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Your guide to core hallmarks of cancer and emerging hallmarks and enabling characteristics.
Updated March 8, 2023
In early 2000, Professors Hanahan and Weinberg proposed that when cells progress towards a neoplastic state, they acquire distinctive capabilities1. These were termed hallmarks of cancer and formed a useful framework to help understand tumor pathogenesis. The hallmarks of cancer include sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis. And they are all underpinned by genome instability and mutation.
Later in 2011, the same authors published an update to reflect advances in understanding and to include reprogramming of energy metabolism, avoiding immune destruction, tumor-promoting inflammation, and evading immune destruction2. In 2022, Hanahan further updated the existing framework, adding four more emerging hallmarks and enabling characteristics3.
Here we review the latest version of the framework and provide the relevant markers and tools to study these important hallmarks of cancer.
|Nucleotide excision repair||ERCC1-XPF||ERCC1 – XPF is an essential endonuclease for DNA damage repair. It is also involved in DNA interstrand crosslink and double-strand break repair.|
|XPA||XPA is a Zinc finger protein responsible for DNA damage repair.|
|TFIID||TFIID is a complex that binds to the TATA box in the core promoter of the gene.|
|Base excision repair||APEX1/APEX2||APEX are nucleases involved in DNA repair.|
|PNKP||PNKP catalyzes 5’- kinase and 3’ – phosphatases activity|
|FEN1||FEN1 is an endonuclease that removes 5’ overhanging flaps in DNA repair.|
|Double-strand break (DSB) repair||Gamma H2AX||Gamma H2AX is a component of histone octamer in the nucleosome. It is phosphorylated after DNA damage.|
|XRCC4||XRCC4 functions with DNA ligase IV and DNA-dependent protein kinase to repair DNA DSB.|
|BRCA1||BRCA genes are one of the widely studied tumor suppressor proteins that regulate DNA repair and cell cycle|
|53bp1||53bp1 binds to damaged chromatin and promotes DNA repair.|
|Kap1||Kap1 is a key regulator of normal development and differentiation.|
|DNA mismatch repair||Msh2/Msh6||Msh2 and Msh6 form MutSα which binds to the site of the mismatch base.|
|Msh2/Msh3||Msh2 and Msh3 form MutSβ which participates in insertion/deletion loop repair.|
|PMS2||Forms heterodimers with MLH1 to form MutLα.|
Tumor cells can achieve unlimited replicative potential by synthesizing high telomerase enzyme levels or via a recombination-based mechanism. This prevents telomere shortening, which leads to senescence and f. However, many cancer cells have been shown to possess short telomeres.
Key targets include the telomere maintenance machinery and signaling pathways, such as Wnt and Hippo.
If you need a cell proliferation or cell cycle assay, please refer to our in-depth guide.
|Telomere maintenance and regulation||hTERT||hTRET is the major component of telomerase activity. Telomerase has been identified as a diagnostic marker for various types of cancer.|
|The Shelterin complex is a core of six proteins integral for telomere function.|
|p53 signaling||TP53 (p53)||p53, called the “guardian of the genome”, is the key regulator of gene expression.|
|MDM2||MDM2 is a proto-oncogene and plays an important p53 regulation. It is the primary inhibitor of p53 transcriptional activation. MDM2 activity is tightly controlled by post-translational modifications.|
|p14ARF/p19ARF||p14ARF is a tumor suppressor gene that binds to the MDM2-p53 complex and prevents the degradation of p53.|
|E2F-1||E2F-1 is the transcription factor of the p53 pathway that regulates by initiating p14ARF transcription.|
To overcome growth inhibition from normal homeostatic signals, cancer cells lack response to external growth-inhibitory signals. Cancer cells resist apoptotic control that allows tight control over cell death and proliferative cell growth.
Apoptosis allows the removal of cells undergoing excessive proliferation to limit cell number and remove diseased cells, while autophagy is a cellular recycling system that removes abnormal proteins and cytoplasmic contents and promotes regeneration. Cancer cells resist apoptotic signaling to prevent cell death and promote autophagy to increase growth and overcome nutrient-limiting conditions.
|Tumor suppressors||Rb1||Retinoblastoma regulates the cell cycle and plays an important role in cellular differentiation.|
|TP53 (p53)||p53, called the “guardian of the genome”, is the key regulator of gene expression. It is also an established marker for cancer diagnosis.|
|APC||APC regulates tumor growth by suppressing Wnt signaling. It also plays an important role in cell adhesion and migration.|
|BRCA1, BRCA2||BRCA is one of the widely studied tumor suppressor proteins that regulate DNA repair and cell cycle|
|PTEN||PTEN is a key regulator of cellular activities. It regulates PI3K-AKT-mTOR signaling through its lipid phosphatase activity.|
|WT1, WT2||Wilms tumor protein is a transcription factor important for normal cellular development and survival. WT1 plays both oncogenic and tumor suppressor roles.|
|NF1, NF2||Neurofibromin is a tumor suppressor that negatively regulates the Ras pathway.|
Resisting cell death hallmark refers to cancer cells preventing apoptosis through intrinsic mechanisms rather than a lack of response to external stimuli. Cancer cells may contain mutations that prevent damage detection or apoptotic signaling within the cell.
Apoptosis is characterized by several features, including cell shrinkage, membrane blebbing, chromosome condensation (pyknosis), nuclear fragmentation (karyorrhexis), DNA laddering, and the eventual engulfment of the cell by phagosomes.
Autophagy is essential in allowing cells to survive in response to multiple stress conditions. Tumor cells exploit this autophagic mechanism to overcome nutrient-limiting conditions and facilitate tumor growth. Autophagy can modulate the tumor microenvironment by promoting angiogenesis, supplying nutrients, and modulating the inflammatory response.
Caspases, Bcl-2, and p53 are among the key apoptotic signaling proteins frequently downregulated, mutated, or bypassed in cancer cells. Key autophagy targets include proteasomal and lysosomal pathways, such as MAPK, ATG, and p62.
To learn more about apoptosis, check out our resources: our in-depth guide to apoptosis, apoptosis in cancer signaling poster, our protocol for induction of apoptosis in cells, and our apoptosis assay and marker guide.
To learn more about autophagy, see our guide to autophagy.
Due to their excessive growth, cancer cells require high levels of energy and nutrients with the ability to survive in hypoxic environments, as tumors can be poorly vascularized. To meet these needs, many of the cellular metabolic pathways are altered in cancer. The Warburg effect concerns the altered glycolytic metabolism in cancer cells, where pyruvate is diverted from the Krebs cycle to lactate production under oxygen conditions. Cancer cells can also increase glutamine metabolism to promote cell proliferation.
To find the right assay for studying metabolism, explore our cellular metabolism assays.
|Hypoxia||HIF1α/ HIF2a / HIF1β||HIF is a heterodimeric DNA binding transcription factor that regulates a broad range of cellular systems to hypoxia.|
|CAIX||CAIX is a mediator of hypoxia-induced stress response in a cancer cell.|
|AP-1/c-jun||The AP-1 transcription factor family is important in tumor progression and development.|
|GLUT-1||GLUT1 levels can be elevated in hypoxia and can be used to indicate the degree of hypoxia.|
|Glycolysis||Tomm20||TOMM20 and GAPDH have been shown to be upregulated in various types of cancer, and it is necessary to metabolize glutamine.|
|V-ATPase||V-ATPase expression is shown to be upregulated in cancer cells.|
|GAPDH||GAPDH and Tom20 have been shown to be upregulated in various types of cancer and can be used as a marker.|
|Mitochondrial metabolism||COX IV||COX IV is used as a marker for the inner mitochondrial marker.|
|VDAC1/Porin||VDAC1/Porin is used as a marker for the outer mitochondrial marker.|
|ATPase Beta||The beta subunit has a crucial role in the structural and functional maturation of Na+/K+-ATPase.|
Growth of the vascular network is important for metastasis as cancer cells require a sufficient supply of nutrients and oxygen, as well as a means of waste removal. This is achieved by angiogenesis and lymphangiogenesis, respectively.
The human immune system protects against foreign pathogens and diseases, but it also plays an important role in clearing the body’s unhealthy and ailing cells. As such, the immune system can also recognize and eliminate cancer cells.
T cells can selectively recognize and kill pathogens or unhealthy cells by orchestrating a coordinated immune response that encompasses innate and adaptive responses.
Explore our resources on cancer immunotherapy: an overview of various strategies used in immunotherapy, our immune checkpoint pathway, tools to study tumor resistance, and the webinar on immune evasion in ovarian cancer.
Signaling within the tumor microenvironment (TME) operates to hijack the immune cells to promote tumor survival. The immune cells in the TME secrete factors that allow growth and metastasis rather than recognizing and destroying the cancerous cells.
Important inflammatory mechanisms corrupted by the tumor include NF-κB, immune checkpoint signaling, and inflammasome signaling. The inflammasome promotes the cleavage of caspase-1 and subsequent cleavage of pro-inflammatory cytokines IL-1β and IL-18.
|NF-κB signaling||NF-κB||NF-κB is a transcription factor that plays an important role in the regulation of cytokines. Dysregulation of NF-κB is linked to inflammatory, autoimmune diseases and cancer.|
|IKK Beta||IKK beta is part of the IKK complex, a negative regulator of transcription factor NF-κB.|
|Tumor-associated macrophages||CD68||CD68 is a key marker for recognizing M1 and M2 macrophages in tumor tissue.|
|CD163||CD163 is a scavenger receptor upregulated in macrophages in an anti-inflammatory environment.|
|iNOS||iNOS is one of the major markers of M1 tumor-associated macrophages.|
Normal cells depend on tightly-regulated cell cycle control to proliferate and maintain tissue homeostasis. This cycle is disrupted in cancer. Cancer cells release and respond to growth factors to stimulate growth, such as epidermal growth factor (EGF/ EGFR signaling).
Cell proliferation can be used to assess normal cell health, to measure responses to toxic insults, or as a prognostic and diagnostic tool in several cancers. The available markers typically look at DNA levels or synthesis, cellular metabolism, or proliferation-specific proteins. See our cell proliferation guide for the most common methods to mark and score cell proliferation.
Tissue invasion is the process of allowing tumor cells to expand into nearby tissues. Metastasis is the process of tumor cells migrating from the primary tumor site to a new distant location and establishing secondary tumors. The well-documented epithelial-to-mesenchymal transition is a key process in these mechanisms, allowing uninhibited cell division and metabolic adaptations that enable cell survival under nutrient-limiting and stress conditions.
These cancer mechanisms involve extensive changes to cell-cell and cell-matrix interactions and cellular transformation to allow invasion and migration, including targets such as Collagen and CEACAM1.
|ECM||Hyaluronan||Hyaluronan is a glycosaminoglycan found in the extracellular matrix (ECM). HA is dramatically increased in most malignancies.|
|Versican||Versican is expressed by cancer cells or stromal cells and plays a wide role in invasion and metastasis.|
|Collagen IV||Collagen IV is essential for tumor angiogenesis by modulating cell growth and proliferation.|
|Adhesion molecules||CEACAM1||CEACAM1 is down-regulated in several cancers. L-Form CEACAM1 has tumor suppressive function, and its dysregulation is found in the early carcinogenic process.|
|DCC||DCC is a transmembrane receptor for netrins. It promotes apoptosis in the absence of netrin ligands.|
|E-Cadherin||E-Cadherin regulates morphogenic processes like cell-cell recognition, cytoskeleton regulation, and surface adhesion.|
|Secreted factors||Tenascin C||Tenascin C interacts with ECM proteoglycans. It can interfere with the tumor suppressor activity of fibronectin.|
|Fibrinogen||Fibrin deposits occur in the stroma of many cancer types and affect the progression of tumor cells|
|Periostin||Periostin is a secreted adhesion-related protein expressed in the periosteum and periodontal ligaments and plays a role in tumorigenesis.|
In the latest 2022 review, Hanahan proposes four emerging hallmarks and enabling characteristics: unlocking phenotypic plasticity, nonmutational epigenetic reprogramming, polymorphic microbiomes, and senescent cells3. He reviews existing evidence for the proposed emerging hallmarks, suggesting they might become incorporated into the core hallmarks of cancer in the future.
Cancer cells unlock phenotypic plasticity - a capability restricted in normal cells – to enable different versions of disrupted differentiation, which can, in turn, facilitate cancer initiation and progression. Evidence suggests that phenotypic plasticity is a critical component of cancer pathogenesis3. Hanahan argues that such plasticity represents a discrete hallmark capability that differs in regulation and cellular phenotype from the previously established core hallmarks of cancer3.
In his review, he distinguishes three main types of phenotypic plasticity: dedifferentiation of mature cells back to progenitor states, blocked differentiation to freeze cancer cells in progenitor/stem cell states, and transdifferentiation into alternative cell lineages. All these plasticity types occur in multiple cancer types during tumor formation, progression, and therapy response.
Colon cancer is a typical example of dedifferentiation plasticity, with two transcription factors, HOXA5 and SMAD4, being highly expressed in differentiating epithelial cells and lost in advanced colon carcinomas. Examples of blocked differentiation include retinoic acid α nuclear receptor (RAR alpha) in acute promyelocytic leukemia, HDAC in acute myeloid leukemia, SOX10 in melanoma, and alpha-ketoglutarate in pancreatic cancer3. Finally, transdifferentiation is well described in pancreatic ductal adenocarcinoma (PDAC), where pancreatic acinar cells transdifferentiate into ductal cell phenotype – the process involving two transcription factors, PTF1A and MIST1.
Hanahan introduces the term “nonmutational epigenetic reprogramming” – an independent mechanism of genome reprogramming based on epigenetically regulated changes in gene expression3. This well-established epigenetic mechanism mediates embryonic development, tissue differentiation, and homeostasis.
Non-mutational epigenetic reprogramming can occur through microenvironmental mechanisms, such as hypoxia or epithelial-to-mesenchymal transition (EMT). Thus, hypoxia can lead to hypermethylation by reducing the activity of TET demethylases. EMT is responsible for mediating the reversible induction of cancer cell invasiveness at the borders of solid tumors. To better understand how EMT contributes to tumor progression, watch our EMT webinar with a leading cancer researcher, Professor Jean Paul Thierry.
Epigenetic regulatory heterogeneity plays an important role in non-mutational epigenetic reprogramming. A great example is the linker histone H1.0, which is dynamically expressed and repressed in subpopulations of cancer cells in several cancer types. Cancer cell populations with repressed H1.0 exhibit stem cell characteristics and an increased cancer-initiating capacity; they are also linked to poor prognosis in patients4.
All these examples support the hypothesis that epigenetic reprogramming can assist in the acquisition of hallmarks of cancer during cancer development and progression.
To read more about the role of various epigenetics targets and pathways in cancer, please refer to our cancer epigenetics guide.
Polymorphic variability in microbiomes – collections of microorganisms residing within our bodies – can significantly influence cancer phenotypes, development, and progression.
Hanahan builds the case for polymorphic microbiomes to represent a discrete enabling characteristic that impacts the acquisition of hallmarks of cancer, facilitating or protecting against different cancer types3. The most significant line of evidence involves the study showing the existence of cancer-protective and cancer-promoting microbiomes, which can modulate the incidence and pathogenesis of colon cancer. Also, the gut microbiome composition influences the immune system, affecting anti-tumor immunity and response to immunotherapy in patients with melanoma5.
Interestingly, the emerging hallmark of polymorphic microbiomes appears to intersect with those established hallmarks of genome instability and mutation and tumor-promoting inflammation.
Cellular senescence is an irreversible mechanism of cell cycle arrest, which likely developed as a safeguard to maintain tissue homeostasis. This mechanism shuts down the cell division cycle, initiates cell morphology and metabolism changes, and activates senescence-associated secretory phenotype (SASP). Senescence can be triggered by various external and internal stimuli, including nutrient deprivation, DNA damage, damage to organelles and cellular infrastructure, and imbalance in cellular signaling.
Although senescent cells normally act as a defense against neoplasia, in some instances, they may stimulate tumor development and progression. Thus, SASP cytokines and growth factors released by senescent cells can be tumor suppressive or oncogenic in different contexts, cell types, and tumors6. In the latest review, Hanahan makes a convincing proposal to consider adding senescent cells to the functionally significant cells of TME.