The Metabolism of Cell Growth and Proliferation

Why Is Metabolism Important to an Understanding of Cancer?

At its heart, cancer is a disease of abnormal proliferation. Proliferation represents a distinct metabolic challenge: cells must replicate all of their proteins, lipids, and nucleic acids to generate a daughter cell. This process requires vast inputs of energy and raw materials. As a result, proliferating cells take up large quantities of nutrients in order to engage the biosynthetic reactions that support cell growth. Unsurprisingly, cancer cells exhibit many of the metabolic features that are characteristic of proliferating cells. Given the logical association between metabolism and proliferation, it is not surprising that the study of cancer metabolism could yield diagnostic and therapeutic opportunities. Indeed, the propensity of cancer cells to take up high levels of glucose has been exploited clinically. For example, uptake of radioactive glucose analogs, measured by positron emission tomography, can be used to diagnose and monitor glucose-avid tumors.

Over the past decade, the study of cancer has become increasingly entwined with the study of cellular metabolism. Altered metabolism is a common feature of cancer cells, and most oncogene and tumor suppressor pathways directly regulate cellular metabolic pathways. Moreover, cancer-associated metabolic alterations can in turn signal to alter the expression or activity of oncogenic pathways. This chapter provides an overview of the metabolic changes associated with cancer cells, the genetic mechanisms that regulate tumor metabolism, and the clinical implications of altered cancer metabolism.

The Metabolic Requirements of Cell Proliferation

Proliferating and nonproliferating cells have very different metabolic requirements. Nonproliferating cells must maintain an ample supply of energy to fuel basic cellular processes, such as maintaining ion gradients and supporting transcription and translation, in addition to fulfilling their tissue-specific biological roles. This energy, usually in the form of adenosine triphosphate (ATP), is generated primarily in the mitochondrion. The basic building blocks of carbohydrates, lipids, and proteins (glucose, fatty acids, and amino acids, respectively) can be broken down in the tricarboxylic acid (TCA) cycle and the NADH generated by their oxidation used to produce ATP using oxidative phosphorylation (OXPHOS) ( Figure 13-1 ). This efficient conversion of nutrients into energy permits relatively low levels of nutrients to support the metabolic demands of quiescent cells.

In contrast, proliferating cells face the challenge of doubling all of the macromolecules in the cell in order to divide into two daughter cells. Cells must produce abundant nucleic acids, amino acids, and fatty acids in order to synthesize the DNA, proteins, and membranes that are necessary to replicate themselves. The process of synthesizing macromolecules, known as anabolism or anabolic metabolism , requires three main inputs that are critical for the process of cell growth:

• 1.

  Substrates. Extensive intracellular metabolic networks ensure that mammalian cells can generate many of the metabolic building blocks required for growth from relatively few inputs. For example, humans can synthesize 11 amino acids (which are consequently known as the nonessential amino acids) by rearranging the nitrogen and carbon backbones of other dietary inputs. (The other 9 amino acids are considered “essential” amino acids, as they cannot be synthesized in human cells.) In most cell types, glucose is the most important substrate for anabolic metabolism, because by-products of glucose breakdown (or catabolism) can contribute to the production of nonessential amino acids as well as nucleic acids and lipids. Most reduced nitrogen utilized for cell growth is taken into cells as glutamine. Glutamine plays a critical role providing nitrogen for nucleotide and nonessential amino acid synthesis. Like glucose, glutamine can also provide carbon units for fatty acid synthesis. Given their importance as central metabolites bridging both anabolic and catabolic pathways, glucose and glutamine are major fuels for cell proliferation and are discussed in detail later in this chapter. Cell proliferation requires numerous other substrates, including essential amino acids and a variety of vitamins and minerals, which will not be discussed (for more reading, see Ref. ).

• 2.

  Chemical energy. Intramolecular bonds store large amounts of energy. Consequently, catabolism of macromolecules releases energy, which is harnessed either directly by driving the production of ATP or indirectly by reducing the electron carrier NAD ⁺ to NADH, which in turn fuels ATP production via OXPHOS. Conversely, macromolecular synthesis requires extensive energy input. Separately, protein translation and DNA replication consume significant amounts of ATP. In contrast to nonproliferating cells, a significant portion of the ATP required to sustain cell proliferation is produced by glycolysis, which is upregulated in rapidly dividing cells.

• 3.

  Reducing equivalents. Just as many catabolic reactions involve the oxidation, or removal of electrons from metabolites to electron carriers such as NADP ⁺ or NAD ⁺ , several anabolic reactions require the input of electrons as reducing agents to forge intramolecular bonds. Reducing equivalents, primarily in the form of NADPH, carry these electrons for use in anabolic pathways. Fatty acid, nucleic acid, and nonessential amino acid synthesis consume large amounts of NADPH. The relative levels of NADPH/NADP ⁺ and NADH/NAD ⁺ often reflect the “redox” status of the cell—the extent to which the cell has more reduced electron carriers (higher NAD(P)H/NAD(P) ⁺ ratio) or more oxidized electron carriers (lower NAD(P)H/NAD(P) ⁺ ratio). Cellular redox balance is tightly controlled by many factors and can contribute to cancer growth, proliferation, and survival in numerous ways. For proliferating cells, a large fraction of NADPH and NADH may be generated by glucose catabolism.

Regulation of Cell Metabolism

During conditions of nutrient scarcity, anabolic programs are suppressed in favor of energy-generating catabolic pathways. Conversely, multiple cellular metabolic pathways must be coordinately rewired to support cell proliferation. Elevated nutrient uptake—particularly of glucose and glutamine—provides the substrates for cell growth. Rapid catabolism of glucose via glycolysis produces sufficient ATP and NADPH to support energy-dependent anabolic reactions while generating the metabolic intermediates that will be critical for macromolecular biosynthesis. Similarly, catabolism of glutamine will help maintain bioenergetics while ensuring adequate substrates for cell growth. Multiple regulatory mechanisms ensure that energy production and macromolecular biosynthesis are appropriately balanced with the metabolic needs of the cell.

Unicellular organisms are directly exposed to the environment and any fluctuations in nutrient availability that may occur. Consequently, these organisms have elegant mechanisms to sense the available nutrients and rewire their metabolism accordingly. Thus, for unicellular organisms, nutrient availability directly controls the signals that regulate growth and proliferation. If a unicellular organism is in an environment with abundant nutrients, these nutrients will directly activate signaling pathways that instruct the cell to engage anabolic metabolic pathways and to undergo cell division. Conversely, conditions of low nutrient availability will halt cell growth and division. Cells will engage catabolic pathways in order to produce energy to survive through the period of scarcity ( Figure 13-2 ).

In contrast, metazoans have complex organ systems that maintain a relatively constant level of extracellular nutrients throughout the body. Cell-autonomous metabolic regulation would be catastrophic, as well-fed cells might undergo aberrant growth and proliferation. Thus, nutrient availability alone cannot determine whether cells engage anabolic or catabolic pathways; the metabolism of individual cells must be aligned with the needs of the organism as a whole. This coordination is largely achieved through extracellular growth factors that regulate nutrient uptake and utilization. Binding to receptors on the cell surface, growth factors stimulate intracellular signal-transduction cascades that regulate many facets of nutrient uptake and metabolism. In particular, growth factor signaling enables cells to take up high levels of nutrients such as glucose and glutamine and to engage in anabolic pathways supporting cell growth (see Figure 13-2 ). In this manner, systemic signals can target individual cells (expressing the proper growth factor receptor) to induce specific activities, thereby ensuring that the behavior of individual cells is tailored to the needs of the entire organism. In the absence of growth factor signaling, metazoan cells are largely quiescent, maintaining homeostasis by the efficient degradation of the limited nutrients they are directed to take up. By engaging oxidative, catabolic pathways that preserve intracellular energy levels, these cells are able to survive and fulfill their allotted functions.

Most cells have the capacity to engage both anabolic and catabolic pathways, and the balance between the two—the balance that guards against inappropriate cell proliferation while maintaining cell survival—is carefully regulated by a number of factors. External signals such as growth factors instruct the cell to grow, activating anabolic reactions and driving nutrient uptake accordingly. The activities of key metabolic enzymes that determine whether metabolites enter anabolic or catabolic pathways are regulated by posttranslational modifications, most commonly phosphorylation triggered by growth factor signaling. Metabolites themselves function as critical allosteric regulators of metabolic enzyme activity, increasing or inhibiting flux through metabolic pathways according to the needs of the cell. For example, signals of abundant energy stores such as ATP and NADH can allosterically inhibit multiple enzymes that otherwise promote the channeling of metabolites into catabolic, energy-producing pathways. This allows for the diversion of metabolites toward anabolic processes when energy supply is plentiful. Similarly, both metabolic cues and growth factor signaling pathways can regulate the expression levels of metabolic enzymes. Through these mechanisms, cells carefully control the activation of anabolic or catabolic metabolic programs and ensure that during cell proliferation, metabolic pathways are coordinately rewired to support cell growth.

A major hallmark of cancer is the development of cell-autonomous regulation of cell growth. Through multiple genetic events, including activation of growth factor signal transduction pathways (oncogenes) and loss of inhibitory signals (tumor suppressors), cancer cells circumvent dependence on external growth factor signaling. Constitutive activation of growth factor signaling pathways ensures that cancer cells are not subject to the normal regulation of metazoan cells. Consequently, cancer cells exhibit metabolic transformation, taking up high levels of nutrients and engaging in proliferative metabolism to support unchecked cell growth.

The Metabolic Profile of Cancer Cells

It is increasingly clear that cancer cells exhibit metabolic phenotypes that are similar to rapidly proliferating normal cells, with the major difference that the metabolic alterations in cancer cells stem from oncogenic cell-autonomous signaling, rather than the appropriate result of specific growth signals originating outside the cell. The characteristic metabolic features of rapidly proliferating tumor cells include elevated glucose uptake and glycolysis, increased glutamine uptake and utilization, and enhanced lipid and nucleotide biosynthesis. This section discusses the common metabolic signatures of tumor cells and how these metabolic alterations may support tumor growth.

Aerobic Glycolysis: The Warburg Effect

The most well-known and prevalent metabolic change associated with cancer cells is the enhanced uptake and metabolism of glucose, often referred to as the Warburg effect . In 1926, Otto Warburg noted that rapidly proliferating ascites cancer cells take up high levels of glucose and produce large amounts of lactate, even in the presence of oxygen. This finding was not intuitive: Work begun by Louis Pasteur had shown a clear inverse relationship between oxygen availability and the rate of glucose fermentation to lactate. The ability of eukaryotic cells to switch between anaerobic energy production through fermentation to aerobic energy production through oxidative phosphorylation depending on the presence of oxygen is known as the Pasteur effect ( Figure 13-3 ). In cancer cells in the presence of oxygen, one might expect glucose to be metabolized to pyruvate, which would then be completely oxidized in the mitochondrion to produce ATP through the oxygen-dependent process of oxidative phosphorylation (see Figure 13-1 ). The surprising observation that cancer cells converted pyruvate to lactate despite abundant oxygen availability—the process of aerobic glycolysis—led Warburg to speculate that mitochondrial function is impaired in tumor cells, forcing a reliance on glycolytic metabolism.

We now know that most cancers do not exhibit impaired mitochondrial energy production. Moreover, research increasingly suggests that mitochondrial metabolic pathways are not simply catabolic and energy-producing; they may also play a critical role supporting anabolic biosynthetic pathways, as discussed later. These findings indicate that high aerobic glycolysis is not the secondary result of a metabolic failure, but rather a specific adaptation that promotes cell growth. Despite the centrality of the Warburg effect to cancer cell metabolism, there is still some debate as to how aerobic glycolysis confers a proliferative advantage to tumor cells.

The Paradox of the Warburg Effect

Glycolysis comprises a series of reactions that convert 1 molecule of glucose to 2 molecules of pyruvate, generating 2 molecules each of ATP and NADH ( Figure 13-4 ). If oxygen is present, pyruvate is converted to acetyl-CoA in the mitochondrion, and acetyl-CoA is oxidized by the TCA cycle, producing 1 molecule of GTP and four pairs of high-energy electrons that will be used to fuel OXPHOS (3 molecules of NADH and 1 molecule of FADH ₂ ). As the glycolytic reactions occur in the cytosol, the reducing equivalents of the NADH generated by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) must be transferred to the mitochondrion in order for their energy to be harnessed into ATP. The malate-aspartate shuttle and the glycerol-3-phosphate shuttle transfer the electrons into the mitochondrion for oxidation by the electron transport chain. All together, this process of complete glucose oxidation produces 38 molecules of ATP. Thus, when oxygen is present, glucose can be efficiently converted to ATP while regenerating the NAD ⁺ required to maintain glycolysis.

If NADH is not oxidized to NAD ⁺ , the subsequent depletion of NAD ⁺ will inhibit GAPDH and block glycolysis. To avoid this, under anaerobic conditions lactate dehydrogenase (LDH) reduces pyruvate to lactate, consuming NADH and producing NAD ⁺ . In total, therefore, anaerobic glycolysis yields two molecules of ATP and two molecules of lactate that are secreted from the cell.

The paradox of the Warburg effect is that cells convert pyruvate to lactate even in the presence of oxygen. If glucose oxidation produces 19 times more ATP than anaerobic glycolysis, what benefit could a cell gain from choosing the less efficient route? And why would a cell that is growing rapidly throw away valuable carbon units in the form of lactate? Warburg’s studies described an astonishing rate of lactate production in ascites tumor cells—cells produced up to 30% of their dry weight in lactic acid per hour. Although it is now known that this is a much higher rate than for most tumors, it is clear that aerobic glycolysis occurs at a high rate in many tumor cells. There are several potential explanations for how tumors could sustain such apparently wasteful metabolism and the benefits that rapid aerobic glycolysis might bestow:

• 1.

  Aerobic glycolysis is a source of rapid ATP generation. According to Warburg’s calculations, the high rate of glucose consumption enabled cells to produce approximately the same amount of ATP through fermentation as through respiration. Similarly, a series of studies demonstrated that stimulating cells to proliferate increased ATP turnover, as expected; however, the increased ATP demand was met entirely by increased glycolytic flux and not by any increase in ATP production by oxidative phosphorylation. Thus, the relative inefficiency of ATP production may be counterbalanced by the high rate of glucose consumption. Furthermore, tumor cells may extract more than two molecules of ATP per molecule of glucose: Several studies demonstrate that cytosolic-mitochondrial NADH shuttles are active in tumor cells, indicating that tumors can oxidize GAPDH-derived NADH in the mitochondrion to produce ATP. In addition, glycolysis generates cytosolic ATP very rapidly: The conversion of glucose to lactate can take mere seconds. This could benefit tumor cells undergoing high rates of protein, lipid, and nucleotide biosynthesis. Thus, as long as glucose supplies are not limited, high levels of glycolysis may provide an advantageously rapid and plentiful source of ATP.

• 2.

  Adaptation to hypoxia. Cells in solid tumors experience notoriously harsh and varied metabolic conditions as tumor growth disrupts the normal tissue architecture. Blood vessels inside the tumors are often dysfunctional or nonexistent. Consequently, tumor cells commonly experience periods of intermittent hypoxia. Survival in hypoxia requires metabolic adaptations, including increasing glycolysis and downregulating mitochondrial fuel oxidation. A cell already primed with these metabolic adaptations would be more likely to survive inside a solid tumor. Indeed, cells cultured ex vivo from solid tumors often display high lactate production even when well oxygenated, suggesting that elevated glycolysis is a fundamental feature that either predisposes cells toward tumor formation or is selected for early in tumor development. It is worth noting that because hypoxia typically results from inadequate blood supply, cells rarely experience hypoxia without concomitant deprivation of nutrients such as glucose that are also provided by the vasculature. Thus, although glycolytic metabolism may increase the likelihood of survival during periods of intermittent hypoxia, sustained hypoxia will likely negatively influence cell growth regardless of metabolic adaptations.

• 3.

  Acidification of microenvironment. There is some evidence that the apparently wasteful secretion of lactate may itself confer a selective advantage to tumor development. Cells export lactate through the family of H ⁺ -linked monocarboxylate transporters. By acidifying the microenvironment, lactate export may promote the death of normal cells and extracellular matrix degradation to enhance tumor invasion. In support of this model, studies suggest that acidic conditions can stimulate tumor cell invasion in vitro, and in vivo interventions to increase pH can reduce spontaneous metastases. Intriguingly, some studies suggest that lactate may also be used as a fuel source. Lactate exported from hypoxic cells can be used as a substrate for oxidative metabolism in normoxic cells within the same tumor, preserving glucose for the hypoxic cells while providing fuel for the normoxic cells. Thus, lactate production may not be an unfortunately costly by-product of glycolytic metabolism, but may also serve pro-tumorigenic roles. Indeed, blocking conversion of pyruvate to lactate by suppressing LDH expression can impair tumorigenesis. Clearly, the production of lactate is critical for cancer cell growth, whether by maintaining oxidized NAD ⁺ to promote glycolysis, acidifying tumor surroundings to promote cancer cell survival, or providing a means for efficient substrate allocation in a metabolically diverse microenvironment.