It is humbling, indeed, to realize that even a simplified diagram of the “big picture” of cell signaling consists of numerous types of membrane receptors, cytoplasmic systems, and nuclear factors (see Signal Transduction—The Big Picture on page xx). These systems and the cell cycle regulators are discussed in Section 3.

Normal energy production uses glucose in the cytoplasm and pyruvic acid in the mitochondria. Under anaerobic conditions, glycolysis predominates. Otto Warburg (1930, 1956a, 1956b) first observed that cancer cells can carry out “aerobic glycolysis” to get more glucose into the cell and compensate for the much lower efficiency of ATP production than that by mitochondrial oxidative phosphorylation.

The basis of the positron emission tomography (PET) scan is that cancer cells can sequester fluorodeoxyglucose and “light up” the cancer. It is an example of how cancers achieve a proliferative advantage. This is accomplished at least in part by upregulating glucose transporters such as GLUT1 (Jones and Thompson, 2009). Glycolysis is also upregulated by RAS oncoprotein and hypoxia, which drive up levels of HIF1α and HIF2α transcription factors (Semenza, 2010a, 2010b).

Excess glycolysis also allows glycolytic intermediates to incorporate nucleosides and amino acids that are needed for the growth of new cells. Some cancers actually have a hybrid complementary energy system: glucose-dependent (“Warburg effect”) cells that secrete lactate and other cells that use the lactate and the citric acid cycle (Kennedy and Dewhirst, 2010). This flexibility is particularly helpful because tumors can have fluctuating degrees of hypoxia due to metabolic instability and abnormal neovasculature.

Gliomas and other human cancers can develop gain-of-function mutations in the isocitrate dehydrogenase 1/2 (IDH) enzymes, producing elevated oxidation and stability of the HIF1 transcription factors (Reitman and Yan, 2010; Yen et al., 2010). Glutamine (GLU) is a nonessential amino acid that is consumed by proliferating cells far more than other amino acids. Glutaminase removes an amide group that produces glutamic acid, which, in turn, loses its amine group to form alpha-ketoglutarate.

Ultimately, GLU contributes to the supply of oxaloacetate, citrate, NADPH, and carbon molecules—all critical to mitochondrial energy production. GLU is sequestered in the cell by solute carrier (SLC) transporters and serves as a nitrogen donor for nucleotide synthesis mediated by the c-MYC oncogene. GLU also activates mTORC1, the mammalian target of rapamycin complex 1. Taken together, these observations form a strong argument that cancer cells can be highly GLU dependent (“addicted”) and inhibiting GLU-mediated metabolism in the cancer cells can be a highly effective therapeutic tactic (Wise and Thompson, 2010).

Normal tissue growth and repair depends on cells responding to negative growth signals and knowing when to stop when they come into contact with each other. Keloids are macroscopic examples of wounds that “heal too much” when this recognition does not occur. One of the earliest microscopic signs of cell transformation is this “heaping up” of cells (Figure 2.3).

There are several ways in which normal cells respond to negative growth signals. Merlin, STK11, and TGFb are three examples of growth controls that may be mutated in various cancers (Hanahan and Weinberg, 2011). Merlin, the neurofibromatosis 2 (NF2) gene product, promotes contact inhibition and cell-to-cell bonds by coupling cell surface adhesion molecules (e.g., E-cadherin) to transmembrane receptor tyrosine kinases (RTKs) (e.g., the epidermal growth factor receptor [EGFR]) and blocks mitogenic signals (Curto et al., 2007).

Serine/threonine kinase 11 (STK11), also known as liver kinase B1 (LKB1) or renal carcinoma antigen NY-REN-19, is an epithelial polarity protein that maintains epithelial structural integrity. Germ-line mutations in this gene have been associated with Peutz-Jeghers syndrome, an autosomal dominant disorder characterized by the growth of polyps in the gastrointestinal tract, pigmented macules on the skin and mouth, and other neoplasms. Somatic mutations of the LKB1 gene have been found in lung, cervical, breast, intestinal, testicular, pancreatic, and skin cancers. LKB1 can block MYC oncogene function (Partanen et al., 2009).

Transforming growth factor beta (TGF-β) is a multipurpose cytokine activity that exists in three isoforms. It is part of a superfamily that includes inhibins, activin, anti-Müllerian hormone, bone morphogenic protein, decapentaplegic, and Vg-related protein 1. TGF-β is known for its antiproliferative effects (Ikushima and Miyazono, 2010). However, in many late-stage tumors, TGF-β signaling changes from suppression of cell proliferation and instead activates a cellular program, termed the epithelial-to-mesenchymal transition (EMT) (see EMT and Metastasis on p. xx.), seen in high-grade malignancy.

Displaying a useful graphic depiction of the highly complex network of microenvironmental signaling interactions remains a great challenge, made even more difficult due to tumor progression and the

interplay between neoplastic and supporting stromal cells. This model becomes even more complex as cancers metastasize.

Virtually, every tumor deposit contains inflammatory cells ranging from sparse infiltrations detectable only with cell type-specific antibodies to dense cellular infiltrates apparent by standard light microscopy (DeNardo et al., 2010). These inflammatory cells supply a whole array of molecules: growth and survival factors, proangiogenic factors, matrix-modifying enzymes that facilitate invasion and metastasis, and inducers of EMT and reactive mutagenic oxygen species (Grivennikov et al., 2010). It is quite possible that some of the beneficial effects of chemotherapy actually may be due to decreasing the activity of sensitive, tumor-infiltrating inflammatory cells even when the cancer cells themselves have acquired chemotherapy resistance.

Leukocytes (macrophages, mast cells, neutrophils, and T/B lymphocytes) can be found in most neoplastic lesions and promote tumor progression (i.e., tumors are wounds that never heal) (Dvorak, 1986; Schäfer and Werner, 2008). In the course of normal wound healing and fighting infections, immune inflammatory cells appear transiently and regress in contrast with the chronic inflammation of cancer, which is associated with fibrosis, scarring, aberrant angiogenesis, and neoplasia (Grivennikov and Greten, 2010) (Figure 2.4).

Immune inflammatory cells can release a variety of cytokines that have either tumor-promoting or tumor-killing effects:

All of the above cytokines are potential targets for cancer therapeutic agents. Tumor-infiltrating myeloid cells, expressing macrophage marker CD11b and neutrophil marker Gr1, suppress CTL and natural killer (NK) cell activity. Recruitment of these myeloid elements may be doubly damaging by directly promoting angiogenesis and tumor progression as well as affording a means to evade immune destruction. Another strategy, therefore, may be to inhibit these promoter cells rather than the CSCs.

Many cancers, such as squamous cancer of the lung, demonstrate spontaneous necrosis. This is more complicated. During necrosis, in contrast to apoptosis, cells self-destruct and release their contents into the local tissue microenvironment. This results in tumor-promoting inflammation, increased angiogenesis, and proliferation due to cytokines such as IL-1α (Hanahan and Weinberg, 2011).

Cell death by necrosis appears to be under genetic control in some circumstances and, rather than being a random and undirected process, explains why some necrotic cancers can act in a very aggressive fashion and produce a whole array of paraneoplastic complications such as fever, anorexia, and fatigue (Galluzzi and Kroemer, 2008). Tumor lysis syndrome is the most extreme example of the harm that can occur when tumors die too rapidly and liberate toxic, life-threatening organic acids, potassium, and so forth. Finding ways to limit cellular necrosis could be a very effective strategy to limit cancer acceleration.

Many negative feedback loops are in place to limit the growth of normal cells. These include tumor suppressors CDK-interacting protein and kinase-inhibitory protein (CIP and KIP) and inhibitor of kinase 4/alternative reading frame (INK4a/ARF). The CIP/KIP family includes the genes p21, p27, and p57. They halt cell cycle in G1 phase, by binding to, and inactivating, cyclin-CDK complexes. p21 is activated by p53 (which, in turn, is triggered by DNA damage, e.g., due to radiation). p27 is activated by TGF-β, a growth inhibitor. The INK4a/ARF family includes p16INK4a, which binds to CDK4 and arrests the cell cycle in G1 phase, and p19ARF, which prevents p53 degradation. However, these “stop signals” can be ignored by cancer cells (Figure 2.5), leading to unregulated growth (e.g., RAS mutations with decreased RAS GTPase activity in cellular events that are constantly in the “on” position rather than temporary and short lived) (Wertz and Dixit, 2010).

Telomerase, the specialized DNA polymerase, which adds repeat segments to the ends of telomeric DNA, is low or absent in normal cells but is expressed at significant levels in human cancer cells.

Senescence is one way that cancer cells may “hibernate.” Senescence limits tumor progression and could represent a strategy to increase survival using tactics such as TP53 reactivation, inhibition of c-MYC in addicted tumors, or treatment with cyclin-dependent kinase (CDK) inhibitors.

The loss of TP53-mediated surveillance of genomic integrity may permit cells to survive chromosomal breakage-fusion-bridge (BFB) cycles, which increase genome mutability. Delayed acquisition of telomerase function first serves to generate tumor-promoting mutations, whereas its later activation stabilizes the mutant genome and then confers “immortality.” This is another example of the “bipolar” nature of cellular processes whereby early on a cancer may develop as a result of the absence of a certain function and later the cancer may be promoted by the acquisition of that same function.

Telomerase and its catalytic reverse transcriptase (hTERT) are found at multiple sites along chromosomes, not just at the telomeres. This results in other functions: telomerase is a cofactor of beta-catenin/LEF transcription and amplifies WNT signaling (Park et al., 2009). It also can enhance cell proliferation, RNA polymerase, resistance to apoptosis, and DNA damage repair
(Maida et al., 2009). This recurring theme of multifunctionality adds complexity to the design of new clinical strategies but could also be exploited to great effect because a telomerase inhibitor could attack cancers in a variety of ways. A number of agents have been developed to inhibit telomerase activity and/or modulate its expression: imetelstat (GRN163L 13-mer oligonucleotide), tertomotide (GV1001 peptide vaccine), BIBR15329 (nucleoside inhibitor shortening TTAGGG repeats) (Puri and Girard, 2013). To date, however, early trials have not identified a setting in which this therapeutic strategy has produced tangible consistent benefit.

Multistep tumor progression can be portrayed as a sequence of clonal evolution. Given the billions of mitotic events that occur in our body, it is no surprise that additional enabling mutations can occur by chance in an already vulnerable DNA “portfolio.” In addition to frank mutation, tumor suppressor genes can be inactivated through “epigenetic” DNA methylation switching and histone modifications (Berdasco and Esteller, 2010). The extraordinary ability of genome maintenance systems to detect and repair defects in the DNA ensures that rates of spontaneous mutation are usually very low during each cell generation (Hanahan and Weinberg, 2011); however, in the course of acquiring the multiple mutant genes needed to orchestrate tumorigenesis, cancer cells often increase the rates of mutation (Negrini et al., 2010; Salk et al., 2010). These genetic changes are brought about through a breakdown in the genomic maintenance machinery.

In addition, the accumulation of mutations can be accelerated by compromising the surveillance systems that normally monitor genomic integrity and force genetically damaged cells into either senescence or apoptosis (Jackson and Bartek, 2009) (Figure 2.5). The role of TP53 is central here, leading to its being called the “guardian of the genome” (Lane, 1992). Tumor suppressors detect and repair damaged DNA. They also inactivate or intercept mutagenic molecules before they have damaged the DNA (Ciccia and Elledge, 2010; Negrini et al., 2010).