Dysregulation of the mevalonate pathway promotes transformation

Abstract

The importance of cancer metabolism has been appreciated for many years, but the intricacies of how metabolic pathways interconnect with oncogenic signaling are not fully understood. With a clear understanding of how metabolism contributes to tumorigenesis, we will be better able to integrate the targeting of these fundamental biochemical pathways into patient care. The mevalonate (MVA) pathway, paced by its rate-limiting enzyme, hydroxymethylglutaryl coenzyme A reductase (HMGCR), is required for the generation of several fundamental end-products including cholesterol and isoprenoids. Despite years of extensive research from the perspective of cardiovascular disease, the contribution of a dysregulated MVA pathway to human cancer remains largely unexplored. We address this issue directly by showing that dysregulation of the MVA pathway, achieved by ectopic expression of either full-length HMGCR or its novel splice variant, promotes transformation. Ectopic HMGCR accentuates growth of transformed and nontransformed cells under anchorage-independent conditions or as xenografts in immunocompromised mice and, importantly, cooperates with RAS to drive the transformation of primary mouse embryonic fibroblasts cells. We further explore whether the MVA pathway may play a role in the etiology of human cancers and show that high mRNA levels of HMGCR and additional MVA pathway genes correlate with poor prognosis in a meta-analysis of six microarray datasets of primary breast cancer. Taken together, our results suggest that HMGCR is a candidate metabolic oncogene and provide a molecular rationale for further exploring the statin family of HMGCR inhibitors as anticancer agents.

Fig. 1.

HMGCR-FL and HMGCR-D13 transcript levels are upregulated in response to lovastatin exposure. (A) A schematic representation of HMGCR-FL and its splice variant, HMGCR-D13, at the genomic, transcript, and protein levels with real-time PCR primers used to assess total endogenous HMGCR and each of HMGCR-FL and HMGCR-D13 specifically. (B) mRNA from HepG2 cells was harvested for real-time PCR analysis of basal transcript expression to show that HMGCR-FL is expressed at higher levels than HMGCR-D13. mRNA was also harvested from HepG2 cells exposed to either ethanol vehicle control or 20 μM lovastatin for the times indicated. Transcript expression of total endogenous HMGCR (C), HMGCR-FL (D) and HMGCR-D13 (E) all increased over time after lovastatin exposure. Data is expressed as log ₂ ratios of expression in lovastatin-exposed cells compared to ethanol-treated cells. * p < 0.05; one sample t-test comparing to 0, i.e., no induced expression. All experiments were performed a minimum of three times and data represent means and standard deviations.

Fig. 2.

Ectopic expression of cHMGCR-FL and cHMGCR-D13 in HepG2 cells. (A) Schematic of the ectopic HMGCR constructs. (B) Confirmation of cHMGCR-FL and cHMGCR-D13 ectopic expression by real-time PCR assessing both HMGCR-FL (left) and HMGCR-D13 (right) transcript expression. * p < 0.05; student’s t-test comparing ectopic construct expressing cell lines to the GFP control. Data represent means and standard deviations. (C) Real-time PCR analysis of endogenous HMGCR transcript levels in HepG2 cells expressing the indicated constructs and exposed to vehicle control or 20 μM lovastatin for 16 hours. * p < 0.05; student’s t-test comparing lovastatin-exposed cells to ethanol-exposed cells. Data represent means and standard deviations. (D) Immunoblot using Upstate/Millipore antibody #07-572 detecting HMGCR protein expression in the indicated HepG2 cell lines exposed to vehicle control or 20 μM lovastatin for 48 hours prior to being harvested for protein lysates as described in the materials and methods. 1—High molecular weight signal corresponding to oligomerized HMGCR; 2—Approximately 100 kDa band corresponding to endogenous HMGCR; 3—Approximately 60 kDa band corresponding to ectopic cHMGCR-FL; 4—Approximately 55 kDa band corresponding to ectopic cHMGCR-D13. Tubulin was assayed as a loading control. All experiments were performed a minimum of three times and one representative example is shown.

Fig. 3.

Dysregulation of the MVA pathway by ectopic cHMGCR-FL and cHMGCR-D13 expression increases transformation of HepG2 cells. HepG2 cells expressing the indicated constructs were seeded in proliferation assays (A) or for cell cycle analysis by fixed PI (B) but no difference in growth rate or cell cycle was observed. (C) Similarly, cells exposed to 2 μM doxorubicin or 10 nM taxol were not protected from cell death by expression of either cHMGCR-FL or cHMGCR-D13, whereas BCL2 inhibited cell death significantly. Cell death was assessed by measurement of subdiploid DNA content (% pre-G1) in fixed PI assays combined with flow cytometry. * p < 0.05; student’s t-test comparing ectopic construct expressing cell lines to the GFP control. Experiments were performed a minimum of three times and data represent means and standard deviations. (D) When seeded in soft agar assays, HepG2 cells expressing either cHMGCR-FL or cHMGCR-D13 formed more colonies than cells expressing the empty GFP vector control, which was normalized to 1 in each experiment. * p < 0.05; one sample t-test comparing ectopic construct expressing cell lines to the GFP control. Experiments were performed a minimum of three times and data represent means and standard deviations. To address transformation in vivo, the flanks of sublethally irradiated SCID mice were injected with HepG2 cells ectopically expressing the empty GFP vector and either cHMGCR-FL (E) or cHMGCR-D13 (F). In each case, the cHMGCR construct-expressing cells grew larger tumors more quickly than the GFP cells. * p < 0.05; student’s t-test comparing ectopic construct expressing cell lines to the GFP control. Data represent means and standard errors of the mean.

Fig. 4.

Dysregulation of the MVA pathway demonstrates the oncogenic potential of HMGCR. (A) Ectopic expression of either cHMGCR-FL or cHMGCR-D13 in transformed MCF7 cells potentiated anchorage-independent growth in soft agar. * p < 0.05; one sample t-test compared to normalized control. These experiments were performed a minimum of three times and data represent means and standard deviations. Representative images of plate quadrants are shown (magnification 1.6×). (B) Nontransformed MCF10A cells formed significantly more colonies in soft agar when ectopically expressing either cHMGCR-FL or cHMGCR-D13. * p < 0.05; one sample t-test compared to normalized control. These experiments were performed a minimum of three times and data represent means and standard deviations. Representative images of plate quadrants are shown (magnification 1.6×). (C) cHMGCR-FL and cHMGCR-D13 constructs in MSCV-YFP vectors were introduced into either normal murine bone marrow or fetal liver cells. Cells were plated in methylcellulose media containing myeloid cytokines. Three independent experiments (two with bone marrow and one with fetal liver) yielded similar results in which cHMGCR-FL increased, and cHMGCR-D13 decreased, myeloid colony formation. One representative experiment is shown; data represent means and standard deviations between duplicate plates. (D) Primary MEFs were infected with retroviral constructs carrying the indicated genes and drug-selected. Transformed foci formed after approximately three weeks and were counted and imaged. Two to four plates of each gene combination were scored and representative images are shown.

Fig. 5.

High mRNA levels of MVA pathway genes correlate with poor prognosis and reduced survival of breast cancer patients. A meta-analysis of six primary breast cancer datasets encompassing 865 patients was performed to evaluate a relationship between patient prognosis and the mRNA expression of HMGCR, hydroxymethylglutaryl coenzyme A synthase 1 (HMGCS1), mevalonate diphosphate decarboxylase (MVD), farnesyl pyrophosphate synthase (FDPS), acetoacetyl-CoA thiolase 2 (ACAT2), and mevalonate kinase (MVK). Kaplan–Meier analysis demonstrates that higher mRNA levels of five out of six gene products correlates with poor prognosis and decreased patient survival.