Chapter 26: Cancer Cell Biology

Cancers are characterized by unregulated cell division, avoidance of cell death, tissue invasion, and the ability to metastasize. A neoplasm is benign when it grows in an unregulated fashion without tissue invasion. The presence of unregulated growth and tissue invasion is characteristic of malignant neoplasms. Cancers are named based on their origin: those derived from epithelial tissue are called carcinomas, those derived from mesenchymal tissues are sarcomas, and those derived from hematopoietic tissue are leukemias, lymphomas, and plasma cell dyscrasias (including multiple myeloma).

Cancers nearly always arise as a consequence of genetic alterations, the vast majority of which begin in a single cell and therefore are monoclonal in origin. However, because a wide variety of genetic and epigenetic changes can occur in different cells within malignant tumors over time, most cancers are characterized by marked heterogeneity in the populations of cells. This heterogeneity significantly complicates the treatment of most cancers because it is likely that there are subsets of cells that will be resistant to therapy and will therefore survive and proliferate even if the majority of cells are killed.

A few cancers appear to, at least initially, be primarily driven by an alteration in a dominant gene that produces uncontrolled cell proliferation. Examples include chronic myeloid leukemia (abl), about half of melanomas (braf), Burkitt’s lymphoma (c-myc), and subsets of lung adenocarcinomas (egfr, alk, ros1, and ret). The genes that can promote cell growth when altered are often called oncogenes. They were first identified as critical elements of viruses that cause animal tumors; it was subsequently found that the viral genes had normal counterparts with important functions in the cell and had been captured and mutated by viruses as they passed from host to host.

However, the vast majority of human cancers are characterized by a multiple-step process involving many genetic abnormalities, each of which contributes to the loss of control of cell proliferation and differentiation and the acquisition of capabilities, such as tissue invasion, the ability to metastasize, and angiogenesis. These properties are not found in the normal adult cell from which the tumor is derived. Indeed, normal cells have a large number of safeguards against uncontrolled proliferation and invasion. Many cancers go through recognizable steps of progressively more abnormal phenotypes: hyperplasia, to adenoma, to dysplasia, to carcinoma in situ, to invasive cancer with the ability to metastasize (Table 26-1). For most cancers, these changes occur over a prolonged period of time, usually many years.

In most organs, only primitive undifferentiated cells are capable of proliferating and the cells lose the capacity to proliferate as they differentiate and acquire functional capability. The expansion of the primitive cells is linked to some functional need in the host through receptors that receive signals from the local environment or through hormonal and other influences delivered by the vascular supply. In the absence of such signals, the cells are at rest. The signals that keep the primitive cells at rest remain incompletely understood. These signals must be environmental, based on the observations that a regenerating liver stops growing when it has replaced the portion that has been surgically removed after partial hepatectomy and regenerating bone marrow stops growing when the peripheral blood counts return to normal. Cancer cells clearly have lost responsiveness to such controls and do not recognize when they have overgrown the niche normally occupied by the organ from which they are derived. A better understanding of the mechanisms of growth regulation is evolving.

Normal cells have a number of control mechanisms that are targeted by specific genetic alterations in cancer. Critical proteins in these control processes that are frequently mutated or otherwise inactivated in cancers are called tumor-suppressor genes. Examples include p53 and Rb (discussed below). The progression of a cell through the cell division cycle is regulated at a number of checkpoints by a wide array of genes. In the first phase, G₁, preparations are made to replicate the genetic material. The cell stops before entering the DNA synthesis phase, or S phase, to take inventory. Are we ready to replicate our DNA? Is the DNA repair machinery in place to fix any mutations that are detected? Are the DNA replicating enzymes available? Is there an adequate supply of nucleotides? Is there sufficient energy? The main brake on the process is the retinoblastoma protein, Rb. When the cell determines that it is prepared to move ahead, sequential activation of cyclin-dependent kinases (CDKs) results in the inactivation of the brake, Rb, by phosphorylation. Phosphorylated Rb releases the S phase–regulating transcription factor, E2F/DP1, and genes required for S phase progression are expressed. If the cell determines that it is unready to move ahead with DNA replication, a number of inhibitors are capable of blocking the action of the CDKs, including p21^Cip2/Waf1, p16^Ink4a, and p27^Kip1. Nearly every cancer has one or more genetic lesions in the G₁ checkpoint that permits progression to S phase.

At the end of S phase, when the cell has exactly duplicated its DNA content, a second inventory is taken at the S checkpoint. Have all of the chromosomes been fully duplicated? Were any segments of DNA copied more than once? Do we have the right number of chromosomes and the right amount of DNA? If so, the cell proceeds to G₂, in which the cell prepares for division by synthesizing mitotic spindle and other proteins needed to produce two daughter cells. When DNA damage is detected, the p53 pathway is normally activated. Called the guardian of the genome, p53 is a transcription factor that is normally present in the cell in very low levels. Its level is generally regulated through its rapid turnover. Normally, p53 is bound to mdm2, a ubiquitin ligase, that both inhibits p53 transcriptional activation and also targets p53 for degradation in the proteasome. When damage is sensed, the ATM (ataxia-telangiectasia mutated) pathway is activated; ATM phosphorylates mdm2, which no longer binds to p53, and p53 then stops cell cycle progression, directs the synthesis of repair enzymes, or if the damage is too great, initiates apoptosis of the cell to prevent the propagation of a damaged cell (Fig. 26-1).

A second method of activating p53 involves the induction of p14^ARF by hyperproliferative signals from oncogenes. p14^ARF competes with p53 for binding to mdm2, allowing p53 to escape the effects of mdm2 and accumulate in the cell. Then p53 stops cell cycle progression by activating CDK inhibitors such as p21 and/or initiating the apoptosis pathway. Not surprisingly given its critical role in controlling cell cycle progression, mutations in the gene for p53 on chromosome 17p are found in more than 50% of human cancers. Most commonly these mutations are acquired in the malignant tissue in one allele and the second allele is deleted, leaving the cell unprotected from DNA-damaging agents or oncogenes. Some environmental exposures produce signature mutations in p53; for example, aflatoxin exposure leads to mutation of arginine to serine at codon 249 and leads to hepatocellular carcinoma. In rare instances, p53 mutations are in the germline (Li-Fraumeni syndrome) and produce a familial cancer syndrome. The absence of p53 leads to chromosome instability and the accumulation of DNA damage including the acquisition of properties that give the abnormal cell a proliferative and survival advantage. Like Rb dysfunction, most cancers have mutations that disable the p53 pathway. Indeed, the importance of p53 and Rb in the development of cancer is underscored by the neoplastic transformation mechanism of human papillomavirus. This virus has two main oncogenes, E6 and E7. E6 acts to increase the rapid turnover of p53, and E7 acts to inhibit Rb function; inhibition of these two targets is required for transformation of epithelial cells.

Another cell cycle checkpoint exists when the cell is undergoing division, the spindle checkpoint. The details of this checkpoint are still being discovered; however, it appears that if the spindle apparatus does not properly align the chromosomes for division, if the chromosome number is abnormal (i.e., greater or less than 4n), or if the centromeres are not properly paired with their duplicated partners, then the cell initiates a cell death pathway to prevent the production of aneuploid progeny (having an altered number of chromosomes). Abnormalities in the spindle checkpoint facilitate the development of aneuploidy. In some tumors, aneuploidy is a predominant genetic feature. In others, a defect in the cells’ ability to repair errors in the DNA due to mutations in genes coding for the proteins critical for mismatched DNA repair is the primary genetic lesion. This is usually detected by finding alterations in repeat sequences of DNA (called microsatellites) or microsatellite instability in malignant cells. In general, tumors either have defects in chromosome number or microsatellite instability, but not both. Defects that lead to cancer include abnormal cell cycle checkpoints, inadequate DNA repair, and failure to preserve genome integrity.

Efforts are under way to therapeutically restore the defects in cell cycle regulation that characterize cancer, although this remains a challenging problem because it is much more difficult to restore normal biologic function than to inhibit abnormal function of proteins driving cell proliferation, such as oncogenes.

The fundamental cellular defects that create a malignant neoplasm act at the cellular level. However, that is not the entire story. Cancers behave as organs that have lost their specialized function and stopped responding to signals that normally limit their growth. Human cancers usually become clinically detectable when a primary mass is at least 1 cm in diameter—such a mass consists of about 10⁹ cells. More commonly, patients present with tumors that are 10¹⁰ cells or greater. A lethal tumor burden is about 10¹² to 10¹³ cells. If all tumor cells were dividing at the time of diagnosis, patients would reach a lethal tumor burden in a very short time. However, human tumors grow by Gompertzian kinetics—this means that not every daughter cell produced by a cell division is itself capable of dividing. The growth fraction of a tumor declines exponentially with time. The growth fraction of the first malignant cell is 100%, and by the time a patient presents for medical care, the growth fraction is 2–3% or less. This fraction is similar to the growth fraction of normal bone marrow and normal intestinal epithelium, the most highly proliferative normal tissues in the human body, a fact that may explain the dose-limiting toxicities of agents that target dividing cells.

The implication of these data is that the tumor is slowing its own growth over time. How does it do this? The tumor cells have multiple genetic lesions that tend to promote proliferation, yet by the time the tumor is clinically detectable, its capacity for proliferation has declined. We need to better understand how a tumor slows its own growth. A number of factors are known to contribute to the failure of tumor cells to proliferate in vivo. Some cells are hypoxemic and have inadequate supply of nutrients and energy. Some have sustained too much genetic damage to complete the cell cycle but have lost the capacity to undergo apoptosis and therefore survive but do not proliferate. However, an important subset is not actively dividing but retains the capacity to divide and can start dividing again under certain conditions such as when the tumor mass is reduced by treatments. Just as the bone marrow increases its rate of proliferation in response to bone marrow–damaging agents, the tumor also seems to sense when tumor cell numbers have been reduced and can respond by increasing growth rate. However, the critical difference is that the marrow stops growing when it has reached its production goals, whereas tumors do not.

Additional tumor cell vulnerabilities are likely to be detected when we learn more about how normal cells respond to “stop” signals from their environment and why and how tumor cells fail to heed such signals.

When normal cells are placed in culture in vitro, most are not capable of sustained growth. Fibroblasts are an exception to this rule. When they are cultured, fibroblasts may divide 30–50 times and then they undergo what has been termed a “crisis” during which the majority of cells stop dividing (usually due to an increase in p21 expression, a CDK inhibitor), many die, and a small fraction emerge that have acquired genetic changes that permit their uncontrolled growth. The cessation of growth of normal cells in culture has been termed “senescence,” and whether this phenomenon is relevant to any physiologic event in vivo is debated.

Among the cellular changes during in vitro propagation is telomere shortening. DNA polymerase is unable to replicate the tips of chromosomes, resulting in the loss of DNA at the specialized ends of chromosomes (called telomeres) with each replication cycle. At birth, human telomeres are 15- to 20-kb pairs long and are composed of tandem repeats of a six-nucleotide sequence (TTAGGG) that associates with specialized telomere-binding proteins to form a T-loop structure that protects the ends of chromosomes from being mistakenly recognized as damaged. The loss of telomeric repeats with each cell division cycle causes gradual telomere shortening, leading to growth arrest (called senescence) when one or more critically short telomeres trigger a p53-regulated DNA-damage checkpoint response. Cells can bypass this growth arrest if pRb and p53 are nonfunctional, but cell death usually ensues when the unprotected ends of chromosomes lead to chromosome fusions or other catastrophic DNA rearrangements. The ability to bypass telomere-based growth limitations is thought to be a critical step in the evolution of most malignancies. This occurs by the reactivation of telomerase expression in cancer cells. Telomerase is an enzyme that adds TTAGGG repeats onto the 3′ ends of chromosomes. It contains a catalytic subunit with reverse transcriptase activity (hTERT) and an RNA component that provides the template for telomere extension. Most normal somatic cells do not express sufficient telomerase to prevent telomere attrition with each cell division. Exceptions include stem cells (such as those found in hematopoietic tissues, gut and skin epithelium, and germ cells) that require extensive cell division to maintain tissue homeostasis. More than 90% of human cancers express high levels of telomerase that prevent telomere shortening to critical levels and allow indefinite cell proliferation. In vitro experiments indicate that inhibition of telomerase activity leads to tumor cell apoptosis. Major efforts are under way to develop methods to inhibit telomerase activity in cancer cells. For example, the protein component of telomerase (hTERT) may act as one of the most widely expressed tumor-associated antigens and be targeted by vaccine approaches.

Although most of the functions of telomerase relate to cell division, it also has several other effects including interfering with the differentiated functions of at least certain stem cells, although the impact on differentiated function of normal non-stem cells is less clear. Nevertheless, a major growth industry in medical research has been discovering an association between short telomeres and human diseases ranging from diabetes and coronary artery disease to Alzheimer’s disease. The picture is further complicated by the fact that rare genetic defects in the telomerase enzyme seem to cause pulmonary fibrosis, aplastic anemia, or dyskeratosis congenita (characterized by abnormalities in skin, nails, and oral mucosa with increased risk for certain malignancies) but not defects in nutrient absorption in the gut, a site that might be presumed to be highly sensitive to defective cell proliferation. Much remains to be learned about how telomere shortening and telomere maintenance are related to human illness in general and cancer in particular.

Signals that affect cell behavior come from adjacent cells, the stroma in which the cells are located, hormonal signals that originate remotely, and from the cells themselves (autocrine signaling). These signals generally exert their influence on the receiving cell through activation of signal transduction pathways that have as their end result the induction of activated transcription factors that mediate a change in cell behavior or function or the acquisition of effector machinery to accomplish a new task. Although signal transduction pathways can lead to a wide variety of outcomes, many such pathways rely on cascades of signals that sequentially activate different proteins or glycoproteins and lipids or glycolipids, and the activation steps often involve the addition or removal of one or more phosphate groups on a downstream target. Other chemical changes can result from signal transduction pathways, but phosphorylation and dephosphorylation play a major role. The proteins that add phosphate groups to proteins are called kinases. There are two major distinct classes of kinases; one class acts on tyrosine residues, and the other acts on serine/threonine residues. The tyrosine kinases often play critical roles in signal transduction pathways; they may be receptor tyrosine kinases, or they may be linked to other cell-surface receptors through associated docking proteins (Fig. 26-2).

Normally, tyrosine kinase activity is short-lived and reversed by protein tyrosine phosphatases (PTPs). However, in many human cancers, tyrosine kinases or components of their downstream pathways are activated by mutation, gene amplification, or chromosomal translocations. Because these pathways regulate proliferation, survival, migration, and angiogenesis, they have been identified as important targets for cancer therapeutics.

Inhibition of kinase activity is effective in the treatment of a number of neoplasms. Lung cancers with mutations in the epidermal growth factor receptor are highly responsive to erlotinib and gefitinib (Table 26-2). Lung cancers with activation of anaplastic lymphoma kinase (ALK) or ROS1 by translocations respond to crizotinib, an ALK and ROS1 inhibitor. A BRAF inhibitor is highly effective in melanomas and thyroid cancers in which BRAF is mutated. Targeting a protein (MEK) downstream of BRAF also has activity against BRAF mutant melanomas. Janus kinase inhibitors are active in myeloproliferative syndromes in which JAK2 activation is a pathogenetic event. Imatinib (which targets a number of tyrosine kinases) is an effective agent in tumors that have translocations of the c-Abl and BCR gene (such as chronic myeloid leukemia), mutant c-Kit (gastrointestinal stromal cell tumors), or mutant platelet-derived growth factor receptor (PDGFR; chronic myelomonocytic leukemia); second-generation inhibitors of BCR-Abl, dasatinib, and nilotinib are even more effective. The third-generation agent bosutinib has activity in some patients who have progressed on other inhibitors, whereas the third-generation agent ponatinib has activity against the T315I mutation, which is resistant to the other agents. Sorafenib and sunitinib, agents that inhibit a large number of kinases, have shown antitumor activity in a number of malignancies, including renal cell cancer (RCC) (both), hepatocellular carcinoma (sorafenib), thyroid cancer (sorafenib), gastrointestinal stromal tumor (GIST) (sunitinib), and pancreatic neuroendocrine tumors (sunitinib). Inhibitors of the mammalian target of rapamycin (mTOR) are active in RCC, pancreatic neuroendocrine tumors, and breast cancer. The list of active agents and treatment indications is growing rapidly. These new agents have ushered in a new era of personalized therapy. It is becoming more routine for resected tumors to be assessed for specific molecular changes that predict response and to have clinical decision-making guided by those results.

However, none of these therapies has yet been curative by themselves for any malignancy, although prolonged periods of disease control lasting many years frequently occur in chronic myeloid leukemia. The reasons for the failure to cure are not completely defined, although resistance to the treatment ultimately develops in most patients. In some tumors, resistance to kinase inhibitors is related to an acquired mutation in the target kinase that inhibits drug binding. Many of these kinase inhibitors act as competitive inhibitors of the ATP-binding pocket. ATP is the phosphate donor in these phosphorylation reactions. Mutation in the BCR-ABL kinase in the ATP-binding pocket (such as the threonine to isoleucine change at codon 315 [T315I]) can prevent imatinib binding. Other resistance mechanisms include altering other signal transduction pathways to bypass the inhibited pathway. As resistance mechanisms become better defined, rational strategies to overcome resistance will emerge. In addition, many kinase inhibitors are less specific for an oncogenic target than was hoped, and toxicities related to off-target inhibition of kinases limit the use of the agent at a dose that would optimally inhibit the cancer-relevant kinase.

Targeted agents can also be used to deliver highly toxic compounds. An important component of the technology for developing effective conjugates is the design of the linker between the two, which needs to be stable. Currently approved antibody drug conjugates include brentuximab vedotin, which links the microtubule toxin monomethyl auristatin E (MMAE) to an antibody targeting the cell surface antigen CD30, which is expressed on a number of malignant cells but especially in Hodgkin’s disease and anaplastic lymphoma. The linker in this case is cleavable, which allows diffusion of the drug out of the cell after delivery. The second approved conjugate is ado-trastuzumab emtansine, which links the microtubule formation inhibitor mertansine and the monoclonal antibody trastuzumab targeted against human epidermal growth factor receptor 2 (HER2) on breast cancer cells. In this case, the linker is noncleavable, thus trapping the chemotherapeutic agent within the cells. There are theoretical pluses and minuses to having either cleavable or noncleavable linkers, and it is likely that both will be used in future developments of antibody-drug conjugates.

Another strategy to enhance the antitumor effects of targeted agents is to use them in rational combinations with each other and in empiric combinations with chemotherapy agents that kill cells in ways distinct from targeted agents. Combinations of trastuzumab (a monoclonal antibody that targets the HER2 receptor [member of the epidermal growth factor receptor (EGFR) family]) with chemotherapy have significant activity against breast and stomach cancers that have high levels of expression of the HER2 protein. The activity of trastuzumab and chemotherapy can be enhanced further by combinations with another targeted monoclonal antibody (pertuzumab), which prevents dimerization of the HER2 receptor with other HER family members including HER3.

Although targeted therapies have not yet resulted in cures when used alone, their use in the adjuvant setting and when combined with other effective treatments has substantially increased the fraction of patients cured. For example, the addition of rituximab, an anti-CD20 antibody, to combination chemotherapy in patients with diffuse large B cell lymphoma improves cure rates by 15–20%. The addition of trastuzumab, antibody to HER2, to combination chemotherapy in the adjuvant treatment of HER2-positive breast cancer reduces relapse rates by 50%.

A major effort is under way to develop targeted therapies for mutations in the ras family of genes, which are the most common mutations in oncogenes in cancers (especially kras) but have proved to be very difficult targets for a number of reasons related to how RAS proteins are activated and inactivated. Targeted therapies against proteins downstream of RAS (including mitogen-activated protein [MAP] kinase and ERK) are currently being studied, both individually and in combination. A large number of inhibitors of phospholipid signaling pathways such as the phosphatidylinositol-3-kinase (PI3K) and phospholipase C-gamma pathways, which are involved in a large number of cellular processes that are important in cancer development and progression, are being evaluated. The targeting of a variety of other pathways that are activated in malignant cells, such as the MET pathway, hedgehog pathway, and various angiogenesis pathways, is also being explored.

One of the strategies for new drug development is to take advantage of so-called oncogene addiction. This situation (Fig. 26-3) is created when a tumor cell develops an activating mutation in an oncogene that becomes a dominant pathway for survival and growth with reduced contributions from other pathways, even when there may be abnormalities in those pathways. This dependency on a single pathway creates a cell that is vulnerable to inhibitors of that oncogene pathway. For example, cells harboring mutations in BRAF are very sensitive to MEK inhibitors that inhibit downstream signaling in the BRAF pathway.

Targeting proteins critical for transcription of proteins vital for malignant cell survival or proliferation provides another potential target for treating cancers. The transcription factor nuclear factor-κB (NF-κB) is a heterodimer composed of p65 and p50 subunits that associate with an inhibitor, IκB, in the cell cytoplasm. In response to growth factor or cytokine signaling, a multi-subunit kinase called IKK (IκB kinase) phosphorylates IκB and directs its degradation by the ubiquitin/proteasome system. NF-κB, free of its inhibitor, translocates to the nucleus and activates target genes, many of which promote the survival of tumor cells. Novel drugs called proteasome inhibitors block the proteolysis of IκB, thereby preventing NF-κB activation. For unexplained reasons, this is selectively toxic to tumor cells. The antitumor effects of proteasome inhibitors are more complicated and involve the inhibition of the degradation of multiple cellular proteins. Proteasome inhibitors (e.g., bortezomib [Velcade]) have activity in patients with multiple myeloma, including partial and complete remissions. Inhibitors of IKK are also in development, with the hope of more selectively blocking the degradation of IκB, thus “locking” NF-κB in an inhibitory complex and rendering the cancer cell more susceptible to apoptosis-inducing agents. Many other transcription factors are activated by phosphorylation, which can be prevented by tyrosine kinase inhibitors or serine/threonine kinase inhibitors, a number of which are currently in clinical trials.

Estrogen receptors (ERs) and androgen receptors (ARs), members of the steroid hormone family of nuclear receptors, are targets of inhibition by drugs used to treat breast and prostate cancers, respectively. Tamoxifen, a partial agonist and antagonist of ER function, can mediate tumor regression in metastatic breast cancer and can prevent disease recurrence in the adjuvant setting. Tamoxifen binds to the ER and modulates its transcriptional activity, inhibiting activity in the breast but promoting activity in bone and uterine epithelium. Selective ER modulators (SERMs) have been developed with the hope of a more beneficial modulation of ER activity, i.e., antiestrogenic activity in the breast, uterus, and ovary, but estrogenic for bone, brain, and cardiovascular tissues. Aromatase inhibitors, which block the conversion of androgens to estrogens in breast and subcutaneous fat tissues, have demonstrated improved clinical efficacy compared with tamoxifen and are often used as first-line therapy in patients with ER-positive disease. A number of approaches have been developed for blocking androgen stimulation of prostate cancer, including decreasing production (e.g., orchiectomy, luteinizing hormone–releasing hormone agonists or antagonists, estrogens, ketoconazole, and inhibitors of enzymes such as CYP17 involved in androgen production) and AR blockers (Chap. 38).

The concepts of oncogene addiction and synthetic lethality have spurred new drug development targeting oncogene- and tumor-suppressor pathways. As discussed earlier in this chapter and outlined in Fig. 26-3, cancer cells can become dependent on signaling pathways containing activated oncogenes; this can effect proliferation (i.e., mutated Kras, Braf, overexpressed Myc, or activated tyrosine kinases), DNA repair (loss of BRCA1 or BRCA2 gene function), survival (overexpression of Bcl-2 or NF-κB), cell metabolism (as occurs when mutant Kras enhances glucose uptake and aerobic glycolysis), and perhaps angiogenesis (production of VEGF in response to HIF-2α in RCC). In such cases, targeted inhibition of the pathway can lead to specific killing of the cancer cells. However, targeting defects in tumor-suppressor genes has been much more difficult, both because the target of mutation is often deleted and because it is much more difficult to restore normal function than to inhibit abnormal function of a protein. Synthetic lethality occurs when loss of function in either of two genes alone has limited effects on cell survival but loss of function in both genes leads to cell death. Identifying genes that have a synthetic lethal relationship to tumor-suppressor pathways that have been mutated in tumor cells may allow targeting of proteins required uniquely by those cells (Fig. 26-3). Several examples of this have been identified. For instance, cells with mutations in the BRCA1 or BRCA2 tumor-suppressor genes (e.g., a subset of breast and ovarian cancers) are unable to repair DNA damage by homologous recombination. PARP are a family of proteins important for single-strand break (SSB) DNA repair. PARP inhibition results in selective killing of cancer cells with BRCA1 or BRCA2 loss. Preliminary trials have suggested some effectiveness of PARP inhibition, especially in combination with chemotherapy; clinical trials are ongoing. The concept of synthetic lethality provides a framework for genetic screens to identify other synthetic lethal combinations involving known tumor-suppressor genes and development of novel therapeutic agents to target dependent pathways.

Chromatin structure regulates the hierarchical order of sequential gene transcription that governs differentiation and tissue homeostasis. Disruption of chromatin remodeling (the process of modifying chromatin structure to control exposure of specific genes to transcriptional proteins, thereby controlling the expression of those genes) leads to aberrant gene expression and can induce proliferation of undifferentiated cells. Epigenetics is defined as changes that alter the pattern of gene expression that persist across at least one cell division but are not caused by changes in the DNA code. Epigenetic changes include alterations of chromatin structure mediated by methylation of cytosine residues in CpG dinucleotides, modification of histones by acetylation or methylation, or changes in higher-order chromosome structure (Fig. 26-4). The transcriptional regulatory regions of active genes often contain a high frequency of CpG dinucleotides (referred to as CpG islands), which are normally unmethylated. Expression of these genes is controlled by transient association with repressor or activator proteins that regulate transcriptional activation. However, hypermethylation of promoter regions is a common mechanism by which tumor-suppressor loci are epigenetically silenced in cancer cells. Thus one allele may be inactivated by mutation or deletion (as occurs in loss of heterozygosity), while expression of the other allele is epigenetically silenced, usually by methylation.

Acetylation of the amino terminus of the core histones H3 and H4 induces an open chromatin conformation that promotes transcription initiation. Histone acetylases are components of coactivator complexes recruited to promoter/enhancer regions by sequence-specific transcription factors during the activation of genes (Fig. 26-4). Histone deacetylases (HDACs; at least 17 are encoded in the human genome) are recruited to genes by transcriptional repressors and prevent the initiation of gene transcription. Methylated cytosine residues in promoter regions become associated with methyl cytosine–binding proteins that recruit protein complexes with HDAC activity. The balance between permissive and inhibitory chromatin structure is therefore largely determined by the activity of transcription factors in modulating the “histone code” and the methylation status of the genetic regulatory elements of genes.

The pattern of gene transcription is aberrant in all human cancers, and in many cases, epigenetic events are responsible. Unlike genetic events that alter DNA primary structure (e.g., deletions), epigenetic changes are potentially reversible and appear amenable to therapeutic intervention. In certain human cancers, including pancreatic cancer and multiple myeloma, the p16^Ink4a promoter is inactivated by methylation, thus permitting the unchecked activity of CDK4/cyclin D and rendering pRb nonfunctional. In sporadic forms of renal, breast, and colon cancer, the von Hippel–Lindau (VHL), breast cancer 1 (BRCA1), and serine/threonine kinase 11 (STK11) genes, respectively, are epigenetically silenced. Other targeted genes include the p15^Ink4b CDK inhibitor, glutathione-S-transferase (which detoxifies reactive oxygen species), and the E-cadherin molecule (important for junction formation between epithelial cells). Epigenetic silencing can occur in premalignant lesions and can affect genes involved in DNA repair, thus predisposing to further genetic damage. Examples include MLH1 (mut L homologue) in hereditary nonpolyposis colon cancer (HNPCC, also called Lynch’s syndrome), which is critical for repair of mismatched bases that occur during DNA synthesis, and O⁶-methylguanine-DNA methyltransferase, which removes alkylated guanine adducts from DNA and is often silenced in colon, lung, and lymphoid tumors.

Human leukemias often have chromosomal translocations that code for novel fusion proteins with enzymatic activities that alter chromatin structure. The promyelocytic leukemia–retinoic acid receptor (PML-RAR) fusion protein, generated by the t(15;17) observed in most cases of acute promyelocytic leukemia (APL), binds to promoters containing retinoic acid response elements and recruits HDAC to these promoters, effectively inhibiting gene expression. This arrests differentiation at the promyelocyte stage and promotes tumor cell proliferation and survival. Treatment with pharmacologic doses of all-trans retinoic acid (ATRA), the ligand for RARα, results in the release of HDAC activity and the recruitment of coactivators, which overcome the differentiation block. This induced differentiation of APL cells has improved treatment of these patients but also has led to a novel treatment toxicity when newly differentiated tumor cells infiltrate the lungs. However, ATRA represents a treatment paradigm for the reversal of epigenetic changes in cancer. For other leukemia-associated fusion proteins, such as acute myeloid leukemia (AML)-eight-twenty-one (ETO) and the MLL fusion proteins seen in AML and acute lymphocytic leukemia, no ligand is known. Therefore, efforts are ongoing to determine the structural basis for interactions between translocation fusion proteins and chromatin-remodeling proteins and to use this information to rationally design small molecules that will disrupt specific protein-protein associations, although this has proven to be technically difficult. Drugs that block the enzymatic activity of HDAC are being tested. HDAC inhibitors have demonstrated antitumor activity in clinical studies against cutaneous T cell lymphoma (e.g., vorinostat) and some solid tumors. HDAC inhibitors may target cancer cells via a number of mechanisms, including upregulation of death receptors (DR4/5, FAS, and their ligands) and p21^Cip1/Waf1, as well as inhibition of cell cycle checkpoints.

Efforts are also under way to reverse the hypermethylation of CpG islands that characterizes many malignancies. Drugs that induce DNA demethylation, such as 5-aza-2′-deoxycytidine, can lead to reexpression of silenced genes in cancer cells with restoration of function, and 5-aza-2′-deoxycytidine is approved for use in myelodysplastic syndrome (MDS). However, 5-aza-2′-deoxycytidine has limited aqueous solubility and is myelosuppressive. Other inhibitors of DNA methyltransferases are in development. In ongoing clinical trials, inhibitors of DNA methylation are being combined with HDAC inhibitors. The hope is that by reversing coexisting epigenetic changes, the deregulated patterns of gene transcription in cancer cells will be at least partially reversed.

Epigenetic gene regulation can also occur via micro-RNAs or long non-coding RNAs (lncRNAs). MicroRNAs are short (average 22 nucleotides in length) RNA molecules that silence gene expression after transcription by binding and inhibiting the translation or promoting the degradation of mRNA transcripts. It is estimated that more than 1000 microRNAs are encoded in the human genome. Each tissue has a distinctive repertoire of microRNA expression, and this pattern is altered in specific ways in cancers. However, specific correlations between microRNA expression and tumor biology and clinical behavior are just now emerging. Therapies targeting microRNAs are not currently at hand but represent a novel area of treatment development. lncRNAs are longer than 200 nucleotides and compose the largest group of noncoding RNAs. Some of them have been shown to play important roles in gene regulation. The potential for altering these RNAs for therapeutic benefit is an area of active investigation, although much more needs to be learned before this will be feasible.

Tissue homeostasis requires a balance between the death of aged, terminally differentiated cells or severely damaged cells and their renewal by proliferation of committed progenitors. Genetic damage to growth-regulating genes of stem cells could lead to catastrophic results for the host as a whole. Thus, genetic events causing activation of oncogenes or loss of tumor suppressors, which would be predicted to lead to unregulated cell proliferation unless corrected, usually activate signal transduction pathways that block aberrant cell proliferation. These pathways can lead to a form of programmed cell death (apoptosis) or irreversible growth arrest (senescence). Much as a panoply of intra- and extracellular signals impinge upon the core cell cycle machinery to regulate cell division, so too are these signals transmitted to a core enzymatic machinery that regulates cell death and survival.

Apoptosis is induced by two main pathways (Fig. 26-5). The extrinsic pathway of apoptosis is activated by cross-linking members of the tumor necrosis factor (TNF) receptor superfamily, such as CD95 (Fas) and death receptors DR4 and DR5, by their ligands, Fas ligand or TRAIL (TNF-related apoptosis-inducing ligand), respectively. This induces the association of FADD (Fas-associated death domain) and procaspase-8 to death domain motifs of the receptors. Caspase-8 is activated and then cleaves and activates effector caspases-3 and -7, which then target cellular constituents (including caspase-activated DNAse, cytoskeletal proteins, and a number of regulatory proteins), inducing the morphologic appearance characteristic of apoptosis, which pathologists term “karyorrhexis.” The intrinsic pathway of apoptosis is initiated by the release of cytochrome c and SMAC (second mitochondrial activator of caspases) from the mitochondrial intermembrane space in response to a variety of noxious stimuli, including DNA damage, loss of adherence to the extracellular matrix (ECM), oncogene-induced proliferation, and growth factor deprivation. Upon release into the cytoplasm, cytochrome c associates with dATP, procaspase-9, and the adaptor protein APAF-1, leading to the sequential activation of caspase-9 and effector caspases. SMAC binds to and blocks the function of inhibitor of apoptosis proteins (IAP), negative regulators of caspase activation.

The release of apoptosis-inducing proteins from the mitochondria is regulated by pro- and antiapoptotic members of the Bcl-2 family. Antiapoptotic members (e.g., Bcl-2, Bcl-XL, and Mcl-1) associate with the mitochondrial outer membrane via their carboxyl termini, exposing to the cytoplasm a hydrophobic binding pocket composed of Bcl-2 homology (BH) domains 1, 2, and 3 that is crucial for their activity. Perturbations of normal physiologic processes in specific cellular compartments lead to the activation of BH3-only proapoptotic family members (such as Bad, Bim, Bid, Puma, Noxa, and others) that can alter the conformation of the outer-membrane proteins Bax and Bak, which then oligomerize to form pores in the mitochondrial outer membrane resulting in cytochrome c release. If proteins composed only of BH3 domains are sequestered by Bcl-2, Bcl-XL, or Mcl-1, pores do not form and apoptosis-inducing proteins are not released from the mitochondria. The ratio of levels of antiapoptotic Bcl-2 family members and the levels of proapoptotic BH3-only proteins at the mitochondrial membrane determines the activation state of the intrinsic pathway. The mitochondrion must therefore be recognized not only as an organelle with vital roles in intermediary metabolism and oxidative phosphorylation but also as a central regulatory structure of the apoptotic process.

The evolution of tumor cells to a more malignant phenotype requires the acquisition of genetic changes that subvert apoptosis pathways and promote cancer cell survival and resistance to anticancer therapies. However, cancer cells may be more vulnerable than normal cells to therapeutic interventions that target the apoptosis pathways that cancer cells depend on. For instance, overexpression of Bcl-2 as a result of the t(14;18) translocation contributes to follicular lymphoma. Upregulation of Bcl-2 expression is also observed in prostate, breast, and lung cancers and melanoma. Targeting of antiapoptotic Bcl-2 family members has been accomplished by the identification of several low-molecular-weight compounds that bind to the hydrophobic pockets of either Bcl-2 or Bcl-XL and block their ability to associate with death-inducing BH3-only proteins. These compounds inhibit the antiapoptotic activities of Bcl-2 and Bcl-XL at nanomolar concentrations in the laboratory and are entering clinical trials.

Preclinical studies targeting death receptors DR4 and DR5 have demonstrated that recombinant, soluble, human TRAIL or humanized monoclonal antibodies with agonist activity against DR4 or DR5 can induce apoptosis of tumor cells while sparing normal cells. The mechanisms for this selectivity may include expression of decoy receptors or elevated levels of intracellular inhibitors (such as FLIP, which competes with caspase-8 for FADD) by normal cells but not tumor cells. Synergy has been shown between TRAIL-induced apoptosis and chemotherapeutic agents. For instance, some colon cancers encode mutated Bax protein as a result of mismatch repair (MMR) defects and are resistant to TRAIL. However, upregulation of Bak by chemotherapy restores the ability of TRAIL to activate the mitochondrial pathway of apoptosis. However, clinical studies have not yet shown significant activity of approaches targeting the TRAIL pathway.

Many of the signal transduction pathways perturbed in cancer promote tumor cell survival (Fig. 26-5). These include activation of the PI3K/Akt pathway, increased levels of the NF-κB transcription factor, and epigenetic silencing of genes such as APAF-1 and caspase-8. Each of these pathways is a target for therapeutic agents that, in addition to affecting cancer cell proliferation or gene expression, may render cancer cells more susceptible to apoptosis, thus promoting synergy when combined with other chemotherapeutic agents.

Some tumor cells resist drug-induced apoptosis by expression of one or more members of the ABC family of ATP-dependent efflux pumps that mediate the multidrug-resistance (MDR) phenotype. The prototype, P-glycoprotein (PGP), spans the plasma membrane 12 times and has two ATP-binding sites. Hydrophobic drugs (e.g., anthracyclines and vinca alkaloids) are recognized by PGP as they enter the cell and are pumped out. Numerous clinical studies have failed to demonstrate that drug resistance can be overcome using inhibitors of PGP. However, ABC transporters have different substrate specificities, and inhibition of a single family member may not be sufficient to overcome the MDR phenotype. Efforts to reverse PGP-mediated drug resistance continue.

Cells, including cancer cells, can also undergo other mechanisms of cell death including autophagy (degradation of proteins and organelles by lysosomal proteases) and necrosis (digestion of cellular components and rupturing of the cell membrane). Necrosis usually occurs in response to external forces resulting in release of cellular components, which leads to inflammation and damage to surrounding tissues. Although necrosis was thought to be unprogrammed, evidence now suggests that at least some aspects may be programmed. The exact role of necrosis in cancer cell death in various settings is still being determined. In addition to its role in cell death, autophagy can serve as a homeostatic mechanism to promote survival for the cell by recycling cellular components to provide necessary energy. The mechanisms that control the balance between enhancing survival versus leading to cell death are still not fully understood. Autophagy appears to play conflicting roles in the development and survival of cancer. Early in the carcinogenic process, it can act as a tumor suppressor by preventing the cell from accumulating abnormal proteins and organelles. However, in established tumors, it may serve as a mechanism of survival for cancer cells when they are stressed by damage such as from chemotherapy. Inhibition of this process can enhance the sensitivity of cancer cells to chemotherapy. Better understanding of the factors that control the survival-promoting versus death-inducing aspects of autophagy is required in order to know how to best manipulate it for therapeutic benefit.

The metastatic process accounts for the vast majority of deaths from solid tumors, and therefore, an understanding of this process is critical. The biology of metastasis is complex and requires multiple steps. The three major features of tissue invasion are cell adhesion to the basement membrane, local proteolysis of the membrane, and movement of the cell through the rent in the membrane and the ECM. Cells that lose contact with the ECM normally undergo programmed cell death (anoikis), and this process has to be suppressed in cells that metastasize. Another process important for metastasizing epithelial cancer cells is epithelial-mesenchymal transition (EMT). This is a process by which cells lose their epithelial properties and gain mesenchymal properties. This normally occurs during the developmental process in embryos, allowing cells to migrate to their appropriate destinations in the embryo. It also occurs in wound healing, tissue regeneration, and fibrotic reactions, but in all of these processes, cells stop proliferating when the process is complete. Malignant cells that metastasize undergo EMT as an important step in that process but retain the capacity for unregulated proliferation. Malignant cells that gain access to the circulation must then repeat those steps at a remote site, find a hospitable niche in a foreign tissue, avoid detection by host defenses, and induce the growth of new blood vessels. The rate-limiting step for metastasis is the ability for tumor cells to survive and expand in the novel microenvironment of the metastatic site, and multiple host-tumor interactions determine the ultimate outcome (Fig. 26-6). Few drugs have been developed to attempt to directly target the process of metastasis, in part because the specifics of the critical steps in the process that would be potentially good targets for drugs are still being identified. However, a number of potential targets are known. HER2 can enhance the metastatic potential of breast cancer cells, and as discussed above, the monoclonal antibody trastuzumab, which targets HER2, improves survival in the adjuvant setting for HER2-positive breast cancer patients. Other potential targets that increase metastatic potential of cells in preclinical studies include HIF-1 and -2, transcription factors induced by hypoxia within tumors; growth factors (e.g., cMET and VEGFR); oncogenes (e.g., SRC); adhesion molecules (e.g., focal adhesion kinase [FAK]); ECM proteins (e.g., matrix metalloproteinases-1 and -2); and inflammatory molecules (e.g., COX-2).

The metastatic phenotype is likely restricted to a small fraction of tumor cells (Fig. 26-6). A number of genetic and epigenetic changes are required for tumor cells to be able to metastasize, including activation of metastasis-promoting genes and inhibition of genes that suppress the metastatic ability. Cells with metastatic capability frequently express chemokine receptors that are likely important in the metastatic process. A number of candidate metastasis-suppressor genes have been identified, including genes coding for proteins that enhance apoptosis, suppress cell division, are involved in the interactions of cells with each other or the ECM, or suppress cell migration. The loss of function of these genes enhances metastasis. Gene expression profiling is being used to study the metastatic process and other properties of tumor cells that may predict susceptibilities.

An example of the ability of malignant cells to survive and grow in a novel microenvironment is bone metastasis. Bone metastases are extremely painful, cause fractures of weight-bearing bones, can lead to hypercalcemia, and are a major cause of morbidity for cancer patients. Osteoclasts and their monocyte-derived precursors express the surface receptor RANK (receptor activator of NF-κB), which is required for terminal differentiation and activation of osteoclasts. Osteoblasts and other stromal cells express RANK ligand (RANKL), as both a membrane-bound and soluble cytokine. Osteoprotegerin (OPG), a soluble receptor for RANKL produced by stromal cells, acts as a decoy receptor to inhibit RANK activation. The relative balance of RANKL and OPG determines the activation state of RANK on osteoclasts. Many tumors increase osteoclast activity by secretion of substances such as parathyroid hormone (PTH), PTH-related peptide, interleukin (IL)-1, or Mip1 that perturb the homeostatic balance of bone remodeling by increasing RANK signaling. One example is multiple myeloma, where tumor cell–stromal cell interactions activate osteoclasts and inhibit osteoblasts, leading to the development of multiple lytic bone lesions. Inhibition of RANKL by an antibody (denosumab) can prevent further bone destruction. Bisphosphonates are also effective inhibitors of osteoclast function that are used in the treatment of cancer patients with bone metastases.

Only a small proportion of the cells within a tumor are capable of initiating colonies in vitro or forming tumors at high efficiency when injected into immunocompromised NOD/SCID mice. Acute and chronic myeloid leukemias (AML and CML) have a small population of cells (<1%) that have properties of stem cells, such as unlimited self-renewal and the capacity to cause leukemia when serially transplanted in mice. These cells have an undifferentiated phenotype (Thy1−CD34+CD38– and do not express other differentiation markers) and resemble normal stem cells in many ways, but are no longer under homeostatic control (Fig. 26-7). Solid tumors may also contain a population of stem cells. Cancer stem cells, like their normal counterparts, have unlimited proliferative capacity and paradoxically traverse the cell cycle at a very slow rate; cancer growth occurs largely due to expansion of the stem cell pool, the unregulated proliferation of an amplifying population, and failure of apoptosis pathways (Fig. 26-7). Slow cell cycle progression and high levels of expression of antiapoptotic Bcl-2 family members and drug efflux pumps of the MDR family render cancer stem cells less vulnerable to cancer chemotherapy or radiation therapy. Implicit in the cancer stem cell hypothesis is the idea that failure to cure most human cancers is due to the fact that current therapeutic agents do not kill the stem cells. If cancer stem cells can be identified and isolated, then aberrant signaling pathways that distinguish these cells from normal tissue stem cells can be identified and targeted. Evidence that cells with stem cell properties can arise from other epithelial cells within the cancer by processes such as epithelial mesenchymal transition also implies that it is essential to treat all of the cancer cells, and not just those with current stem cell-like properties, in order to eliminate the self-renewing cancer cell population. The exact nature of cancer stem cells remains an area of investigation. One of the unanswered questions is the exact origin of cancer stem cells for the different cancers.

Cancer cells, and especially stem cells, have the capacity for significant plasticity, allowing them to alter multiple aspects of cell biology in response to external factors (e.g., chemotherapy, inflammation, immune response). Thus, a major problem in cancer therapy is that malignancies have a wide spectrum of mechanisms for both initial and adaptive resistance to treatments. These include inhibiting drug delivery to the cancer cells, blocking drug uptake and retention, increasing drug metabolism, altering levels of target proteins, acquiring mutations in target proteins, modifying metabolism and cell signaling pathways, using alternate signaling pathways, adjusting the cell replication process including mechanisms by which the cell deals with DNA damage, inhibiting apoptosis, and evading the immune system. Thus, most metastatic cancers (except those curable with chemotherapy such as germ cell tumors) eventually become resistant to the therapy being used. Overcoming resistance is a major area of research.

One of the distinguishing characteristics of cancer cells is that they have altered metabolism as compared with normal cells in supporting survival and their high rates of proliferation. These cells must focus a significant fraction of their energy resources on synthesis of proteins and other molecules while still maintaining sufficient ATP production to survive and grow. Although normal proliferating cells also have similar needs, there are differences in how cancer cells metabolize glucose and a number of other compounds, including glutamine, as compared to normal cells. Many cancer cells use aerobic glycolysis (the Warburg effect) (Fig. 26-8) to metabolize glucose, leading to increased lactic acid production, whereas normal cells use oxidative phosphorylation in mitochondria under aerobic conditions, a much more efficient process. One consequence is increased glucose uptake by cancer cells, a fact used in fluorodeoxyglucose (FDG) positron emission tomography (PET) scanning to detect tumors. A number of proteins in cancer cells, including CMYC, HIF1, RAS, p53, pRB, and AKT, are all involved in modulating glycolytic processes and controlling the Warburg effect. Although these pathways remain difficult to target therapeutically, both the PI3 kinase pathway with signaling through mTOR and the AMP-activated kinase (AMPK) pathway, which inhibits mTOR complex 1 (mTORC1; a protein complex that includes mTOR), are important in controlling the glycolytic process and thus provide potential targets for inhibiting this process. The inefficient utilization of glucose also leads to a need for alternative metabolic pathways for other compounds as well, one of which is glutamine. Similar to glucose, this provides both a source for structural molecules as well as energy production. Glutamine is also inefficiently used by cancer cells.

Mutations in genes involved in the metastatic process occur in a number of cancers. Among the most frequently found to date are mutations in isocitrate dehydrogenases 1 and 2 (IDH1 and IDH2). These have been most commonly seen in gliomas, AML, and intrahepatic cholangiocarcinomas. These mutations lead to the production of an oncometabolite (2-hydroxyglutarate [2HG]) instead of the normal product α-ketoglutarate. Although the exact mechanisms of oncogenesis by 2HG are still being elucidated, α-ketoglutarate is a key cofactor for a number of dioxygenases involved in controlling DNA methylation. 2HG can act as a competitive inhibitor for α-ketoglutarate, leading to alterations in methylation status (primarily hypermethylation) of genes (epigenetic changes) that can have profound effects on a number of cellular processes including differentiation. Inhibitors of mutant IDH1 and IDH2 are being developed.

Much needs to be learned about the specific differences in metabolism between cancer cells and normal cells; however, modulators of metabolism are being tested clinically. The first of these is the antidiabetic agent metformin, both alone and in combination with chemotherapeutic agents. Metformin inhibits gluconeogenesis and may have direct effects on tumor cells by activating the 5′-adenosine monophosphate-activated kinase (AMPK), a serine/threonine protein kinase that is downstream of the LKB1 tumor suppressor, and thus inhibiting mTORC1. This leads to decreased protein synthesis and proliferation. A second approach being tested involves dichloracetate (DCA), an inhibitor of pyruvate dehydrogenase kinase (PDK). PDK inhibits pyruvate dehydrogenase in cancer cells, leading to a switch from mitochondrial oxidative phosphorylation of glucose to cytoplasmic glycolysis (the Warburg effect). By blocking PDK, DCA inhibits glycolysis. Additional approaches targeting tumor metabolism will likely emerge.

Tumors consist not only of malignant cells but also of a complex microenvironment including many other types of cells (e.g., inflammatory cells), ECM, secreted factors (e.g., growth factors), reactive oxygen and nitrogen species, mechanical factors, blood vessels, and lymphatics. This microenvironment is not static but rather is dynamic and continually evolving. Both the complexity and dynamic nature of the microenvironment enhance the difficulty of treating tumors. There are also a number of mechanisms by which the microenvironment can contribute to resistance to anticancer therapies.

One of the critical elements of tumor cell proliferation is delivery of oxygen, nutrients, and circulating factors important for growth and survival. The diffusion limit for oxygen in tissues is ~100–200 μm, and thus, a critical aspect in the growth of tumors is the development of new blood vessels, or angiogenesis. The growth of primary and metastatic tumors to larger than a few millimeters requires the recruitment of blood vessels and vascular endothelial cells to support their metabolic requirements. Thus, a critical element in growth of primary tumors and formation of metastatic sites is the angiogenic switch: the ability of the tumor to promote the formation of new capillaries from preexisting host vessels. The angiogenic switch is a phase in tumor development when the dynamic balance of pro- and antiangiogenic factors is tipped in favor of vessel formation by the effects of the tumor on its immediate environment. Stimuli for tumor angiogenesis include hypoxemia, inflammation, and genetic lesions in oncogenes or tumor suppressors that alter tumor cell gene expression. Angiogenesis consists of several steps, including the stimulation of endothelial cells (ECs) by growth factors, degradation of the ECM by proteases, proliferation and migration of ECs into the tumor, and the eventual formation of new capillary tubes.

Tumor blood vessels are not normal; they have chaotic architecture and blood flow. Due to an imbalance of angiogenic regulators such as VEGF and angiopoietins (see below), tumor vessels are tortuous and dilated with an uneven diameter, excessive branching, and shunting. Tumor blood flow is variable, with areas of hypoxemia and acidosis leading to the selection of variants that are resistant to hypoxemia-induced apoptosis (often due to the loss of p53 expression). Tumor vessel walls have numerous openings, widened interendothelial junctions, and discontinuous or absent basement membrane; this contributes to the high vascular permeability of these vessels and, together with lack of functional intratumoral lymphatics, causes increased interstitial pressure within the tumor (which also interferes with the delivery of therapeutics to the tumor; Figs. 26-9, 26-10, and 26-11). Tumor blood vessels lack perivascular cells such as pericytes and smooth-muscle cells that normally regulate flow in response to tissue metabolic needs.

Unlike normal blood vessels, the vascular lining of tumor vessels is not a homogeneous layer of ECs but often consists of a mosaic of ECs and tumor cells with upregulated genes seen in ECs and vessel formation that can occur in hypoxic conditions because of their plasticity; the concept of cancer cell–derived vascular channels, which may be lined by ECM secreted by the tumor cells, is referred to as vascular mimicry. During tumor angiogenesis, ECs are highly proliferative and express a number of plasma membrane proteins that are characteristic of activated endothelium, including growth factor receptors and adhesion molecules such as integrins.

Tumors use a number of mechanisms to promote vascularization, subverting normal angiogenic processes for this purpose (Fig. 26-9). Primary or metastatic tumor cells sometimes arise in proximity to host blood vessels and grow around these vessels, parasitizing nutrients by co-opting the local blood supply. However, most tumor blood vessels arise by the process of sprouting, in which tumors secrete trophic angiogenic molecules, the most potent being vascular endothelial growth factors (VEGF), that induce the proliferation and migration of host ECs into the tumor. Sprouting in normal and pathogenic angiogenesis is regulated by three families of transmembrane receptor tyrosine kinases (RTKs) expressed on ECs and their ligands (VEGFs, angiopoietins, ephrins; Fig. 26-10), which are produced by tumor cells, inflammatory cells, or stromal cells in the tumor microenvironment.

When tumor cells arise in or metastasize to an avascular area, they grow to a size limited by hypoxemia and nutrient deprivation. Hypoxemia, a key regulator of tumor angiogenesis, causes the transcriptional induction of the gene encoding VEGF. VEGF and its receptors are required for embryonic vasculogenesis (development of new blood vessels when none preexist) and normal (wound healing, corpus luteum formation) and pathologic angiogenesis (tumor angiogenesis, inflammatory conditions such as rheumatoid arthritis). VEGF-A is a heparin-binding glycoprotein with at least four isoforms (splice variants) that regulates blood vessel formation by binding to the RTKs VEGFR1 and VEGFR2, which are expressed on all ECs in addition to a subset of hematopoietic cells (Fig. 26-9). VEGFR2 regulates EC proliferation, migration, and survival, whereas VEGFR1 may act as an antagonist of R2 in ECs but is probably also important for angioblast differentiation during embryogenesis. Tumor vessels may be more dependent on VEGFR signaling for growth and survival than normal ECs. Although VEGF signaling is a critical initiator of angiogenesis, this is a complex process regulated by additional signaling pathways (Fig. 26-10). The angiopoietin, Ang1, produced by stromal cells, binds to the EC RTK Tie2 and promotes the interaction of ECs with the ECM and perivascular cells, such as pericytes and smooth-muscle cells, to form tight, nonleaky vessels. Platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) help to recruit these perivascular cells. Ang1 is required for maintaining the quiescence and stability of mature blood vessels and prevents the vascular permeability normally induced by VEGF and inflammatory cytokines.

For tumor cell–derived VEGF to initiate sprouting from host vessels, the stability conferred by the Ang1/Tie2 pathway must be perturbed; this occurs by the secretion of Ang2 by ECs that are undergoing active remodeling. Ang2 binds to Tie2 and is a competitive inhibitor of Ang1 action: under the influence of Ang2, preexisting blood vessels become more responsive to remodeling signals, with less adherence of ECs to stroma and associated perivascular cells and more responsiveness to VEGF. Therefore, Ang2 is required at early stages of tumor angiogenesis for destabilizing the vasculature by making host ECs more sensitive to angiogenic signals. Because tumor ECs are blocked by Ang2, there is no stabilization by the Ang1/Tie2 interaction, and tumor blood vessels are leaky, hemorrhagic, and have poor association of ECs with underlying stroma. Sprouting tumor ECs express high levels of the transmembrane protein ephrin-B2 and its receptor, the RTK EPH, whose signaling appears to work with the angiopoietins during vessel remodeling. During embryogenesis, EPH receptors are expressed on the endothelium of primordial venous vessels while the transmembrane ligand ephrin-B2 is expressed by cells of primordial arteries; the reciprocal expression may regulate differentiation and patterning of the vasculature.

A number of ubiquitously expressed host molecules play critical roles in normal and pathologic angiogenesis. Proangiogenic cytokines, chemokines, and growth factors secreted by stromal cells or inflammatory cells make important contributions to neovascularization, including bFGF, transforming growth factor α (TGF-α), TNF-α, and IL-8. In contrast to normal endothelium, angiogenic endothelium overexpresses specific members of the integrin family of ECM-binding proteins that mediate EC adhesion, migration, and survival. Specifically, expression of integrins α_vβ₃, α_vβ₅, and α₅β₁ mediates spreading and migration of ECs and is required for angiogenesis induced by VEGF and bFGF, which in turn can upregulate EC integrin expression. The α_vβ₃ integrin physically associates with VEGFR2 in the plasma membrane and promotes signal transduction from each receptor to promote EC proliferation (via focal adhesion kinase, src, PI3K, and other pathways) and survival (by inhibition of p53 and increasing the Bcl-2/Bax expression ratio). In addition, α_vβ₃ forms cell-surface complexes with matrix metalloproteinases (MMPs), zinc-requiring proteases that cleave ECM proteins, leading to enhanced EC migration and the release of heparin-binding growth factors, including VEGF and bFGF. EC adhesion molecules can be upregulated (i.e., by VEGF, TNF-α) or downregulated (by TGF-β); this, together with chaotic blood flow, explains poor leukocyte-endothelial interactions in tumor blood vessels and may help tumor cells avoid immune surveillance.

Lymphatic vessels also exist within tumors. Development of tumor lymphatics is associated with expression of VEGFR3 and its ligands VEGF-C and VEGF-D. The role of these vessels in tumor cell metastasis to regional lymph nodes remains to be determined. However, VEGF-C levels correlate significantly with metastasis to regional lymph nodes in lung, prostate, and colorectal cancers.

Angiogenesis inhibitors function by targeting the critical molecular pathways involved in EC proliferation, migration, and/or survival, many of which are unique to the activated endothelium in tumors. Inhibition of growth factor and adhesion-dependent signaling pathways can induce EC apoptosis with concomitant inhibition of tumor growth. Different types of tumors can use distinct combinations of molecular mechanisms to activate the angiogenic switch. Therefore, it is doubtful that a single antiangiogenic strategy will suffice for all human cancers; rather, a number of agents or combinations of agents will be needed, depending on distinct programs of angiogenesis used by different human cancers. Despite this, experimental data indicate that for some tumor types, blockade of a single growth factor (e.g., VEGF) may inhibit tumor-induced vascular growth.

Bevacizumab, an antibody that binds VEGF, appears to potentiate the effects of a number of different types of active chemotherapeutic regimens used to treat a variety of different tumor types including colon cancer, lung cancer, cervical cancer, and RCC.

Bevacizumab is administered IV every 2–3 weeks (its half-life is nearly 20 days) and is generally well tolerated. Hypertension is the most common side effect of inhibitors of VEGF (or its receptors), but can be treated with antihypertensive agents and rarely requires discontinuation of therapy. Rare but serious potential risks include arterial thromboembolic events, including stroke and myocardial infarction, and hemorrhage. Another serious complication is bowel perforation, which has been observed in 1–3% of patients (mainly those with colon and ovarian cancers). Inhibition of wound healing is also seen.

Several small-molecule inhibitors (SMIs) that target VEGFR tyrosine kinase activity but are also inhibitory to other kinases have also been approved to treat certain cancers. Sunitinib (see above and Table 26-2) has activity directed against mutant c-Kit receptors (approved for GIST), but also targets VEGFR and PDGFR, and has shown significant antitumor activity against metastatic RCC, presumably on the basis of its antiangiogenic activity. Similarly, sorafenib, originally developed as a Raf kinase inhibitor but with potent activity against VEGFR and PDGFR, has activity against RCC, thyroid cancer, and hepatocellular cancer. Other inhibitors of VEGFR approved for the treatment of RCC include axitinib and pazopanib.

The success in targeting tumor angiogenesis has led to enhanced enthusiasm for the development of drugs that target other aspects of the angiogenic process; some of these therapeutic approaches are outlined in Fig. 26-12.

Cancers have a number of mechanisms that allow them to evade detection and elimination by the immune system. These include downregulation of cell surface proteins involved in immune recognition (including MHC proteins and tumor-specific antigens), expression of other cell surface proteins that inhibit immune function (including members of the B7 family of proteins such as PD-L1), secretion of proteins and other molecules that are immunosuppressive, recruitment and expansion of immunosuppressive cells such as regulatory T cells, and induction of T cell tolerance. In addition, the inflammatory effects of some of the immune mediator cells in the tumor microenvironment (especially tissue-associated macrophages and myeloid-derived suppressor cells) can suppress T cell responses to the tumor as well as stimulate inflammation that can enhance tumor growth.

Immunotherapy approaches to treat cancer aimed at activating the immune response against tumors using immunostimulatory molecules such as interferons, IL-2, and monoclonal antibodies have had some successes. Another approach that has shown particular clinical promise is the targeting of proteins or cells (such as regulatory T cells) involved in normal homeostatic control to prevent autoimmune damage to the host but that malignant cells and their stroma can also use to inhibit the immune response directed against them. The approach that is furthest along clinically has involved targeting CTLA-4, PD-1, and PDL-1, co-inhibitory molecules that are expressed on the surface of cancer cells, cells of the immune system, and/or stromal cells and are involved in inhibiting the immune response against cancer (Fig. 26-13). Monoclonal antibodies directed against CTLA-4 and PD-1 are approved for the treatment of melanoma, and additional antibodies targeting PD-1 or PDL-1 have shown activity against melanoma, RCC, and lung cancer and continue to be evaluated against other malignancies as well. Combination approaches targeting more than one protein or involving other anticancer approaches (targeted agents, chemotherapy, radiation therapy) are also being explored and have shown promise in early studies. An important aspect of these approaches is balancing sufficient release of the negative control of the immune response to allow immune-mediated attack on the tumors while not allowing too much release and inducing severe autoimmune effects (such as against skin, thyroid, pituitary gland, or the gastrointestinal tract).

The explosion of information on tumor cell biology, metastasis, and tumor-host interactions (including angiogenesis and immune evasion by tumors) has ushered in a new era of rational targeted therapy for cancer. Furthermore, it has become clear that specific molecular factors detected in individual tumors (specific gene mutations, gene-expression profiles, microRNA expression, overexpression of specific proteins) can be used to tailor therapy and maximize antitumor effects.

Acknowledgment

Robert G. Fenton contributed to this chapter in prior editions, and important material from those prior chapters has been included here.