Inhibiting delta-6 desaturase activity suppresses tumor growth in mice

Abstract

Recent studies have shown that a tumor-supportive microenvironment is characterized by high levels of pro-inflammatory and pro-angiogenic eicosanoids derived from omega-6 (n-6) arachidonic acid (AA). Although the metabolic pathways (COX, LOX, and P450) that generate these n-6 AA eicosanoids have been targeted, the role of endogenous AA production in tumorigenesis remains unexplored. Delta-6 desaturase (D6D) is the rate-limiting enzyme responsible for the synthesis of n-6 AA and increased D6D activity can lead to enhanced n-6 AA production. Here, we show that D6D activity is upregulated during melanoma and lung tumor growth and that suppressing D6D activity, either by RNAi knockdown or a specific D6D inhibitor, dramatically reduces tumor growth. Accordingly, the content of AA and AA-derived tumor-promoting metabolites is significantly decreased. Angiogenesis and inflammatory status are also reduced. These results identify D6D as a key factor for tumor growth and as a potential target for cancer therapy and prevention.

Figure 1. D6D activity is higher in tumor tissue than in adjacent non-tumor tissue.

(a) The activity of biomarkers of D6D in B16 melanoma. Upper: Gas chromatography showing the differences in LA, ETA and AA content between B16 melanoma and adjacent non-tumor tissue. Lower: The ratios of ETA/LA and AA/LA indicate D6D enzyme activity. **P<0.01, n = 4. (b) Activity of biomarkers of D6D in LLC tumors. Upper: Gas chromatography showing the differences in LA, ETA and AA content between LLC tumor and adjacent non-tumor tissue. Lower: The ratios of ETA/LA and AA/LA indicate D6D enzyme activity. **P<0.01, n = 4. (c) D6D mRNA levels in tumor and adjacent non-tumor tissue. **P<0.01, n = 3. (d) Western blot showing D6D protein levels in tumor and adjacent non-tumor tissue. (e) Correlation of the activity of biomarkers of D6D (based on ETA/LA and AA/LA ratios) with tumor size. (f) Correlation of D6D mRNA levels with tumor size.

Figure 2. Reducing D6D expression or activity suppresses tumor growth.

(a) Effect of D6D-RNAi knockdown on B16 melanoma growth. **P<0.001; n = 8 for both groups. (b) Effect of the D6D selective inhibitor SC-26196 on B16 melanoma growth. **P<0.0001; control, n = 7; treated, n = 8. (c) Effect of D6D-RNAi knockdown on LLC tumor growth. *P<0.05; n = 8 for both groups. (d) Effect of the D6D selective inhibitor SC-26196 on LLC tumor growth. **P<0.01; control, n = 7; treated, n = 10. (a–d) Upper left: Representative tumor sizes. Upper right: Average tumor weight at the end of the experiment. Lower: Tumor growth rate during the 14-day experimental period.

Figure 3. Decreased D6D reduces the levels of AA and AA-derived eicosanoids in tumor tissue.

Knocking down D6D (with RNAi) or inhibiting D6D (with selective inhibitor SC-26196) decreased (a) AA and (b) AA metabolite levels in mouse models of B16 melanoma and LLC. *P<0.05; **P<0.01; n = 3–5.

Figure 4. Decreased D6D suppresses tumor angiogenesis and inflammation.

(a) SC-26196, a D6D selective inhibitor, at 100 µM significantly inhibited basic fibroblast growth factor (bFGF)-induced angiogenesis in a Matrigel plug assay (**P<0.01; n = 3). (b, c) Immunohistological staining of microvessel density in tumors. Knocking down D6D (with RNAi) or inhibiting D6D (with selective inhibitor SC-26196) reduced angiogenesis in (b) B16 melanoma and (c) LLC tumors (**P<0.01; n = 3). (d) The mRNA expression of the inflammatory factors interleukin-6 (IL-6) and TNF-α is down-regulated in B16 melanoma and LLC lung tumors treated with D6D-RNAi or SC-26196, shown here by percent of control. **P<0.01; n = 4.