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June 19, 2018

Aggressive Brain Cancer Can Be Driven by Tumor Suppressor

Molecularly stained brain cells shows the AMPK target protein (green) in a large glioblastoma brain tumor that grew from transplanted human cancer cells in a mouse brain. Scientists report in Nature Cell Biology that tumor growth was reduced when the researchers genetically inhibited AMPK, suggesting it may be a potential treatment strategy for lethal and mostly untreatable brain cancers like high-grade glioblastoma. [Cincinnati Children's]

  • AMP-activated protein kinase (AMPK) is involved in regulating cell metabolism and is largely thought to play a suppressive role in cancer. Research by scientists at the Cincinnati Children’s Cancer and Blood Diseases Institute has now indicated that the protein may represent a key driver of aggressive brain cancers, including glioblastoma (GBM). Their studies showed that blocking AMPK reduced the growth of GBM stem cells (GSCs) transplanted into the cerebral cortex of experimental mice, leading to increased survival. The results hint at the feasibility of using AMPK inhibition as a treatment for GBM in human patients, if safe, targeted drugs can be developed.

    "AMPK is considered to play a suppressive role in cancer because it inhibits cancer-promoting enzymes like mammalian target of rapamycin (mTOR) and acetyl Co-A carboxylase (ACC)," says senior investigator Biplab Dasgupta, Ph.D., at the Division of Oncology at Cincinnati Chidren’s. "Our study uses analysis of The Cancer Genome Atlas to show that AMPK proteins are highly expressed in lethal human glioblastoma and that inhibiting AMPK by genetic means shrinks brain tumors and prolongs survival in mice. It also shows that deleting AMPK from the whole body of adult mice is safe for the animals."

    The researchers report their findings in Nature Cell Biology, in a paper entitled “AMP Kinase Promotes Glioblastoma Bioenergetics and Tumour Growth.”

    AMPK normally works as a bioenergetics sensor that helps to regulate cell metabolism and stress, and while the protein has been found to play a suppressive role in some cancers, a potential oncogenic role has been suggested for activated AMPK in astrocytic tumors of the brain, the authors write. “Contrary to early pharmacological studies, some recent genetic studies showed that in some contexts, AMPK provides survival advantage critical for tumour growth.”

    It was while they were searching The Cancer Genome Atlas (TCGA) database for metabolic kinases that are differentially expressed in cancers that the team discovered that expression of AMPK catalytic subunits α1, β1, and γ1 was upregulated in GBM, compared with normal brain tissue, and also compared with lower-grade gliomas (LGG), although higher expression of activated AMPK (pAMPK) also correlated with poorer patient survival in LGG. “This is reminiscent of other genes (such as HIF1α and CREB1) that are highly expressed (sometimes uniformly) across GBM relative to LGG, and are not prognostic but are important for GBM pathogenesis,” the authors write. Additional analysis of data highlighted high levels of pAMPK in GBM tumors compared with normal brain tissue, and in primary GBM stem cell lines (GSCs) isolated from fresh tumors. Further evaluation suggested that the high AMPK activity in GSCs was due to oncogenesis-associated stress (OAS), which effectively activated AMPK, “suggesting a link between OAS and pAMPK in GMB.”

    In an initial series of in vitro tests the team showed that the viability of primary GSC lines could be reduced using a number of different approaches to genetically deplete AMPK. Inhibiting upstream kinases that activate AMPK also reduced GSC line viability, “consistent with the AMPK activation pathway being important for GBM,” the team notes. In contrast, however, the viability of more long-established GBM serum lines wasn’t affected by AMPK subunit deletion.

    AMPK activation during metabolic and oncogenic stress usually protects cells, but studies showed that mimicking metabolic stress in AMPK-silenced GSCs didn’t increase cell death. “In fact, unlike long-established GBM serum lines, primary GSCs remained viable under stress,” they write. “Thus, stress-tolerant primary lines are reliable surrogates of the tumor in maintaining an AMPK-dependent stress adaptation for survival in vitro."

    Initial in vivo studies showed that AMPK-depleted GSC tumors grew more slowly following transplantation into the cerebral cortex of experimental mice, and the animals also survived for longer. “The AMP-silenced tumours were smaller and showed reduced proliferation and increased apoptosis compared to controls,” the team writes. "It remains to be seen if inhibiting AMPK in combination with standard of care therapy prolongs survival even further," Dasgupta adds.

    In contrast, there was no effect of AMPK silencing on the growth of GBM serum line-derived transplants. “Why the established GBM serum lines remained viable and formed tumours independent of AMPK is unclear,” the team acknowledges. “Decades of culture could have altered the genetic, epigenetic, and metabolic landscape of these lines, allowing them to adapt and evolve AMPK-independent growth and survival pathways.”

    Further in vitro studies suggested that the effects of AMPK silencing in GSCs were not related to mTOR regulation. “…AMPK silencing did not increase mTORC1 activity, and, consistent with previous work, the mTORC1 inhibitor rapamycin failed to protect AMPK-silenced GSCs,” the team states. “…we postulated that AMPK-depleted GSCs probably died not due to energy drainage, but to a deficit in energy production.”

    In fact, RNA sequencing studies showed that the pathway relating to cellular metabolism bioenergetics—glycolysis and mitochondrial function—was most significantly downregulated in AMPK-depleted GSCs. Functional analyses confirmed that both glycolysis and mitochondrial respiration were reduced in AMPK-silenced GSCs and tumors, although, significantly, not in long-established GBM serum lines. "Together, our findings indicate the presence of an AMPK-regulated transcriptional program that is important for GBM bioenergetics.”

    In stressed—i.e., exercised—skeletal muscle, activated AMPK cooperates with cAMP response element binding protein-1 (CREB1) to promote glucose metabolism. In subsequent in vitro tests, the researchers demonstrated that oncogenic stress also chronically activates AMPK in GSCs, which effectively “co-opts the AMPK-CREB1 pathway to coordinate tumour bioenergetics through the transcription factors HIF1α and GABPA.”

    Given the results indicating that AMPK may represent a target for GBM, the researchers investigated the effect of deleting AMPK systemically in an animal model. They found that “remarkably,” adult mice with systemic AMPK knockdown lived a normal lifespan, with no obvious metabolic effects, “underscoring the potential use of AMPK inhibitors in the treatment of GBM and perhaps other human cancers where AMPK is activated.”

    Although the studies suggest that pharmacological inhibition of AMPK could represent a therapeutic option for human glioblastoma, years of research will still be needed to verify if the findings are clinically relevant. "We are hopeful our studies will encourage pharmaceutical companies to screen for AMPK inhibitors," Dasgupta notes.

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