human mammary fibroblasts from normal human breast tissue were used for in vitro co-culture experiments with cancer cells, targeted lipidomics by mass spectrometry, or RNAseq. Conditioned media from those cells was used to determine the effects on cancer cell proliferation, migration, invasion, and multicellular tumor spheroid growth and invasion.
Results: We found that deletion or inhibition of the S1P-producing enzyme SphK2 in mice suppressed syngeneic breast tumor growth and lung metastasis by creating an anti-tumor microenvironment. Inhibition or deletion of SphK2 decreased S1P levels in tissue, increased ceramides, including C16-ceramide and its target, the tumor suppressor p53, specifically in stromal fibroblasts, limiting their activation to cancer-associated fibroblasts (CAFs). Their restrained production of tumor-promoting factors, including cytokines, growth factors, and ECM-degrading enzymes, resulted in an unfavorable host tumor microenvironment that restricted cancer establishment, growth, and metastasis. Deletion of p53 in SphK2-deficient fibroblasts reversed these effects and enhanced the expression of pro-tumorigenic factors, enabled their activation to CAFs, and promoted tumor growth and invasion.
Conclusions: Our in vitro and in vivo data support direct CAF-tumor interactions as predominant drivers for tumor growth, independent of immune cells. In summary, we uncovered a novel role of SphK2 in regulating non-cell-autonomous, tumor-suppressive functions of p53 in stromal fibroblasts, restricting the generation of tumor-promoting CAFs. This work may lead to new strategies to target SphK2 in the tumor microenvironment and enhance therapeutic efficacy in breast cancer treatment.
This work was supported by the National Institutes of Health (R01 GM043880 to S.S.) and the Department of Defense (W81XWH2010434 to S.S). M.A.M. was supported by the Ruth L. Kirschstein Individual Predoctoral National Service Award (F31 CA220798). The authors acknowledge the Virginia Commonwealth University Lipidomics/Metabolomics, Cancer Mouse Models, Flow Cytometry, and Microscopy Shared Resources (supported in part by funding from the NIH-NCI Cancer Center Support Grant P30 CA016059).
Speakers
Cynthia Weigel