The Hallmarks of are ‘acquired functional capabilities that allow cancer cells to survive, proliferate, and disseminate’. The peer-reviewed article was originally published in 2000 in the journal ‘Cell’ and discussed the six hallmarks. It was updated in 2011 to add the enabling and emerging characteristics. Below follows a summary of the hallmarks, the emerging and enabling characteristics and a summary of the authors’ discussion on the importance of tumour microenvironment and potential for therapeutic treatment.

Sustaining of proliferative signalling Normal tissues control cell proliferation to allow of cell numbers. Growth factors bind to cell surface receptors and control the progression of a cell through the cell cycle. Tumour cells alter this in a number of ways: by producing their own growth factors, by sending signalling to other cells in the microenvironment to produce growth factors, by increasing numbers of cell surface receptors to become hyperresponsive to growth factors, by altering structure of receptors so that they fire without external stimulation and by producing active components to trigger the proliferative circuitry further downstream. They can also undergo in enzymes (eg GTPase) that break down proliferative signals and so disrupt negative feedback loops. Interestingly, hyperexpression of certain growth factors leads to cell , where the cell can no longer divide. This means for cancer cells to continue proliferating,there must be a balance where levels are elevated to the extent that cells can continue to proliferate yet without triggering the senescence circuitry.

Evading growth suppressors Growth suppressors limit cell size and number increases. They govern whether the cell proliferates or whether senescence and apoptotic programmes (more on this later) are activated. RB and TP53 proteins are key growth suppressors. The RB protein takes into account a variety of intra and extra-cellular protein signalling, determining whether the cell cycle continues. Absence of the RB protein results in constant cell proliferation. TP53 receives intracellular inputs from stress or abnormality sensors. Examples of these inputs are excessive damage to the genome, abnormal levels of nucleotides, poor oxygenation or glucose levels or suboptimal growth promoting signals. TP53 halts the cell cycle until conditions normalise, or if there is irreversible damage, triggers . A second mechanism suppressing growth involves the binding of cell surface growth receptors when cells come into contact, reducing cell responsiveness to growth factors. Mutations in tumour cells of the gene coding for the enzyme that carries out this process allow constant growth.

Resisting cell death There are three ways in which cell death can be brought about - apoptosis, autophagy and necrosis. Tumour cells have systems to circumvent each one. Apoptosis, or ‘’, is triggered by physiologic stresses such as elevated oncogene signalling or DNA damage as a result of hyper-proliferation. The cell’s proteases break down cellular proteins which are then absorbed by neighbouring and specialised phagocytic cells. Most commonly in cancer cells, apoptosis is halted due to the loss of the TP53 tumour suppressor function. However, it may also be stopped by increasing expression of antiapoptotic regulators, downregulating proapoptotic factors or inhibiting extracellular signals. Autophagy is induced by cellular distress, arising in situations such as nutrient deficiency, radiotherapy or injection of cytotoxic drugs. Specialised cellular vesicles digest the cell's own organelles, providing metabolites for cell survival. Issues arise over whether autophagy is tumour suppressing or enhancing. Mice where the alleles involved in the autophagocytic machinery had been suppressed showed an increased risk of tumour growth. However, it also appears to allow later stage cancer cells to go into a state of reversible dormancy when exposed to cytotoxic chemicals (eg chemotherapy), explaining the return of many malignant tumours even after treatment. Necrosis is a form of cell death where the cell becomes bloated and explodes, releasing its contents into the surrounding micro environment. This includes bioactive substances that stimulate neighbouring cells to proliferate. Inflammatory signals are also released so that immune cells can remove the necrotic debris. As discussed below, the inflammatory response carries tumour-enhancing properties.

Enabling replicative immortality For a tumour to develop, cells must be able to replicate indefinitely (replicative immortality). Under normal circumstances, senescence and apoptosis act as barriers to replicative immortality. The main mechanism inducing senescence involves : sections of nucleotides at the ends of chromosomes, protecting chromosomal DNA which shorten with each cell proliferation. The length of the normally determines the number of replication cycles. Around 90% of immortalised cells express , an enzyme that lengthens the telomere. Telomerase may also aid in tumour growth by being involved in enhancing cell proliferation or increasing resistance to apoptosis. In cancer cells, absence of TP53 (DNA damage detector) means that the shortened telomere goes undetected. This may enhance the rate of in cancer cells due to the lack of chromosomal protection, allowing mutations in oncogenes and tumour suppressor genes which increase tumour growth.

Inducing As in all tissues, tumours require vasculature to provide nutrients and oxygen and to remove metabolic waste products. Angiogenesis, the sprouting of new blood vessels, can be switched on and off. It is mainly controlled by the proteins VEGF-A, which stimulates angiogenesis, and TSP-1, which suppresses it. Both bind to cell surface receptors on endothelial cells. Tumour vasculature shows a number of abnormalities, such as high capillary sprouting, high vessel branching, distorted vessels and leaky, erratic blood flow. They have a low pericyte coverage (vascular auxiliary supportive cells). Interestingly there appears to be a diverse pattern of vasculature delending on tumour type, where some are hypo and others are hyper vascularised.

Activating invasion and The key ways in which tumours appear to become metastatic is through downregulating the expression of cell to cell and cell to extra-cellular matrix receptors. Adhesion molecules associated with cell migration, such as , are upregulated. Metastasis takes place in two phases - first, physical dissemination of the cells to distant tissues. Second, the cells must adapt to the foreign tissue micro environment. Invasion is caused by the loss of adherens junctions, the change of cell shape from polygonal and epithelial to spindly and fibroblastic, the expression of matrix degrading enzymes, increased motility and increased resistance to apoptosis. Expression of these characteristics is thought to be brought about by the tumour microenvironment. This means when cells migrate to new environments, they lose this signalling and may also lose invasive capability.

Enabling characteristics: characteristics which make the hallmarks possible.

Genome instability The acquisition of hallmark capabilities requires either mutation or a change in gene expression. Cancer cells show increased mutation rate, as this increases the chance of the tumour progressing. Mutation rate can be increased through increased sensitivity to mutagenic agents, a breakdown in genetic surveillance machinery or compromising DNA damage surveillance systems (eg TP53). Loss of the telomere also increases genetic instability.

Tumour-promoting inflammation Tumours appear to be in a state of permanent inflammation, which some suggest is the body’s attempt to remove the cancerous tissue. However, the immune response enhances tumour growth by supplying bioactive molecules to the micro environment, providing growth factors that sustain proliferative signalling and survival factors which limit cell death as well as bringing proangiogenic factors. It also brings ECM modifying enzymes which facilitate angiogenesis, invasion and metastasis. Mutagenic reactive oxygen species are also released. Therefore the body’s immune response to the tumour actively promotes its growth.

Emerging characteristics

Deregulation cellular energetics The energy metabolism in cancer cells appears changed. They appear to favour ‘aerobic ’ as their method of ATP production, using the intermediate to produce molecules necessary for , such as amino acids and nucleotides. As this system is inefficient at producing ATP, expression of glucose transporters is upregulated. This system also allows to continue growing in hypoxic conditions. There also appears to be two phenotypes in some tumours - one relies on the method described above and so secretes lactic acid. The other, better oxygenated population then aerobically respires the lactic acid, creating a symbiotic relationship.

Avoiding immune destruction The majority of incipient cancer cells are removed by our immune systems. Therefore for a tumour to grow, it must evade immune detection. Immunocompromised individuals do show an increased prevalence of cancer, although this may also be due to an increased risk of viral infection. In experiments with mice, tumours grown in immunocompromised mice did not grow in immunocompetent mice, while tumours grown in the immunocompetent mice did grow in the immunocompromised mice. This suggests that the tumours in the immunocompetent mice have evolved some system of evading the immune system that the tumours in the immunocompromised mice have not. Furthermore, patients with certain types of cancer and a higher abundance of killer lymphocytes had a better prognosis than those with a lower abundance.

Tumour microenvironment In the final section, the authors discuss the key cell types in the tumour microenvironment and each of their respective roles. Cancer Cells and Cancer Stem Cells (CSCs) are the cells within a tumour that contain the mutations which drive tumour progression onwards. Cancer stem cells allow new tumour growth and form the bulk of most tumours. They express proteins that are also expressed by non-cancerous stem cells. Endothelial cells are the cells which line all vessels. There are a number of known mechanisms in which normal vascular endothelial cells are converted into tumour-associated endothelial cells, including the ‘angiogenic switch’. Less is known about the mechanism for development of lymph vessels. Due to the high pressure within tumours, internal lymph vessels often collapse. Instead there appear to be growing lymph vessels around the tumour and in the adjacent normal tissue. Pericytes are cells which support endothelial tubing. In tumours they show decreased coverage. This disrupts endothelial homeostasis - as pericytes are known to contribute to antiproliferative signalling - as well as vascular function and integrity. This may facilitate entry of cancer cells into the bloodstream, allowing invasion and metastasis. Immune inflammatory cells contain subclasses that are both tumour antagonising and tumour promoting. These contradictory qualities arise to the dual nature of the immune system - on the one hand, it exists to target and destroy infectious agents but on the other also exists to promote wound healing and clear cellular debris. This second class of immune cells secrete the growth factors, angiogenic growth factors and matrix degrading enzymes that allow tumour growth, progression and metastasis. Cancer associated fibroblasts create the structural foundation that supports tissue, facilitating tumour growth. These cell types therefore facilitate the development of large, structurally advanced tumours. The tumour stroma are the cells which constitute the tumour microenvironment. They appear to be mainly sourced from stem and progenitor cells in bone marrow, but may also arise from proliferation of pre-existing stromal cells and differentiation of stem and progenitor cells in situ.

Therapeutic treatment Treatment could involve specific inhibitors/drugs that prevent acquisition of certain hallmark capabilities. As most hallmark targets are very specific, this minimises off-target effects decreasing toxicity of treatment. However, as many hallmark capabilities arise due to redundant signalling, inhibitory agents may be ineffective at shutting them off. Cells also appear able to evolve to rely less on certain hallmarks - for example, shutting off angiogenesis was shut off resulted in an increase in invasion and metastasis. Therefore for therapeutic success, multiple hallmarks would need to be targeted.

The authors conclude pointing at the areas where significant amounts of investigation remains to be done. For example, into emerging hallmarks and whether they are indeed hallmarks or simply subsets of others. There also remains much to be found out about invasion and metastasis, aerobic glycolysis and cell to cell intercommunication.

References Hanahan D, Weinberg RA [2011] “Hallmarks of cancer: the next generation.” Cell 144(5), 646‐674