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Autophagy represents an important cell-physiologic response that, like apoptosis, normally operates at low, basal levels in cells but can be strongly induced in certain states of cellular stress, the most obvious of which is nutrient deficiency (

,

). The autophagic program enables cells to break down cellular organelles, such as ribosomes and mitochondria, allowing the resulting catabolites to be recycled and thus used for biosynthesis and energy metabolism. As part of this program, intracellular vesicles termed autophagosomes envelope intracellular organelles and then fuse with lysosomes wherein degradation occurs. In this fashion, low-molecular-weight metabolites are generated that support survival in the stressed, nutrient-limited environments experienced by many cancer cells.

Like apoptosis, the autophagy machinery has both regulatory and effector components (

,

). Among the latter are proteins that mediate autophagosome formation and delivery to lysosomes. Of note, recent research has revealed intersections between the regulatory circuits governing autophagy, apoptosis, and cellular homeostasis. For example, the signaling pathway involving the PI3-kinase, AKT, and mTOR kinases, which is stimulated by survival signals to block apoptosis, similarly inhibits autophagy; when survival signals are insufficient, the PI3K signaling pathway is downregulated, with the result that autophagy and/or apoptosis may be induced (

,

,

).

Another interconnection between these two programs resides in the Beclin-1 protein, which has been shown by genetic studies to be necessary for induction of autophagy (

,

,

). Beclin-1 is a member of the BH3-only subfamily of apoptotic regulatory proteins, and its BH3 domain allows it to bind the Bcl-2/Bcl-x_L proteins. Stress-sensor-coupled BH3 proteins can displace Beclin-1 from its association with Bcl-2/Bcl-x_L, enabling the liberated Beclin-1 to trigger autophagy, much as they can release proapoptotic Bax and Bak to trigger apoptosis. Hence, stress-transducing BH3 proteins (e.g., Bid, Bad, Puma, et al.) can induce apoptosis and/or autophagy depending on the physiologic state of the cell.

Mice bearing inactivated alleles of the Beclin-1 gene or of certain other components of the autophagy machinery exhibit increased susceptibility to cancer (

:

). These results suggest that induction of autophagy can serve as a barrier to tumorigenesis that may operate independently of or in concert with apoptosis. Accordingly, autophagy appears to represent yet another barrier that needs to be circumvented during tumor development (

).

Perhaps paradoxically, nutrient starvation, radiotherapy, and certain cytotoxic drugs can induce elevated levels of autophagy that are apparently cytoprotective for cancer cells, impairing rather than accentuating the killing actions of these stress-inducing situations (

,

,

,

). Moreover, severely stressed cancer cells have been shown to shrink via autophagy to a state of reversible dormancy (

,

). This survival response may enable the persistence and eventual regrowth of some late-stage tumors following treatment with potent anticancer agents. Thus, in analogy to TGF-β signaling, which can be tumor suppressing at early stages of tumorigenesis and tumor promoting later on, autophagy seems to have conflicting effects on tumor cells and thus tumor progression (

,

). An important agenda for future research will involve clarifying the genetic and cell-physiologic conditions that dictate when and how autophagy enables cancer cells to survive or causes them to die.

In contrast to apoptosis, in which a dying cell contracts into an almost-invisible corpse that is soon consumed by neighbors, necrotic cells become bloated and explode, releasing their contents into the local tissue microenvironment. Although necrosis has historically been viewed much like organismic death, as a form of system-wide exhaustion and breakdown, the conceptual landscape is changing: cell death by necrosis is clearly under genetic control in some circumstances, rather than being a random and undirected process (

,

).

Perhaps more important, necrotic cell death releases proinflammatory signals into the surrounding tissue microenvironment, in contrast to apoptosis and autophagy, which do not. As a consequence, necrotic cells can recruit inflammatory cells of the immune system (

,

,

), whose dedicated function is to survey the extent of tissue damage and remove associated necrotic debris. In the context of neoplasia, however, multiple lines of evidence indicate that immune inflammatory cells can be actively tumor promoting, given that such cells are capable of fostering angiogenesis, cancer cell proliferation, and invasiveness (see below). Additionally, necrotic cells can release bioactive regulatory factors, such as IL-1α, which can directly stimulate neighboring viable cells to proliferate, with the potential, once again, to facilitate neoplastic progression (

). Consequently, necrotic cell death, while seemingly beneficial in counterbalancing cancer-associated hyperproliferation, may ultimately do more damage than good. Accordingly, incipient neoplasias and potentially invasive and metastatic tumors may gain an advantage by tolerating some degree of necrotic cell death, doing so in order to recruit tumor-promoting inflammatory cells that bring growth-stimulating factors to the surviving cells within these growths.

By 2000, it was widely accepted that cancer cells require unlimited replicative potential in order to generate macroscopic tumors. This capability stands in marked contrast to the behavior of the cells in most normal cell lineages in the body, which are able to pass through only a limited number of successive cell growth-and-division cycles. This limitation has been associated with two distinct barriers to proliferation: senescence, a typically irreversible entrance into a nonproliferative but viable state, and crisis, which involves cell death. Accordingly, when cells are propagated in culture, repeated cycles of cell division lead first to induction of senescence and then, for those cells that succeed in circumventing this barrier, to a crisis phase, in which the great majority of cells in the population die. On rare occasion, cells emerge from a population in crisis and exhibit unlimited replicative potential. This transition has been termed immortalization, a trait that most established cell lines possess by virtue of their ability to proliferate in culture without evidence of either senescence or crisis.

Multiple lines of evidence indicate that telomeres protecting the ends of chromosomes are centrally involved in the capability for unlimited proliferation (

,

). The telomeres, composed of multiple tandem hexanucleotide repeats, shorten progressively in nonimmortalized cells propagated in culture, eventually losing the ability to protect the ends of chromosomal DNAs from end-to-end fusions; such fusions generate unstable dicentric chromosomes whose resolution results in a scrambling of karyotype that threatens cell viability. Accordingly, the length of telomeric DNA in a cell dictates how many successive cell generations its progeny can pass through before telomeres are largely eroded and have consequently lost their protective functions, triggering entrance into crisis.

Telomerase, the specialized DNA polymerase that adds telomere repeat segments to the ends of telomeric DNA, is almost absent in nonimmortalized cells but expressed at functionally significant levels in the vast majority (∼90%) of spontaneously immortalized cells, including human cancer cells. By extending telomeric DNA, telomerase is able to counter the progressive telomere erosion that would otherwise occur in its absence. The presence of telomerase activity, either in spontaneously immortalized cells or in the context of cells engineered to express the enzyme, is correlated with a resistance to induction of both senescence and crisis/apoptosis; conversely, suppression of telomerase activity leads to telomere shortening and to activation of one or the other of these proliferative barriers.

The two barriers to proliferation—senescence and crisis/apoptosis—have been rationalized as crucial anticancer defenses that are hard-wired into our cells, being deployed to impede the outgrowth of clones of preneoplastic and frankly neoplastic cells. According to this thinking, most incipient neoplasias exhaust their endowment of replicative doublings and are stopped in their tracks by one or the other of these barriers. The eventual immortalization of rare variant cells that proceed to form tumors has been attributed to their ability to maintain telomeric DNA at lengths sufficient to avoid triggering senescence or apoptosis, achieved most commonly by upregulating expression of telomerase or, less frequently, via an alternative recombination-based telomere maintenance mechanism. Hence, telomere shortening has come to be viewed as a clocking device that determines the limited replicative potential of normal cells and thus one that must be overcome by cancer cells.

Whereas telomere maintenance has been increasingly substantiated as a condition critical to the neoplastic state, the concept of replication-induced senescence as a general barrier requires refinement and reformulation. (Differences in telomere structure and function in mouse versus human cells have also complicated investigation of the roles of telomeres and telomerase in replicative senescence.) Recent experiments have revealed that the induction of senescence in certain cultured cells can be delayed and possibly eliminated by the use of improved cell culture conditions, suggesting that recently explanted primary cells may be able to proliferate unimpeded in culture up the point of crisis and the associated induction of apoptosis triggered by critically shortened telomeres (

,

,

,

). In contrast, experiments in mice engineered to lack telomerase indicate that the consequently shortened telomeres can shunt premalignant cells into a senescent state that contributes (along with apoptosis) to attenuated tumorigenesis in mice genetically destined to develop particular forms of cancer (

). Such telomerase null mice with highly eroded telomeres exhibit multiorgan dysfunction and abnormalities that include evidence for both senescence and apoptosis, perhaps analogous to the senescence and apoptosis observed in cell culture (

,

).

Of note, and as discussed earlier, a morphologically similar form of cell senescence induced by excessive or unbalanced oncogene signaling is now well documented as a protective mechanism against neoplasia; the possible interconnections of this form of senescence with telomerase and telomeres remain to be ascertained. Thus, cell senescence is emerging conceptually as a protective barrier to neoplastic expansion that can be triggered by various proliferation-associated abnormalities, including high levels of oncogenic signaling and, apparently, subcritical shortening of telomeres.

There is now evidence that clones of incipient cancer cells often experience telomere loss-induced crisis relatively early during the course of multistep tumor progression due to their inability to express significant levels of telomerase. Thus, extensively eroded telomeres have been documented in premalignant growths through the use of fluorescence in situ hybridization (FISH), which has also revealed the end-to-end chromosomal fusions that signal telomere failure and crisis (

,

). These results also suggest that such cells have passed through a substantial number of successive telomere-shortening cell divisions during their evolution from fully normal cells-of-origin. Accordingly, the development of some human neoplasias may be aborted by telomere-induced crisis long before they succeed in becoming macroscopic, frankly neoplastic growths.

In contrast, the absence of TP53-mediated surveillance of genomic integrity may permit other incipient neoplasias to survive initial telomere erosion and attendant chromosomal breakage-fusion-bridge (BFB) cycles. The genomic alterations resulting from these BFB cycles, including deletions and amplifications of chromosomal segments, evidently serve to increase the mutability of the genome, thereby accelerating the acquisition of mutant oncogenes and tumor suppressor genes. The realization that impaired telomere function can actually foster tumor progression has come from the study of mutant mice that lack both p53 and telomerase function (

,

). The proposition that these two defects can cooperatively enhance human tumorigenesis has not yet been directly documented.

Circumstantial support for the importance of transient telomere deficiency in facilitating malignant progression has come, in addition, from comparative analyses of premalignant and malignant lesions in the human breast (

,

). The premalignant lesions did not express significant levels of telomerase and were marked by telomere shortening and nonclonal chromosomal aberrations. In contrast, overt carcinomas exhibited telomerase expression concordantly with the reconstruction of longer telomeres and the fixation (via clonal outgrowth) of the aberrant karyotypes that would seem to have been acquired after telomere failure but before the acquisition of telomerase activity. When portrayed in this way, the delayed acquisition of telomerase function serves to generate tumor-promoting mutations, whereas its subsequent activation stabilizes the mutant genome and confers the unlimited replicative capacity that cancer cells require in order to generate clinically apparent tumors.

Telomerase was discovered because of its ability to elongate and maintain telomeric DNA, and almost all telomerase research has been posited on the notion that its functions are confined to this crucial function. However, in recent years it has become apparent that telomerase exerts functions that are relevant to cell proliferation but unrelated to telomere maintenance. The noncanonical roles of telomerase, and in particular its protein subunit TERT, have been revealed by functional studies in mice and cultured cells; in some cases novel functions have been demonstrated in conditions where the telomerase enzymatic activity has been eliminated (

). Among the growing list of telomere-independent functions of TERT/telomerase is the ability of TERT to amplify signaling by the Wnt pathway, by serving as a cofactor of the β-catenin/LEF transcription factor complex (

). Other ascribed telomere-independent effects include demonstrable enhancement of cell proliferation and/or resistance to apoptosis (

), involvement in DNA-damage repair (

), and RNA-dependent RNA polymerase function (

). Consistent with these broader roles, TERT can be found associated with chromatin at multiple sites along the chromosomes, not just at the telomeres (

,

). Hence, telomere maintenance is proving to be the most prominent of a diverse series of functions to which TERT contributes. The contributions of these additional functions of telomerase to tumorigenesis remain to be fully elucidated.

Like normal tissues, tumors require sustenance in the form of nutrients and oxygen as well as an ability to evacuate metabolic wastes and carbon dioxide. The tumor-associated neovasculature, generated by the process of angiogenesis, addresses these needs. During embryogenesis, the development of the vasculature involves the birth of new endothelial cells and their assembly into tubes (vasculogenesis) in addition to the sprouting (angiogenesis) of new vessels from existing ones. Following this morphogenesis, the normal vasculature becomes largely quiescent. In the adult, as part of physiologic processes such as wound healing and female reproductive cycling, angiogenesis is turned on, but only transiently. In contrast, during tumor progression, an “angiogenic switch” is almost always activated and remains on, causing normally quiescent vasculature to continually sprout new vessels that help sustain expanding neoplastic growths (

).

A compelling body of evidence indicates that the angiogenic switch is governed by countervailing factors that either induce or oppose angiogenesis (

,

). Some of these angiogenic regulators are signaling proteins that bind to stimulatory or inhibitory cell-surface receptors displayed by vascular endothelial cells. The well-known prototypes of angiogenesis inducers and inhibitors are vascular endothelial growth factor-A (VEGF-A) and thrombospondin-1 (TSP-1), respectively.

The VEGF-A gene encodes ligands that are involved in orchestrating new blood vessel growth during embryonic and postnatal development, and then in homeostatic survival of endothelial cells, as well as in physiological and pathological situations in the adult. VEGF signaling via three receptor tyrosine kinases (VEGFR-1–3) is regulated at multiple levels, reflecting this complexity of purpose. Thus, VEGF gene expression can by upregulated both by hypoxia and by oncogene signaling (

,

,

). Additionally, VEGF ligands can be sequestered in the extracellular matrix in latent forms that are subject to release and activation by extracellular matrix-degrading proteases (e.g., MMP-9;

). In addition, other proangiogenic signals, such as members of the fibroblast growth factor (FGF) family, have been implicated in sustaining tumor angiogenesis when their expression is chronically upregulated (

). TSP-1, a key counterbalance in the angiogenic switch, also binds transmembrane receptors displayed by endothelial cells and thereby evokes suppressive signals that can counteract proangiogenic stimuli (

).

The blood vessels produced within tumors by chronically activated angiogenesis and an unbalanced mix of proangiogenic signals are typically aberrant: tumor neovasculature is marked by precocious capillary sprouting, convoluted and excessive vessel branching, distorted and enlarged vessels, erratic blood flow, microhemorrhaging, leakiness, and abnormal levels of endothelial cell proliferation and apoptosis (

,

).

Angiogenesis is induced surprisingly early during the multistage development of invasive cancers both in animal models and in humans. Histological analyses of premalignant, noninvasive lesions, including dysplasias and in situ carcinomas arising in a variety of organs, have revealed the early tripping of the angiogenic switch (

,

). Historically, angiogenesis was envisioned to be important only when rapidly growing macroscopic tumors had formed, but more recent data indicate that angiogenesis also contributes to the microscopic premalignant phase of neoplastic progression, further cementing its status as an integral hallmark of cancer.

The past decade has witnessed an astonishing outpouring of research on angiogenesis. Amid this wealth of new knowledge, we highlight several advances of particular relevance to tumor physiology.

Once angiogenesis has been activated, tumors exhibit diverse patterns of neovascularization. Some tumors, including such highly aggressive types as pancreatic ductal adenocarcinomas, are hypovascularized and replete with stromal “deserts” that are largely avascular and indeed may even be actively antiangiogenic (

). Many other tumors, including human renal and pancreatic neuroendocrine carcinomas, are highly angiogenic and consequently densely vascularized (

,

).

Collectively, such observations suggest an initial tripping of the angiogenic switch during tumor development that is followed by a variable intensity of ongoing neovascularization, the latter being controlled by a complex biological rheostat that involves both the cancer cells and the associated stromal microenvironment (

,

). Of note, the switching mechanism can vary in its form, even though the net result is a common inductive signal (e.g., VEGF). In some tumors, dominant oncogenes operating within tumor cells, such as Ras and Myc, can upregulate expression of angiogenic factors, whereas in others, such inductive signals are produced indirectly by immune inflammatory cells, as discussed below. The direct induction of angiogenesis by oncogenes that also drive proliferative signaling illustrates the important principle that distinct hallmark capabilities can be coregulated by the same transforming agents.

Research in the 1990s revealed that TSP-1 as well as fragments of plasmin (angiostatin) and type 18 collagen (endostatin) can act as endogenous inhibitors of angiogenesis (

,

,

,

,

). The last decade has seen reports of another dozen such agents (

,

,

). Most are proteins, and many are derived by proteolytic cleavage of structural proteins that are not themselves angiogenic regulators. A number of these endogenous inhibitors of angiogenesis can be detected in the circulation of normal mice and humans. The genes encoding several endogenous angiogenesis inhibitors have been deleted from the mouse germline without untoward physiological effects; the growth of autochthonous and implanted tumors, however, is enhanced as a consequence (

,

). By contrast, if the circulating levels of an endogenous inhibitor are genetically increased (e.g., via overexpression in transgenic mice or in xenotransplanted tumors), tumor growth is impaired (

,

); interestingly, wound healing and fat deposition are impaired or accelerated by elevated or ablated expression of such genes (

,

). The data suggest that such endogenous angiogenesis inhibitors serve under normal circumstances as physiologic regulators that modulate transitory angiogenesis during tissue remodeling and wound healing; they may also act as intrinsic barriers to induction and/or persistence of angiogenesis by incipient neoplasias.

Pericytes have long been known as supporting cells that are closely apposed to the outer surfaces of the endothelial tubes in normal tissue vasculature, where they provide important mechanical and physiologic support to the endothelial cells. Tumor-associated vasculature, in contrast, was portrayed as lacking appreciable coverage by these auxiliary cells. However, careful microscopic studies conducted in recent years have revealed that pericytes are associated, albeit loosely, with the neovasculature of most if not all tumors (

,

). More importantly, mechanistic studies discussed below have revealed that pericyte coverage is important for the maintenance of a functional tumor neovasculature.

It is now clear that a repertoire of cell types originating in the bone marrow play crucial roles in pathological angiogenesis (

,

,

,

). These include cells of the innate immune system—notably macrophages, neutrophils, mast cells, and myeloid progenitors—that infiltrate premalignant lesions and progressed tumors and assemble at the margins of such lesions; the peri-tumoral inflammatory cells help to trip the angiogenic switch in previously quiescent tissue and to sustain ongoing angiogenesis associated with tumor growth, in addition to facilitating local invasion, as noted below. In addition, they can help protect the vasculature from the effects of drugs targeting endothelial cell signaling (

). Additionally, several types of bone marrow-derived “vascular progenitor cells” have been observed in certain cases to have migrated into neoplastic lesions and become intercalated into the neovasculature as pericytes or endothelial cells (

,

,

).

In 2000, the mechanisms underlying invasion and metastasis were largely an enigma. It was clear that as carcinomas arising from epithelial tissues progressed to higher pathological grades of malignancy, reflected in local invasion and distant metastasis, the associated cancer cells typically developed alterations in their shape as well as in their attachment to other cells and to the extracellular matrix (ECM). The best characterized alteration involved the loss by carcinoma cells of E-cadherin, a key cell-to-cell adhesion molecule. By forming adherens junctions with adjacent epithelial cells, E-cadherin helps to assemble epithelial cell sheets and maintain the quiescence of the cells within these sheets. Increased expression of E-cadherin was well established as an antagonist of invasion and metastasis, whereas reduction of its expression was known to potentiate these phenotypes. The frequently observed downregulation and occasional mutational inactivation of E-cadherin in human carcinomas provided strong support for its role as a key suppressor of this hallmark capability (

,

).

Additionally, expression of genes encoding other cell-to-cell and cell-to-ECM adhesion molecules is demonstrably altered in some highly aggressive carcinomas, with those favoring cytostasis typically being downregulated. Conversely, adhesion molecules normally associated with the cell migrations that occur during embryogenesis and inflammation are often upregulated. For example, N-cadherin, which is normally expressed in migrating neurons and mesenchymal cells during organogenesis, is upregulated in many invasive carcinoma cells. Beyond the gain and loss of such cell-cell/matrix attachment proteins, the master regulators of invasion and metastasis were largely unknown or, when suspected, lacking in functional validation (

).

The multistep process of invasion and metastasis has been schematized as a sequence of discrete steps, often termed the invasion-metastasis cascade (

,

). This depiction envisions a succession of cell-biologic changes, beginning with local invasion, then intravasation by cancer cells into nearby blood and lymphatic vessels, transit of cancer cells through the lymphatic and hematogenous systems, followed by escape of cancer cells from the lumina of such vessels into the parenchyma of distant tissues (extravasation), the formation of small nodules of cancer cells (micrometastases), and finally the growth of micrometastatic lesions into macroscopic tumors, this last step being termed “colonization.”

Research into the capability for invasion and metastasis has accelerated dramatically over the past decade as powerful new research tools and refined experimental models have become available, and as critical regulatory genes were identified. While still an emerging field replete with major unanswered questions, significant progress has been made in delineating important features of this complex hallmark capability. An admittedly incomplete representation of these advances is highlighted below.