De novo lipogenesis represents a therapeutic target in mutant Kras non-small cell lung cancer

Abstract

Oncogenic Kras mutations are one of the most common alterations in non-small cell lung cancer and are associated with poor response to treatment and reduced survival. Driver oncogenes, such as Kras are now appreciated for their ability to promote tumor growth via up-regulation of anabolic pathways. Therefore, we wanted to identify metabolic vulnerabilities in Kras-mutant lung cancer. Using the Kras ^LSL-G12D lung cancer model, we show that mutant Kras drives a lipogenic gene-expression program. Stable-isotope analysis reveals that mutant Kras promotes de novo fatty acid synthesis in vitro and in vivo. The importance of fatty acid synthesis in Kras-induced tumorigenesis was evident by decreased tumor formation in Kras ^LSL-G12D mice after treatment with a fatty acid synthesis inhibitor. Importantly, with gain and loss of function models of mutant Kras, we demonstrate that mutant Kras potentiates the growth inhibitory effects of several fatty acid synthesis inhibitors. These studies highlight the potential to target mutant Kras tumors by taking advantage of the lipogenic phenotype induced by mutant Kras.-Singh, A., Ruiz, C., Bhalla, K., Haley, J. A., Li, Q. K., Acquaah-Mensah, G., Montal, E., Sudini, K. R., Skoulidis, F., Wistuba, I. I., Papadimitrakopoulou, V., Heymach, J. V., Boros, L. G., Gabrielson, E., Carretero, J., Wong, K.-k., Haley, J. D., Biswal, S., Girnun, G. D. De novo lipogenesis represents a therapeutic target in mutant Kras non-small cell lung cancer.

Keywords: NSCLC; Warburg Effect; cancer metabolism.

Figure 1

Oncogenic Kras^LSL-G12D expressing murine lung tumors up-regulate the transcription of lipogenic genes. A) The heat map showing gene expression patterns of the individual genes from the GSEA set across mouse genotypes (green indicates high expression levels; red indicates low expression levels). Genes represent GSEA from Supplemental Fig. 1A. B) Schematic of enzymes involved in de novo lipogenesis from glucose. CoA, coenzyme A. C–E) Lungs harboring Kras^LSL-G12D tumors demonstrated remarkably higher mRNA expression of Acly (C), Acc (D), and Fasn (E), relative to healthy lung samples from sham-treated wild-type (WT)-C57BL6J mice. Actin was used as a housekeeping gene. All RNA was isolated from normal adjacent lung or Kras^LSL-G12D-induced lung tumors; n = 3. *P < 0.05, **P < 0.005.

Figure 2

Lipogenic gene expression and Kras mutations in patients with lung cancer. A, B) Kaplan-Meier analysis of the Director’s Challenge Consortium data set of human lung tumors showing expression levels of lipogenic genes FASN (A) or ACLY (B) and association with overall survival. C, D) Expression of FASN in tumors from patients with tumors containing wild-type or mutant Kras in the TCGA database (C) or the BATTLE-1 study (D). *P < 0.05, **P < 0.001.

Figure 3

Increased lipogenesis in oncogenic Kras^LSL-G12D-expressing murine lung tumors. A–D) Increased levels of total palmitate (A), stearate (B), and oleate (C) in Kras^LSL-G12D-expressing murine lung tumors relative to sham-treated, nontumor-bearing lung. D) Fractional enrichment of direct palmitate (de novo) as a function of [¹³C] acetate (m + 2 isotopomers). Mice with Kras^LSL-G12D-induced lung tumors and control mice were administered [U₆]–[¹³C]–glucose. After 90 min, mice were euthanized, and lipids were harvested from tumor tissue and saponified, and stable isotope analysis was performed by GC/MS; n = 3. *P < 0.05.

Figure 4

Pharmacologicl inhibition of FASN inhibits Kras^LSL-G12D-induced lung tumorigenesis. Lung tumorigenesis was induced in Kras^LSL-G12D mice using adenoviral Cre-recombinase, as described in Materials and Methods. After 4 wk, mice were treated with vehicle or 10 mg/kg C75. A) Tumor incidence in vehicle-treated and C75-treated Kras^LSL-G12D mice (macroscopic). B) Tumor multiplicity in vehicle-treated and C75-treated Kras^LSL-G12D mice (macroscopic); n = 8. *P < 0.05. C, D) Representative hematoxylin and eosin–stained lung images (×4 and 10) from 2 mice/treatment: vehicle-t (C) and C75- (D) treated Kras^LSL-G12D mice. E) Representative Ki-67–stained lung images (×20) from vehicle- and C75-treated Kras^LSL-G12D mice.

Figure 5

Cells expressing mutant Kras are sensitized to FASN inhibitor-based growth inhibition. A, B) Nontarget shRNA and Kras expressing shRNA A549 (A) and H23 (B) cells were treated with the indicated doses of C75 or GSK2194069, and the cell numbers were determined. C, D) H838 (C) and H1299 (D) cells expressing GFP or Kras^G12V were treated with the indicated doses of C75 or GSK2194069, and the cell numbers were determined using a Countess automated cell counter; n = 3–5. Experiments were performed in RPMI-1640 medium supplemented with 1% FBS. Shown is the percentage of cells after treatment with the indicated drugs compared with vehicle. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Palmitate rescues FASN inhibitor-mediated inhibition of cell growth. A549 (A, C) and H23 (B, D) expressing NTshRNA (left) or shKras (right) were treated with 2.5 μM GSK2194069 or 10 μM C75 alone or in combination with 7.5 μM palmitate. Cell number was determined with a Countess automated cell counter; n = 3. Experiments were performed in RPMI-1640 medium supplemented with 1% FBS. Bovine serum albumin was added as a lipid carrier because of the reduced serum conditions. *P < 0.05, **P < 0.01.