Citric Acid Cycle Unveiled: Understanding its Impact on Cancer Cells — Part 4 of 5
By Eva Fata
This is 4th Part in a 5 Part Series I’ve written comparing cellular respiration in normal cells versus cancer cells. In this part, I will be exploring the citric acid cycle in cancer cells. For all parts, please see the links at the bottom of this article.
Citric Acid Cycle in Cancer Cells
In cancer cells, the citric acid cycle a central metabolic pathway is key for energy production, biosynthesis undergoes different alterations to support their uncontrolled proliferation and survival. Cancer cells redirect the citric acid cycle molecules toward biosynthetic pathways to meet there demands of macromolecule synthesis. Below I will talk about all the different alterations seen in cancer cells.
I will break down the citric acid cycle in cancer cells into these categories fuels feeding the cycle, the effect of oncogenes and tumor suppressors on fuel and cycle usage, common mutations and deregulations of cycle enzymes.
Fuels feeding the citric acid cycle
Glucose
Glucose is the most common fuel source in mammalian cells, it is imported into the cell by glucose transporters (GLUT) and can be used in many ways. In normal cells, glucose is metabolized via glycolysis which produces pyruvate and further enters the citric acid cycle as acetyl-CoA. Progression of the citric acid cycle usually occurs when there is an increased need for energy. However, cancer cells increase their glucose uptake, often through aerobic glycolysis even in the presence of oxygen (Warburg Effect). Tumour cells acquire this additional glucose by stimulating glucose transporters GLUT1 and GLUT3. Not only do cancer cells increase their glucose rate, but they also have a different way of using that glucose. Normal cells oxidize glucose in the mitochondria and produce ATP through oxidative phosphorylation, cancer cells prefer to use aerobic glycolysis even in the presence of oxygen. This leads to the conversion of glucose to lactate however it is less efficient in the way that it only produces 2 ATP per glucose molecule rather than oxidative phosphorylation through the citric acid cycle produces 38 ATP molecules. To recoup with the reduced ATP produced and meet their energy demand cancer cells use alternative fuel sources like glutamine which can be used in the citric acid cycle after undergoing glutaminolysis which will provide cancer cells with additional resources like ATP and NADH to help with energy production and biosynthesis.
Glutamine
Glutamine is the most abundant amino acid in the body and could serve as a significant fuel source for the citric acid cycle. Glutaminolysis is the breakdown of glutamine into glutamate by the enzyme glutaminase (GLS). Glutamate could further be metabolized tino a-KG which helps replenish the citric acid cycle. This is critical for providing cancer cells with the necessary molecules and nutrients to maintain the citric acid cycle flow and support their cellular proliferation. The importance of glutaminolysis in cancer cell proliferation was discovered by Harry Eagle, who saw that certain cancer cells like HeLa cells have a preference for glutamine for their growth. This dependence on glutamine is partially driven by the increased glucose uptake which leads to the need for alternative resources for the citric acid cycle molecules. Cancer cells increase the rate of glutamine transport and enzymes that are involved in glutaminolysis to meet their energy demands. MYC a proto-oncogene is a regulator of glutaminolysis and promotes the expression of different glutame transporters and GLS. Elevated levels of GLS and glutamine transporter can allow cancer cells to get energy and macromolecules from glutamine catabolism which can help contribute to their growth and proliferation.
Fatty acids
Fatty acids enter the citric acid cycle after breaking down through the processes of β-oxidation which takes place in the mitochondria. During β-oxidation, a fatty acid molecule is broken down into two carbon units which generate acetyl-CoA molecules. These acetyl-CoA molecules then enter the citric acid cycle and undergo oxidation to produce energy in the form of ATP. This Acetyl-CoA which is from the fatty acid β-oxidation could also serve as a a starting material for the synthesis of new fatty acids through lipogenesis (the process in which fatty acids are turned into fat). Lipogenesis is crucial for cancer cells because they require a rather abundant supply of lipids for cell membrane formation and other cellular processes. In cancer cells, there is an increased demand for lipids because of their rapid proliferation, and therefore lipogenesis occurs at a higher through the over-expression of these enzymes. This dysregulation allows cancer cells to have elevated lipid requirements for membrane synthesis, signalling pathways and other cellular processes which are necessary for tumour growth and progression.
Enzymes which are involved in lipid synthesis such as ACLY, ACC and FAS are often overexpressed in several types of cancer. For example, ACLY is often upregulated in breast cancer, cervical cancer and many others. ACC is often overexpressed in non-small cell lung cancer, and FAS is upregulated in breast and prostate cancers.
The enzymes involved in fatty acid biosynthesis are adenosine triphosphate citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) all of which play a vital role in regulating lipid metabolism. ACLY converts citrate to cytosolic acetyl-CoA, which is then carboxylated by ACC to form malonyl-CoA. FAS catalyzes the synthesis of fatty acids, mostly palmitate, by combining malonyl-CoA with acetyl-CoA. This process is strongly regulated to meet the lipid demands for rapidly proliferating cancer cells.
Increased activation and overexpression of ACLY, ACC and FAS in tumours are correlated with disease progression and poor prognosis. Due to this, these enzymes are being investigated as potential biomarkers of metastasis, highlighting their importance in cancer biology and their potential to be used as a diagnostic or prognostic indicator.
All of these molecules are fuel sources for cancer cells and contribute to their energy production and biosynthesis.
The Effect of Oncogenes and Tumour Suppressors on Fuel and Cycle Usage
MYC
The proto-oncogene MYC controls many cellular processes, like cell proliferation, metabolism, differentiation and genomic stability, it also is a dominant driver of tumour transformation and progression as well. Abnormal MYC activity can arise from chromosomal translocation, increased mRNA/protein stability and gene amplification which are common in many human cancers.
One of MYC’s key roles is regulating cellular metabolism. It applies control over a wide range of different metabolic pathways such as aerobic glycolysis, glutaminolysis, oxidative phosphorylation amino acid biosynthesis and more. For glutamine metabolism, MYC transcriptionally activates genes and enzymes which are involved in glutaminolysis which makes it a key driver of glutamine metabolism through the citric acid cycle. Furthermore, MYC facilitates the uptake of glutamine transports like ASC amino acid transporter 2 (ASCT2) and system N transporter (SN2).
Once glutamate is produced, the MYC-dependent cancer cells have 2 options to convert it to α-KG to enter into the citric acid cycle. One option is a pathway which is controlled by glutamate dehydrogenase (GLUD) or by aminotransferases which help transfer an amino acid group from an amino acid to a keto acid.
Furthermore, MYC can play a role in managing fatty acid oxidation and channelling its metabolites into the citric acid cycle by acetyl-CoA. MYC expression leads to the increase of fatty acid transporters and fatty acid oxidation genes which facilitates the use of fatty acids as an energy source.
Overall MYC’s involvement in regulating multiple metabolic pathways such as glutamine metabolism emphasizes its significance in tumour cell proliferation and survival which could make it a target for cancer therapy.
HIF
Hypoxia-inducible factors (HIFs) are transcription factors which respond to reduced oxygen availability in the cellular environment. HIFs are composed of oxygen-dependent α-subunits and constitutively expressed β-subunits. Under normal oxygen conditions, the α-subunit are hydroxylated by prolyl hydroxylases (PHD) and subsequently targeted for degradation by the von Hippel-Lindau (VHL) tumour suppressor protein.
In tumours, HIF activation occurs in response to hypoxia caused by genetic alteration (loss of VHL) or inadequate blood vessel formation. When activated HIF directs a metabolic reprogramming which favours the catabolism of glucose through aerobic glycolysis which diverts glucose away from the citric acid cycle.
HIF promotes glycolysis and lactate production by upregulating the expression of glucose transporters (SLC2A1), and glycolytic enzymes. (pyruvate kinase, hexokinase) and lactate dehydrogenase A (LDHA). This metabolic shift is key for tumour cells to generate the energy they need to sustain rapid proliferation under these hypoxic conditions.
Additionally, HIF slows down glucose metabolism through the citric acid cycle which is a process called glucose anaplerosis, which activates pyruvate dehydrogenase kinase 1 (PDK-1) and in turn slows down the pyruvate dehydrogenase (PDH) complex. This prevents the conversion of pyruvate to acetyl-CoA which is a key step in glucose oxidation through the citric acid cycle.
To recuperate for this reduced glucose availability for the citric acid cycle tumour cells with HIF activation will often increase their reliance on alternative fuel sources like glutamine. Now under this hypoxic condition, glutamine becomes a substrate (facilitator for the reaction) for the citric acid cycle, mostly in the form of α-ketoglutarate (α-KG). This then promotes reductive carboxylation reactions which adds a carboxyl group to molecules and uses electrons rather than releasing them. This generates citrate for lipogenesis which helps support the biosyntheic demands for tumour cells.
Overall HIF helps tumour cells to adapt to hypoxic environments by enhancing glycolysis, reducing glucose oxidation through the citric acid cycle and increasing the use of other alternative nutrients like glutamine to help with their growth and survival.
P53
P53 is a quite well-known tumour suppressor and plays a key role as well in regulating various cellular processes like metabolism. The wild-type P53 (normal non-mutated one) helps to maintain balances between cellular bioenergetics and biosynthesis and achieves this by adjusting different metabolic pathways.
One key function of P53 (including any form of P53 protein both wild-type and mutated variants) in metabolism is to slow down glycolysis. It achieves this by reducing the expression of glucose transports GLUT1 and GLUT4 which therefore reduces the consumption of glucose into cells Furthermore, P53 also slows down the activity of different glycolytic enzymes like phosphofructokinase 1 (PFK1) and phosphoglycerate mutase which reduces the flow of molecules through glycolysis.
P53 promotes oxidative phosphorylation. To support this P53 make sure that the availability of anaplerotic substrates (special molecules that our cells use to replenish important ingredients for the citric acid cycle) like glucose and glutamine.
P53 also promotes the use of glutamine in the citric acid cycle by Increasing the expression of glutaminase 2 (GLS2), an enzyme that plays a role in converting glutamine into α-ketoglutarate, which is an important molecule in the TCA cycle.
In solid tumours, P53 is often mutated, and somatic mutation occurs in more than 50% of human malignancies. Additionally the loss of wild-type P53 function because of this mutation further leads to the dysregulation of metabolism by enhancing glycolysis and reducing oxidative phosphorylation in tumour cells. This shift again helps to promote their proliferation and survival.
RAS
RAS particularly KRAS, NRAS, and HRAS genes are often mutated in many cancers and play a role in cancer development and progression. These genes act as signalling molecules (substances that cells use to communicate). RAS activation leads to the activation of different pathways which are involved in cell growth, survival and metabolism. One critical role of RAS in cancer metabolism is the involvement it has in nutrient consumption and use. In certain tumours, RAS activation can trigger certain pathways that allow cancer cells to get essential nutrients from extracellular and intracellular sources.
For example, KRAS-driven pancreatic cancer cells scavenge proteins, like glutamine from their extracellular environment. Once glutamine is consumed it serves as a crucial fuel for the citric acid cycle and sustains the energy demands for rapidly proliferating cancer cells.
Recent studies have shown that the increased number of mutated KRAS alleles is associated with tumour progression and can also enhance glucose anaplerosis to fuel the citric acid cycle. This allows cancer cells to efficiently use glucose-derived carbons for the citric acid cycle which supports their increased metabolic demand during tumour growth and metastasis.
Overall, RAS activation in cancer cells arranges different metabolic adaptations which can promote nutrient consumption and use which further allows for the continuous supply of energy and building blocks which are necessary for cancer cell survival and proliferation.
Common Mutations and Disregulations of Cycle Enzymes
SDH
Succinate dehydrogenase (SDH) plays a crucial role in the citric acid cycle and the electron transport chain (ETC) within the mitochondria. SDH is a heterotetrameric enzyme which means it is a four-part enzyme with the four subunits SDHA, SDHB, SDHC and SDHD. Mutations in the subunits of SDH like SDHA, SDHB, SDHC and SDHD have been seen in hereditary paragangliomas (hPGLs) and pheochromocytomas (PCCs). These mutations put individuals at risk of the development of neuroendocrine tumours. Specifically heterozygous mutations in SDH increase the risk of developing hPGL and PCC.
Mutations in SDH have also been seen in other types of cancer such as gastrointestinal stromal tumours, renal tumours, thyroid tumours, neuroblastoma and testicular seminoma. This shows the significance of SDH dysfunction in cancer development and progression. Altogether, SDH mutations contribute to tumourigenesis by disrupting normal cellular metabolism and energy production pathways.
FH
Fumarate hydratase (FH) is a crucial enzyme also involved in the citric acid cycle by catalyzing the reversible hydration of fumarate to L-malate, which occurs in the mitochondria and is essential for maintaining cellular metabolism and energy production. It is also expressed in the cytoplasm and takes part in different metabolic pathways like the urea cycle and amino acid metabolism.
Heterozygous mutations in the FH gene put individuals at risk of multiple cutaneous and uterine leiomyomas (MCUL) and hereditary leiomyomatosis and renal cell cancer (HLRCC). Furthermore, FH mutations have also been linked to other types of cancer like bladder, breast and testicular.
FH is classified as a tumour suppressor gene. The loss or inactivation of the FH enzyme can disrupt normal cellular metabolism and can contribute to tumorigenesis by changing metabolic pathways and promoting cell proliferation.
IDH
Isocitrate dehydrogenase (IDH) consists of three isoforms IDH1, IDH2 and IDH3. These isoforms play a role in cellular metabolism by catalyzing the conversion of isocitrate to α-ketoglutarate (α-KG), which is an important step in the citric acid cycle.
Mutations in IDH1 and IDH2 are seen in low-grade glioma and glioblastoma and 80% of cases have these mutations. They are also found in other types of cancer such as acute myeloid leukemia (AML).
The accumulation of 2-HG because of IDH1/2 mutations can contribute to tumorigenesis by promoting different epigenetic alterations, inhibiting cellular differentiation and inducing genomic instability.
Targeting the mutant IDH enzymes could be promising as a therapeutic strategy for cancer treatment.
Why should I learn about this?
Targeting the citric acid cycle in cancer has emerged as a promising therapeutic strategy because of the metabolic dependencies and vulnerabilities of cancer cells. Since many tumours rely on glutamine as a fuel source for the citric acid cycle so making the suppression of glutaminolysis can make an attractive therapeutic approach. Furthermore, the different alterations in the citric acid cycle can help with biomarkers for cancer diagnosis, prognosis and treatment response. Studying the citric acid cycle can lead to the identification of different biomarkers for disease detection.
Next Articles in The Series
To explore the previous parts of the series: Part 1, Part 2, Part 3
To explore the next parts of the series: Part 5
