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. 2016 Apr 15;76(8):2406-18.
doi: 10.1158/0008-5472.CAN-15-1646. Epub 2016 Feb 29.

Cyclin E Associates with the Lipogenic Enzyme ATP-Citrate Lyase to Enable Malignant Growth of Breast Cancer Cells

Kimberly S Lucenay  1 , Iman Doostan  1 , Cansu Karakas  1 , Tuyen Bui  1 , Zhiyong Ding  2 , Gordon B Mills  2 , Kelly K Hunt  3 , Khandan Keyomarsi  4
Affiliations

Cyclin E Associates with the Lipogenic Enzyme ATP-Citrate Lyase to Enable Malignant Growth of Breast Cancer Cells

Kimberly S Lucenay et al. Cancer Res. .

Abstract

Cyclin E is altered in nearly a third of invasive breast cancers where it is a powerful independent predictor of survival in women with stage I-III disease. Full-length cyclin E is posttranslationally cleaved into low molecular weight (LMW-E) isoforms, which are tumor-specific and accumulate in the cytoplasm because they lack a nuclear localization sequence. We hypothesized that aberrant localization of cytosolic LMW-E isoforms alters target binding and activation ultimately contributing to LMW-E-induced tumorigenicity. To address this hypothesis, we used a retrovirus-based protein complementation assay to find LMW-E binding proteins in breast cancer, identifying ATP-citrate lyase (ACLY), an enzyme in the de novo lipogenesis pathway, as a novel LMW-E-interacting protein in the cytoplasm. LMW-E upregulated ACLY enzymatic activity, subsequently increasing lipid droplet formation, thereby providing cells with essential building blocks to support growth. ACLY was also required for LMW-E-mediated transformation, migration, and invasion of breast cancer cells in vitro along with tumor growth in vivo In clinical specimens of breast cancer, the absence of LMW-E and low expression of adipophilin (PLIN2), a marker of lipid droplet formation, associated with favorable prognosis, whereas overexpression of both proteins correlated with a markedly worse prognosis. Taken together, our findings establish a novel relationship between LMW-E isoforms of cyclin E and aberrant lipid metabolism pathways in breast cancer tumorigenesis, warranting further investigation in additional malignancies exhibiting their expression. Cancer Res; 76(8); 2406-18. ©2016 AACR.

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Figures

Figure 1
Figure 1. Validation of RePCA clones
(A) RePCA clone #46, #18 and #16 were fixed and counterstained with Dapi. (B) 293T lysates expressing SFB-tagged cyclin E isoforms and myc-ACLY were immunoprecipitated using an anti-Flag antibody. (C) Recombinant GST-ACLY protein was incubated with in vitro transcribed and translated (TnT) cyclin E isoforms or Cdk2 and isolated using Glutathione sepharose beads and subjected to western blot analysis. (D) In vitro kinase assay using recombinant GST-ACLY aa 426-486 and recombinant cyclin E isoforms produced in Sf9 cells alone or in complex with CDK2 were isolated by Cdk2 immunoprecipitation and incubated with GST-ACLY in the presence of 32P-γ-ATP.
Figure 2
Figure 2. LMW-E isoforms affects ACLY enzymatic activity
(A) MCF7 cell lysates ectopically expressing the cyclin E isoforms were used to examine ACLY enzymatic activity by the malate dehydrogenase coupled method. Error bars= SEM from three independent replicates (Student's t-test,**p<0.007). (B) Western blot showing protein expression. (C) 76NE6 lysates containing inducible expression of the LMW-E isoforms increases ACLY enzymatic activity. Error bars= SEM from three independent replicates (Student's t-test,*p<0.03 and p<0.004). (D) Western blot showing protein expression. (E) In vitro transcribed and translated cyclin E isoforms or CDK2 were incubated with purified ACLY protein and subjected to the malate dehydrogenase coupled method. Error bars= SEM from three independent replicates (Student's t-test,**p<0.03).
Figure 3
Figure 3. Cytoplasmic ACLY activity results in lipid droplet accumulation
(A) Western blot showing protein expression from fractionated cells. (B) ACLY activity is shown for at least 3 independent replicates from fractionated 76NE6 cells using the malate dehydrogenase coupled method. Statistical analysis was conducted using the student's t-test. Error bars= SEM (**p<0.008 and *p<0.04). (C) 76NE6 cell containing inducible expression of cyclin E stained with LipidTox and counterstained with Dapi. (D) Lipid droplet formation was quantified before and after addition of doxycycline (n=100). (Student's t-test *p<0.05). (E) Western blot showing protein expression. (F) 76NE6-inducible LMW-E(T2) lysates containing ACLY knockdown to examine ACLY activity. Three independent replicates are shown; error bars=SEM (**p<0.002). (G) 76NE6-inducible LMW-E(T2) cells stained with LipidTox and counterstained with Dapi. (H) Cells were counted before and after addition of doxycycline (n=100). (Student's t-test *p<0.05).
Figure 4
Figure 4. ACLY is required for LMW-E mediated anchorage-independent growth
(A) Western blot showing protein expression. (B) MCF7 lysates ectopically expressing the cyclin E isoforms with ACLY downregulation were used to examine ACLY activity from 2 independent replicates. Error bars=SEM, **p<0.005. (C) Quantitation of colonies formed after 10 days and 30 days in anchorage-independent growth conditions of MCF7 cells ectopically expressing the cyclin E isoforms with ACLY downregulation. Statistical analysis was performed using the student's t-test in 3 independent experiments performed in triplicate (10 days *p<0.04, **p<0.009, ***p<0.0002 and 30 days *p<0.04 and **p<0.0025). (D) Average colony diameter of colonies formed in soft agar conditions. Statistical analysis was performed using the student's t-test; (**p<0.0085) and performed in triplicate with images from representative colonies are shown. (E) CDK2 associated kinase activity, using Histone H1 (HH1) as a substrate.
Figure 5
Figure 5. Inhibition of ACLY reduces migration and invasion in HMECs
(A) 76NE6 and TDCs containing either scrambled shRNA or shRNA toward ACLY reached confluency and scratched with a pipet tip to create a wound. Images were taken at 0 and 24 hours post-scratch. (B) Statistical analysis was conducted using the student's t-test from 3 independent replicates. Error bars=SEM; *p<0.05, **p<0.008. (C) (Top panel) 76NE6 TDCs containing shRNA were harvested and Allophycocyanin was added and analyzed by flow cytometry. (Bottom panel) Proliferation was measured by BrdU incorporation. (D) 76NE6 TDCs with ACLY downregulation were plated on a transwell chamber containing Matrigel and incubated on top of fibronectin-containing media for 24 hours. Invaded cells were stained with crystal violet. Images of 20X magnification were taken with a light microscope. (G) Cells on the bottom of the transwell were collected at 24 hours and counted. Statistical analysis was conducted using the student's t-test from 3 independent replicates. Error bars=SEM, *p<0.05,**p<0.005.
Figure 6
Figure 6. ACLY is required for LMW-E mediated tumor growth
(A) Inhibition of tumor growth in MCF7 cells containing stable overexpression of the cyclin E isoforms and either scrambled shRNA or shRNA targeted to ACLY. Tumors were measured for 10 weeks starting at 3 weeks. Statistical analysis was conducted using the student's t-test. N=5; error bars=SEM; **p<0.005. (B) Representative pictures of shACLY#1 tumors. (C) Representative sections from tumors expressing the cyclin E isoforms were examined for the expression of cyclin E, ACLY, Ki-67 and adipophilin antibodies by immunohistochemical staining. H&E staining revealed the morphology of the tumor. Images are at 20x magnification. (D) Neutral lipid accumulation in tissues from mice tumors. Magnification is 10X for per group. Insets from the images are magnified 40X in order to highlight the lipid-staining morphology.
Figure 7
Figure 7. Analysis of LMWE and/or adipophilin expression in breast cancer patients
(A) Representative images of immunohistochemical staining for adipophilin and correlation with expression of cyclin E are shown (magnification 100x, magnification of inset images, 400x). Specifically, adipophilin (a, b) and corresponding cyclin E (e, f) staining for low adipophilin/LMWE (−); adipophilin (c, d), and corresponding (g, h), for high adipophilin/LMWE (+) expression. (B) Association between adipophilin and cyclin E expression in 100 invasive breast carcinoma tissue specimens, determined using Fisher's exact test. The results revealed that there was a significantly positive correlation between the expression levels of adipophilin and cytoplasmic cyclin E (LMW-E +) expression (p<0.001). Corresponding table shows significant positive correlation of staining of adipophilin and cytoplasmic cyclin E expression in the 100 human breast tumor samples examined. Quantification of adipophilin (C) and cyclin E (D) expression in the different subtypes of invasive breast carcinoma tissue specimens: Frequency distribution illustrating the percentage of cases, evaluated by IHC, falling into each categorical score over the range 0 to 7 (see supplementary Table 5) for luminal A, luminal B, HER-2 (+) and triple negative breast carcinoma (TNBC), statistical significance determined by Fisher's exact test. (E, F) Kaplan-Meier survival plots demonstrating the association between adipophilin and LMWE expression and breast cancer recurrence-free survival in 100 invasive breast carcinoma patients. (G) Kaplan-Meier survival plots demonstrating the association between combined adipophilin and cyclin E expression and breast cancer recurrence-free survival in 100 invasive breast carcinoma patients.
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