American Association for Cancer Research
10780432ccr200269-sup-236038_2_supp_6215147_q8g827.pdf (7.1 MB)

Supplementary Figures from AMPK Activation by Metformin Promotes Survival of Dormant ER+ Breast Cancer Cells

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journal contribution
posted on 2023-03-31, 21:42 authored by Riley A. Hampsch, Jason D. Wells, Nicole A. Traphagen, Charlotte F. McCleery, Jennifer L. Fields, Kevin Shee, Lloye M. Dillon, Darcy B. Pooler, Lionel D. Lewis, Eugene Demidenko, Yina H. Huang, Jonathan D. Marotti, Abigail E. Goen, William B. Kinlaw, Todd W. Miller

Fig. S1 shows that dormant ER+ breast cancer cells retain tumor-initiating capacity. Fig. S2 shows individual tumor growth curves. Fig. S3 shows IHC images for Ki67, cleaved caspase-3 and ER in xenografts. Fig. S4 shows Gene Set Variation Analysis of RNA-seq data from xenografts. Fig. S5 shows overlap of altered genes from RNA-seq data. Fig. S6 shows IHC images for AMPK�2 and P-ACC in xenografts. Fig. S7 shows mTORC1 activation in MCF-7 tumors. Fig. S8 shows a transcriptional signature of FAO in human tumors. Fig. S9 shows mitochondrial content and activity in xenografts. Fig. S10 shows effects of AMPK knockdown on respiration. Fig. S11 shows that inhibition of FAO hastens tumor regression. Fig. S12 shows pharmacologic analysis of metformin. Fig. S13 shows metformin effects in MDA-MB-415 tumors. Fig. S14 shows that metformin prevents tumor regression. Fig. S15 shows that metformin does not alter fasting levels of blood glucose, serum insulin, or serum free fatty acids in mice. Fig. S16 show IHC images of P-ACC in tumors from mice treated with metformin. Fig. S17 shows AMPK effects on HCC-1500 cells and tumors. Fig. S18 shows the effects of high-fat diet on tumor regression. Fig. S19 shows IHC images of Ki67, CPT1α, and P-ACC in xenografts from mice treated with high-fat or low-fat diet.


Mary Kay Foundation


Dartmouth College Norris Cotton Cancer Center




Despite adjuvant endocrine therapy for patients with estrogen receptor alpha (ER)-positive breast cancer, dormant residual disease can persist for years and eventually cause tumor recurrence. We sought to deduce mechanisms underlying the persistence of dormant cancer cells to identify therapeutic strategies. Mimicking the aromatase inhibitor–induced depletion of estrogen levels used to treat patients, we developed preclinical models of dormancy in ER+ breast cancer induced by estrogen withdrawal in mice. We analyzed tumor xenografts and cultured cancer cells for molecular and cellular responses to estrogen withdrawal and drug treatments. Publicly available clinical breast tumor gene expression datasets were analyzed for responses to neoadjuvant endocrine therapy. Dormant breast cancer cells exhibited upregulated 5′ adenosine monophosphate-activated protein kinase (AMPK) levels and activity, and upregulated fatty acid oxidation. While the antidiabetes AMPK-activating drug metformin slowed the estrogen-driven growth of cells and tumors, metformin promoted the persistence of estrogen-deprived cells and tumors through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival. AMPK has context-dependent effects in cancer, cautioning against the widespread use of an AMPK activator across disease settings. The development of therapeutics targeting fat metabolism is warranted in ER+ breast cancer.

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