PPARα activation can help prevent and treat non-small cell lung cancer

Abstract

Non-small cell lung cancer (NSCLC) not amenable to surgical resection has a high mortality rate, due to the ineffectiveness and toxicity of chemotherapy. Thus, there remains an urgent need of efficacious drugs that can combat this disease. In this study, we show that targeting the formation of proangiogenic epoxyeicosatrienoic acids (EET) by the cytochrome P450 arachidonic acid epoxygenases (Cyp2c) represents a new and safe mechanism to treat NSCLC growth and progression. In the transgenic murine K-Ras model and human orthotopic models of NSCLC, we found that Cyp2c44 could be downregulated by activating the transcription factor PPARα with the ligands bezafibrate and Wyeth-14,643. Notably, both treatments reduced primary and metastatic NSCLC growth, tumor angiogenesis, endothelial Cyp2c44 expression, and circulating EET levels. These beneficial effects were independent of the time of administration, whether before or after the onset of primary NSCLC, and they persisted after drug withdrawal, suggesting the benefits were durable. Our findings suggest that strategies to downregulate Cyp2c expression and/or its enzymatic activity may provide a safer and effective strategy to treat NSCLC. Moreover, as bezafibrate is a well-tolerated clinically approved drug used for managing lipidemia, our findings provide an immediate cue for clinical studies to evaluate the utility of PPARα ligands as safe agents for the treatment of lung cancer in humans.

Figure 1. EET analogs promote tumorigenesis

(A) Images of large T antigen/ras/Myc-transformed mouse fibroblast p60.5 cell-derived tumors grown in the mice indicated. Mice were left untreated or administered the EET analogs 2-(13-(3-pentylureido)tridec-8(Z)-enamido)succinic acid (EET Analog 1) or N-isopropyl-N-(5-((2-pivalamidobenzo[d]thiazol-4-yl)oxy)pentyl)heptanamide (EET Analog 2) for 4 days prior injection with tumor cells. Mice were further treated for 2 weeks and then sacrificed. (B, C) Weight (B) and volume (C) of tumors grown in the mice indicated (n=9). Circles show individual values, while bars show mean values. (D, E) Frozen sections of the tumors grown in the mice indicated were stained with anti-mouse CD31 antibodies and their vascularization was quantified as described in Materials and Methods. The values are means ± S.D. of 10 tumors/group with 2 images/tumor analyzed. (F) Primary lung endothelial cells were cultured with or without 14,15-EET or the EET analogs (EET A1 and EET A2) at the indicated concentrations. Cell proliferation was then evaluated as described in Materials and Methods. Values are means ± S.D. of 3 experiments performed in quadruplicate. p<0.05 between untreated WT vs. Cyp2c44KO (*) or untreated vs. treated Cyp2c44KO (**) cells.

Figure 2. Treatment of KRasLA2 mice with Wyeth-14,643

(A) Diagram showing the Wyeth-14,643 treatment protocol used in KRasLA2 mice. The number of mice/group is indicated in parenthesis. (B) Liver/body weight ratio of the KRasLA2 mice described in (A). Circles show individual values, while bars show mean values (C). Levels of EETs and DHETs in the plasma of the KRasLA2 mice described in (A). Values are averages ± SD of the mice indicated. (D) Hepatic microsomal fractions (20 μg/lane) of the KRasLA2 mice indicated were analyzed by Western blot for expression of Cyp2c44. Ponceau staining shows equal loading.

Figure 3. Wyeth-14,643 reduces primary NSCLC growth

Images (A) and hematoxylin and eosin staining (B) of lungs and lung paraffin sections derived from the KRasLA2 mice indicated. At sacrifice, the number (C) and size (D) of tumors (arrowhead) on the lung surface were evaluated. (C) Circles show individual values, while bars show mean values. (D) Distribution of tumor size (diameter) in the KRasLA2 mice indicated (groups 1–3, upper panel; groups 4–6, lower panel). The number in parenthesis indicates tumors analyzed.

Figure 4. Wyeth-14,643 reduces NSCLC angiogenesis and endothelial Cyp2c44 expression

(A, B) Frozen sections of lung tumors from the KrasLA2 mice indicated were stained with anti-mouse CD31 antibodies. Tumor vascularization was quantified as described in the Materials and Methods. The values are means ± SD of 10 tumors/group with 2 images/tumor analyzed. (C, D) Frozen sections of lung tumors from KrasLA2 mice in groups 2 and 3 (C) or groups 4–6 (D) were co-stained with anti-mouse CD31 and anti-mouse Cyp2c44 antibodies. Images were merged to visualize endothelial expression of Cyp2c44. T, tumor; V, vasculature.

Figure 5. Treatment of athymic mice orthotopically injected with human GFP-A549 cells with Wyeth-14,643

(A) Diagram showing the Wyeth-14,643 treatment protocol used in athymic mice. The number in parenthesis indicates mice used in each group. (B) Liver/body weight ratio of the mice described in (A). The circles show individual values, while bars show mean values. (C) Levels of EETs and DHETs in the plasma of the athymic mice described in (A). Values are averages ± SD of the mice indicated.

Figure 6. Wyeth-14,643 reduces the number of primary and metastatic GFP-A549 cell-derived tumors

(A) Lungs from athymic mice untreated (a) or treated with Wyeth-14,643 (b–d) isolated 5 weeks after GFP-A549 cell injection. (B) Number of GFP-positive primary (left lung) and metastatic (right lung and liver) tumors detected in freshly isolated organs 5 weeks after tumor cell injection. Values represent the mean +/− S.D. of the organs indicated.

Figure 7. Wyeth-14,643 reduces the size of primary and metastatic GFP-A549 cell-derived tumors

(A) Images of primary and metastatic tumors visible in the organs of athymic mice untreated (a) or treated with Wyeth-14,643 (b–d) 5 weeks after GFP-A549 cell injection. (B) Tumor area expressed as percentage of area occupied by GFP-positive structures per microscopic field. Values represent the mean +/− s.e.m. of the organs indicated.