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Cancer Metabolism: It's a Brave New World

Mention cellular metabolism to most people and visions of Krebs cycle diagrams and glycolysis come to mind. While some may think of metabolism as a linear pathway, Raymond Gilmour, Ph.D., Discovery Research, Eli Lilly and Company, believes nothing could be further from the truth. He says there are many dynamic interactions within metabolic pathways and “it’s only following the advent of genomic and metabolomic technologies that we’re beginning to understand the detailed regulation of cancer cell metabolism.”

Gilmour co-authored the editorial introduction to a special collection of four original research papers focusing on recent advances in metabolic assay development, patient tumor metabolomics and the identification of novel inhibitors in the April 2017 issue of SLAS Discovery. His interest in cell metabolism goes back to his days as a biochemistry post-doc working on oxidative phosphorylation in bacteria. He points out that all metabolism is based on biochemistry, an understanding of enzyme kinetics and metabolic flux. “A lot of what I learned in bacteria is applicable to tumor metabolism as well,” an area he’s been focused on for the past five years.

Old Discovery Meets New Technology

The link between tumor growth and altered metabolism isn’t new. Dr. Otto Warburg was among the first to observe that cancer cells generate ATP through glycolysis rather than oxidative phosphorylation even when enough oxygen is present, a process he dubbed oxidative glycolysis. He first published his observation, now known as the Warburg effect, in 1924. But it would be nearly a century before scientists had the tools to begin researching the mechanisms behind these altered pathways.

It started in the 1970s with DNA sequencing of bacteria. Once these techniques had been refined and became widely available in the 1980s and 1990s, scientists began working on sequencing entire genomes to try to discover the mutations involved with tumor development and progression.

Thanks to next generation sequencing and programs like The Cancer Genome Atlas (TCGA) project, by the mid-2000s genetic mutations had been identified in nearly all tumor types. “The next question became how do these mutations change the cell in a way that makes it malignant,” Gilmour says. “That’s when people who had been focusing on signaling research started investigating cell metabolism to try to understand this interplay between metabolism, cell signaling and oncogenes.”

This led to the discovery that some oncogenic mutations can increase anabolic growth pathways such as glycolysis and glutaminolysis through up-regulation of expression of key enzymes. In addition, gene amplification of specific metabolic genes was shown to result in overexpression; leading to activation of metabolic pathways. . As Gilmour points out, altered cellular metabolism leads to changes in intracellular and extracellular levels of specific  metabolic substrates like glucose and glutamine, as well as their downstream metabolites, making them ideal readouts for screening.

Measuring Metabolites

If substrates and their metabolites are the ideal readouts, the ideal assay is a rapid, sensitive technique that requires minimal sample preparation and is adaptable to high-throughput formats and automation. So says Donna Leippe from Promega Corporation, Madison, WI. Leippe is lead author on the issue’s paper entitled “Bioluminescent Assays for Glucose and Glutamine Metabolism: High-Throughput Screening for Changes in Extracellular and Intracellular Metabolites.”

She notes that while metabolic profiling and stable isotope labeling provide comprehensive information about changes in multiple metabolites under certain conditions, “they require extensive sample preparation and aren’t well suited to looking at key metabolites under multiple treatment conditions.” Furthermore, the amount of sample needed for bioanalyzers can be large and throughput is limited.

To address these limitations, Leippe and colleagues evaluate the applicability of bioluminescent glucose, lactate, glutamine and glutamate assays for high-throughput screening (HTS). Using two ovarian cancer cell lines with different glutamine dependencies, assays are developed to measure changes in glucose, lactate, glutamine and glutamate levels over time in tumor cells and growth culture. The Library of Pharmacologically Active Compounds (LOPAC) was used as a model library to identify compounds that altered the production of lactate by SKOV-3 cells, the cell line requiring glutamine for growth.

The results of these assays show that they are useful for high-throughput screening of a library with multiple cell lines. The authors point out that while the LOPAC cassette contains 1,280 compounds, the same principles can be applied to screening campaigns of any size. Gilmour says, “The high sensitivity, wide linear range and multiplexing of these assays make them very useful in screening campaigns.”

Eat or Be Eaten

In addition to glycolysis and glutaminolysis, tumor cells use catabolic pathways to their advantage as well. One such pathway is autophagy, where large molecules such as proteins are enveloped in a double membrane, and digested by lysosomes within the cell. The metabolic components then are recycled for use as an energy source.

As Danqing Xu from Roche Pharma Research, Shanghai, China explains, it’s an intricate process involving multiprotein complexes with different enzymatic activity. Many of these proteins have been identified and characterized, including the cysteine protease ATG4, an enzyme necessary to initiate the formation of the double membrane. ATG4 has several different homologues that operate at different stages in autophagy, with ATG4B having the broadest substrate spectrum. Gilmour points out, “This redundancy is a necessary consideration when screening for targeted compounds.” It also makes compounds that inhibit ATG4B attractive targets for drug discovery.

In the issue’s paper entitled “Identification of New ATG4B Inhibitors Based on a Novel High-Throughput Screen Platform,” Xu and colleagues illustrate how a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay measuring ATG4B activity is developed where compounds with ATG4B inhibitory activity show an enhanced FRET signal. Once the assay is optimized, it is adapted into a HTS format. Using a focus library of 57,000 compounds, nearly 700 hits are obtained and a compound with ATG4B inhibitor activity is identified. According to Gilmour, “This elegant FRET-based assay is a robust platform that could be applied for the screening of large compound libraries to identify novel chemical scaffolds.”

A Personalized Approach

As knowledge of the mechanics of tumor cell metabolism expands, new tools and techniques also are being developed to study tumor metabolism in individual patients. Metabolomics is a powerful new tool in the era of personalized medicine. Gilmour says “It has the potential to provide a wealth of data that can be used to identify key metabolic pathways in patients before and after treatment, as well as at relapse.”

Since metabolism is altered in cancer, the metabolic fingerprint left behind in tumor cells is different than in healthy cells. Identifying these differences may lead to the discovery of biomarkers that can lead to earlier detection and diagnosis of disease, he adds.

Perhaps nowhere is early detection more important than in pancreatic ductal adenocarcinoma (PDAC). Symptoms of PDAC usually are vague until the advanced stages of the disease when it may be too late for curative treatment.

As Sandra Rios Peces, lead author of the paper entitled “Untargeted LC-HRMS-Based Metabolomics for Searching New Biomarkers of Pancreatic Ductal Adenocarcinoma: A Pilot Study” explains, the extreme diversity of metabolites in biofluids makes untargeted analysis of serum challenging. Single-liquid chromatographic systems like reverse-phase liquid chromatography (RPLC) don’t adequately separate polar metabolites. Hydrophilic interaction liquid chromatography (HILIC) offers complimentary information to RPLC.

In their paper, Peces and colleagues describe how the combination of RPLC and HILIC coupled to high-resolution mass spectrometry (HRMS) is used to compare the metabolic fingerprints of serum samples from healthy volunteers to those of patients with PDAC. The levels of four phospholipid metabolites were significantly lower in the healthy volunteer group, demonstrating that this combination approach improves the comprehensiveness of untargeted metabolic profiling. Gilmour adds, “This study represents a starting point to better understand the impact of altered metabolism on PDAC development and progression.”

Looking Beyond Cancer

Some drugs traditionally used for the treatment of diseases like diabetes and hyperlipidemia also have been shown to be useful in treating or preventing certain types of cancer. Gilmour says understanding how these molecules target metabolic pathways can be valuable in cancer research.

The monoacylglycerol acyltransferases (MGAT) pathway is one example. It’s an important pathway in the synthesis of triacylglycerol, the primary fat in the human diet. In the paper entitled “Discovery of Human Intestinal MGAT Inhibitors Using High-Throughput Mass Spectrometry,” Ryutaro Adachi and colleagues from Takeda Pharmaceutical Company demonstrate how a rapid-fire mass spectrometry assay in a physiologically relevant setting can be used to screen a large volume of small molecules and identify MGAT inhibitors. According to Gilmour, “These tools and techniques will be valuable to understanding any metabolic situation, whether it’s cancer, obesity or any change in metabolism.

It’s Just the Beginning

With new tools and advances in available technologies, Gilmour believes the future of cancer metabolism research looks promising.

Take imaging, for example. Fluorodeoxyglucose positron emission tomography (FDG-PET) provides direct assessment of glucose uptake and has been used in clinical practice for some time. According to Gilmour newer PET tracers recently have been developed to monitor other metabolites, including glutamine and choline. And nuclear magnetic resonance spectroscopy (NMRS) has advanced to the point researchers can get very high resolution information on patients even during surgery.

There’s also been more interest in the role of the immune system in cancer. “Metabolism plays a very important role in t-cell metabolism and myeloid cell metabolism,” says Gilmour. “Inhibitors targeting key enzymes in the tryptophan metabolic pathways already are being evaluated in the clinic.”

Synthetic lethality is another approach being explored in targeting metabolic pathways. Gilmour cites the example of the ENO1 and ENO2, two genes that code for enolase, an enzyme used in glycolysis. He explains that when ENO1 is deleted in glioblastoma tumors, tumor growth isn’t directly impaired. But the tumor becomes completely dependent on ENO2, making it more sensitive to ENO2 inhibitions.

“The key message,” says Gilmour, “is there’s still a lot to be understood within cancer metabolism and many opportunities to modulate cancer metabolism for therapeutic benefit. I think it’s going to be an exciting field for the foreseeable future.”

Learn More in the April 2017 Issue of SLAS Discovery

 “Special collections like this one are part of SLAS’s commitment to providing research information that is both educational and cutting edge, as well as offering a venue for scientists to present their important data,” says Bob Campbell, editor-in-chief of SLAS Discovery.

Now in its 22nd year of peer-reviewed publication, SLAS Discovery is one of two official SLAS journals. Together, SLAS Discovery and SLAS Technology address the full spectrum of issues that are mission-critical to life sciences professionals, enabling research teams to gain scientific insights; increase productivity; elevate data quality; reduce process cycle times; and enable research and development that otherwise would be impossible.

April 3, 2017