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. 2015 Sep 29;6(29):26909-21.
doi: 10.18632/oncotarget.4817.

Genome-wide RNAi analysis reveals that simultaneous inhibition of specific mevalonate pathway genes potentiates tumor cell death

Aleksandra A Pandyra  1   2   3 , Peter J Mullen  1 , Carolyn A Goard  1   2 , Elke Ericson  4   5 , Piyush Sharma  3 , Manpreet Kalkat  1   2 , Rosemary Yu  1   2 , Janice T Pong  1   2 , Kevin R Brown  4 , Traver Hart  4 , Marinella Gebbia  4 , Karl S Lang  3 , Guri Giaever  4   6 , Corey Nislow  4   6 , Jason Moffat  4 , Linda Z Penn  1   2
Affiliations

Genome-wide RNAi analysis reveals that simultaneous inhibition of specific mevalonate pathway genes potentiates tumor cell death

Aleksandra A Pandyra et al. Oncotarget. .

Abstract

The mevalonate (MVA) pathway is often dysregulated or overexpressed in many cancers suggesting tumor dependency on this classic metabolic pathway. Statins, which target the rate-limiting enzyme of this pathway, 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), are promising agents currently being evaluated in clinical trials for anti-cancer efficacy. To uncover novel targets that potentiate statin-induced apoptosis when knocked down, we carried out a pooled genome-wide short hairpin RNA (shRNA) screen. Genes of the MVA pathway were amongst the top-scoring targets, including sterol regulatory element binding transcription factor 2 (SREBP2), 3-hydroxy-3-methylglutaryl-coenzyme A synthase 1 (HMGCS1) and geranylgeranyl diphosphate synthase 1 (GGPS1). Each gene was independently validated and shown to significantly sensitize A549 cells to statin-induced apoptosis when knocked down. SREBP2 knockdown in lung and breast cancer cells completely abrogated the fluvastatin-induced upregulation of sterol-responsive genes HMGCR and HMGCS1. Knockdown of SREBP2 alone did not affect three-dimensional growth of lung and breast cancer cells, yet in combination with fluvastatin cell growth was disrupted. Taken together, these results show that directly targeting multiple levels of the MVA pathway, including blocking the sterol-feedback loop initiated by statin treatment, is an effective and targetable anti-tumor strategy.

Keywords: SREBP2; feedback inhibition; mevalonate pathway; statins; tumor metabolism.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. A genome-wide dropout screen uncovers putative shRNAs that potentiate fluvastatin-induced cell death
A. A simplified schematic of the mevalonate (MVA) pathway. Double arrows represent multiple steps in the pathway. B. In the pooled, genome-wide dropout screen A549 cells that had been stably transduced with the library of 80K shRNAs were treated with sublethal doses of fluvastatin (4–5 μM) every 3 days, over 12 days. C. Viability as assessed by trypan blue exclusion, relative to control of the fluvastatin-treated replicates. D. Significant hits, HMGCS1, GGPS1 and SREBP2, chosen for follow-up are indicated on the scatterplot (diamonds).
Figure 2
Figure 2. Generation of A549 cell lines stably expressing shRNA constructs targeting genes of the MVA pathway
A-B. RNA and protein lysates were harvested from asynchronously growing A549 cells that express two independent control shRNAs (shControl A, shControl B) or shRNAs targeting SREBP2, GGPS1 or HMGCS1 (shRNA A, shRNA B). Real-time mRNA EXPRESSION data represents the mean ± SD of three independent experiments. Immunoblots are representative of at least three independent experiments. C. Knockdown of SREBP2 and GGPS1 significantly decreased fluvastatin IC50 values. *p < 0.05 (one-way ANOVA with Tukey post-test). Representative dose-response curves assessed using the MTT assay are shown in C. (right panel).
Figure 3
Figure 3. shRNAs targeting MVA pathway gene hits potentiate fluvastatin-induced apoptosis
A. Exposure to fluvastatin (10 μM) for 72 hours of A549 cell lines stably expressing the indicated shRNA significantly induced apoptosis. B. Fluvastatin-induced apoptosis 72 hours post-treatment in the shGGPS1 cell lines was reversed by the concomitant addition of GGPP (10 μM) and not MVA (100 μM) but by both GGPP and MVA in shSREBP2 and shHMGCS1 cell lines. Bars represent the mean ± SD of at least three independent experiments * # p < 0.05 (*one-way ANOVA with Tukey-post test within each shA549 cell line comparing all groups, the fluvastatin-treated group being significantly different from the others, # the fluvastatin-treated group being significantly different than the ethanol and fluvastatin + GGPP groups). C. Protein lysates harvested from A549 cell lines treated with 10 μM of fluvastatin for 72 hours were immunoblotted and probed for PARP expression. Immunoblots are representative of at least three independent experiments.
Figure 4
Figure 4. Stable knockdown of SREBP2 sensitizes breast cancer MDA-MB-231 and MCF7 cells to the pro-apoptotic and anti-proliferative effects of fluvastatin
A-F. Protein lysates harvested from asynchronously growing MDA-MB-231 and MCF7 cells were immunoblotted for SREBP2 protein expression relative to cells transduced with the shControls (A and D respectively). Fluvastatin IC50 values in the MDA-MB-231 and MCF7 cell lines expressing the SREBP2 shRNAs were significantly lower when compared to the control cell lines (B and E respectively). Exposure of representative MDA-MB-231 and MCF7 cell lines to sub-lethal doses of fluvastatin caused a significant increase in apoptosis (C and F respectively). *p < 0.05 (t-test, unpaired, two-tailed, comparison made within each cell line). Immunoblots represent a minimum of two independent experiments.
Figure 5
Figure 5. Knockdown of MVA pathway genes does not sensitize breast and lung cancer cells to the anti-proliferative effects of doxorubicin
A. Proliferation as assessed by measuring the doubling time using the CyQuant cell proliferation assay kit was not significantly different between cell lines expressing the SREBP2 shRNAs and control cell lines in A549, MCF7 and MDA-MB-231 cells (one-way ANOVA with Tukey post test within each shA549 cell line comparing all groups). Bars represent the mean ± SD of at least three independent experiments. B. Doxorubicin IC50 values of the A549 and MCF7 cell lines expressing the SREBP2 shRNAs were not significantly different when compared to the cell lines expressing control shRNAs.
Figure 6
Figure 6. Knockdown of SREBP2 abrogates the sterol-feedback loop and impairs 3D growth upon fluvastatin treatment
Cells were treated for 24 hours with 10 μM of fluvastatin or ethanol control and assessed for expression of A. HMGCR B. HMGCS1 relative to GAPDH in shA549 cell lines. Bars represent the mean ± SD of at least three independent experiments *p < 0.05 (t-test, unpaired, two-tailed, comparison made within each cell line). C. Protein lysates harvested from A549 cell lines treated with 10 μM of fluvastatin (Fluva.) for 24 hours were immunoblotted for SREBP2 expression. Immunoblots are representative of at least three independent experiments. D. Treatment with fluvastatin for 72 hours with the indicated fluvastatin concentrations disrupts A549 acini in shSREBP2 cell lines in 3D culture. Cells were imaged using an AxioObserver and are representative of three independent experiments. Scale bar represents 200 μm in main image and 100 μm in inset.
Figure 7
Figure 7. Knockdown of SREBP2 abrogates the sterol-feedback loop and impairs 3D growth of MCF7 cells upon fluvastatin treatment
A. Cells were treated for 24 hours with 10 μM of fluvastatin or ethanol control and assessed for expression of HMGCR (A), HMGCS1 (B) relative to GAPDH in shMCF7 cell lines. Bars represent the mean ± SD of at least three independent experiments *p < 0.05 (t-test, unpaired, two-tailed, comparison made within each cell line). B. Treatment with fluvastatin for 72 hours with the indicated concentrations disrupts MCF7 acini in the shSREBP2 cell line in 3D culture but not in the shControl cell line. Images are representative of three independent experiments composed of three technical replicates. Scale bar represents 200 μm in main image and 100 μm in inset.
Figure 8
Figure 8. HMGCS1 is amplified in various cancers
Analysis of cancer genomics datasets using cBioPortal demonstrating HMGCS1 genomic alterations across various tumor types.
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