What is Ubiquitination?
Ubiquitination is the process by which a small protein called ubiquitin is covalently attached to target proteins. This modification often marks proteins for degradation by the proteasome, a large protease complex responsible for breaking down unwanted or damaged proteins. The ubiquitin molecules are added in chains, with polyubiquitin chains being the most common signal for degradation.
Beyond protein degradation, ubiquitination also regulates protein activity, localization, and interactions, influencing numerous cellular processes like DNA repair, signal transduction, and cell cycle progression. Dysregulation of ubiquitination can lead to disease, including cancer, by altering these crucial cellular functions.
Ubiquitin-Proteasome System in Cancer
The UPS is central to regulating protein turnover in cells, and its components are tightly controlled to maintain cellular homeostasis. In cancer, alterations in the UPS can either enhance the survival of tumor cells or facilitate their uncontrolled growth. For example, the overexpression of proteasomal subunits or downregulation of deubiquitinating enzymes (DUBs) may provide cancer cells with a survival advantage, enabling them to resist apoptosis and proliferate uncontrollably.
Ubiquitination also plays a vital role in signal transduction pathways, such as those involving growth factors, oncogenes, and tumor suppressors. Through the UPS, cells can adjust their signaling networks, contributing to processes like tumorigenesis, metastasis, and immune evasion.
Ubiquitination and Cancer Pathogenesis
Ubiquitination of Tumor Suppressor Proteins
Tumor suppressor proteins are key guardians of cellular integrity, primarily by regulating cell cycle progression, DNA repair, and apoptosis. Their loss or inactivation is a hallmark of cancer, and their dysfunction is often caused by altered ubiquitination. One of the most well-studied tumor suppressors affected by ubiquitination is p53, a critical protein involved in detecting DNA damage and activating cell cycle checkpoints.
p53 and Ubiquitination
Under normal conditions, p53 levels are tightly controlled by the MDM2 E3 ubiquitin ligase, which facilitates the attachment of polyubiquitin chains to p53, marking it for degradation by the proteasome. This regulation ensures that p53 does not accumulate in healthy cells, where its activity could lead to unnecessary cell cycle arrest or apoptosis. However, in response to DNA damage or other cellular stresses, p53 is stabilized through post-translational modifications, including phosphorylation and acetylation, which disrupt its interaction with MDM2, preventing its ubiquitination and degradation.
In many cancers, MDM2 gene amplification or mutations lead to hyperactivation of MDM2, causing excessive degradation of p53 even in the presence of DNA damage. This allows cancer cells to bypass growth arrest or apoptosis, contributing to uncontrolled cell proliferation and genomic instability. Furthermore, alterations in the regulation of MDM2 itself, such as mutations that reduce its binding affinity to p53 or aberrant expressions of other E3 ligases, can prevent proper regulation of p53, facilitating tumorigenesis. In fact, MDM2-p53 interactions are currently a major target for therapeutic strategies aimed at reactivating p53 in tumors.
Other Tumor Suppressors and Ubiquitination
Beyond p53, other tumor suppressors like RB (retinoblastoma protein) and APC (adenomatous polyposis coli) are also regulated by ubiquitination. For instance, RB, which controls the G1 to S phase transition in the cell cycle, is inactivated in many cancers through phosphorylation-induced ubiquitination, leading to its degradation. Similarly, the APC protein, which regulates chromosomal segregation and the degradation of beta-catenin, is often inactivated in colon cancer through mutations that prevent its normal ubiquitin-mediated degradation function.
Ubiquitination of Oncoproteins
Oncoproteins are proteins that promote tumorigenesis when overexpressed or mutated. These proteins often function as positive regulators of cell cycle progression, survival, and proliferation. Ubiquitination regulates the turnover and activity of key oncoproteins, and alterations in this process can significantly contribute to cancer development.
Ubiquitination of c-Myc
c-Myc, a transcription factor and one of the most well-known oncoproteins, is regulated by ubiquitination to control its levels and activity. In normal cells, c-Myc undergoes ubiquitination by E3 ligases like FBW7, marking it for degradation by the proteasome. This regulation ensures that c-Myc is kept at appropriate levels to avoid excessive cell proliferation. However, in cancer, mutations in FBW7 or deregulation of other ubiquitin ligases can lead to the stabilization and overexpression of c-Myc. This uncontrolled activation of c-Myc is implicated in various cancers, including leukemias, lymphomas, and solid tumors, where it drives unchecked cell growth and survival.
Ras Oncoprotein and Ubiquitination
The Ras family of small GTPases, including HRas, KRas, and NRas, plays a pivotal role in cell signaling by regulating cell proliferation, differentiation, and survival. Mutations in Ras, particularly in KRas, are found in a significant number of cancers, including pancreatic, colorectal, and lung cancers. Ubiquitination modulates the turnover and activity of Ras proteins. The E3 ligase Cbl regulates Ras by promoting its ubiquitination and subsequent degradation, preventing persistent Ras signaling. In cancer, mutations in Ras or dysregulation of its ubiquitination pathways often lead to constitutive activation of Ras signaling, contributing to tumor growth, metastasis, and resistance to apoptosis.
Ubiquitination and Cell Cycle Dysregulation
The proper progression of the cell cycle is essential for maintaining genomic integrity. Ubiquitination serves as a critical mechanism for regulating cell cycle progression, particularly by controlling the activity and stability of cyclins, cyclin-dependent kinases (CDKs), and their inhibitors. Aberrant regulation of these molecules due to defects in ubiquitination can lead to unregulated cell division, a key feature of cancer.
Cyclins and Ubiquitination
Cyclins are proteins that activate CDKs, driving the cell cycle through its various phases. Ubiquitination regulates cyclin degradation, which is essential for transitioning between cell cycle phases. For example, cyclin E, a key regulator of the G1 to S phase transition, is targeted for degradation by the E3 ligase APC/C, which prevents the cell from prematurely entering S phase. In cancers, the dysregulation of this ubiquitin-mediated degradation often results in overexpression of cyclins or their stabilization, driving continuous cell cycle progression even in the absence of proper signals. The overactivation of cyclins, particularly cyclins D and E, is commonly seen in breast, lung, and colorectal cancers.
CDK Inhibitors and Ubiquitination
On the flip side, the regulation of CDK inhibitors, such as p21 and p27, also involves ubiquitination. These inhibitors normally act to block CDK activity and prevent unchecked cell cycle progression. Ubiquitination, often mediated by SCF (Skp1-Cullin-F-box) complex, targets these inhibitors for degradation, allowing cells to progress through the cycle. In cancer, the downregulation of CDK inhibitors through overactive ubiquitination can promote tumor growth by allowing uncontrolled cell cycle entry and progression.
Ubiquitination and DNA Damage Response
Ubiquitination plays an essential role in the DNA damage response (DDR), which ensures that cells either repair damaged DNA or undergo apoptosis if the damage is irreparable. In response to DNA damage, proteins involved in DDR, such as ATM, ATR, and BRCA1, are ubiquitinated to facilitate the activation of repair mechanisms. The dysregulation of these repair proteins due to defective ubiquitination can lead to the accumulation of DNA damage, genome instability, and tumorigenesis.
For example, BRCA1, a key tumor suppressor involved in DNA repair, is regulated by ubiquitination. Mutations that affect its ubiquitination pathway can lead to its degradation or improper function, impairing DNA repair and increasing susceptibility to breast and ovarian cancers. Similarly, the ubiquitination of p53 in the context of DNA damage dictates whether the cell will undergo repair or apoptosis, highlighting the intertwined roles of ubiquitination in both tumor suppressor regulation and the maintenance of genome integrity.
The processes of ubiquitination and deubiquitination occur within the ubiquitin–proteasome system (UPS) (Liu et al., 2024).
Ubiquitination and Cancer Biomarkers
Ubiquitination-Related Proteins as Cancer Biomarkers
Abnormal expression or dysfunction of ubiquitination-related proteins is closely associated with various types of cancer. These proteins include E3 ligases, deubiquitinases (DUBs), and ubiquitination substrates such as tumor suppressor proteins and oncoproteins. The specific alterations in these proteins within cancer make them potential biomarkers for diagnosis, prognosis, and treatment.
E3 Ligases
E3 ligases are key enzymes in the ubiquitination process responsible for attaching ubiquitin molecules to target proteins. Some E3 ligases are abnormally overexpressed or dysfunctional in cancer, leading to the excessive degradation of tumor suppressors or stabilization of oncoproteins. Examples include:
- MDM2: MDM2 is the primary E3 ligase for p53, and its overexpression leads to the ubiquitination and degradation of p53, thereby promoting tumorigenesis. High expression of MDM2 has been associated with poor prognosis in various cancers, including breast cancer, lung cancer, and colorectal cancer.
- SKP2: SKP2 is an E3 ligase that targets the cell cycle inhibitor p27 for degradation, thereby promoting cell proliferation. High expression of SKP2 has been linked to increased invasiveness and poor prognosis in multiple cancers, such as prostate cancer and gastric cancer.
Deubiquitinases (DUBs)
Deubiquitinases (DUBs) stabilize target proteins by removing ubiquitin molecules. Their role in cancer is also critical, as some DUBs are abnormally activated, leading to the stabilization of oncoproteins or the inactivation of tumor suppressors. Examples include:
- USP7: USP7 stabilizes MDM2 and p53 through deubiquitination. Overexpression of USP7 has been implicated in the progression of various cancers.
- USP9X: USP9X stabilizes MCL-1, an anti-apoptotic protein, by removing ubiquitin. Overexpression of USP9X is associated with chemotherapy resistance in several cancers.
Ubiquitination Substrates
The aberrant ubiquitination of substrates, such as p53, PTEN, and MYC, can also serve as cancer biomarkers. Examples include:
- p53 Ubiquitination Status: The level of p53 ubiquitination is closely related to cancer invasiveness and treatment response. Over-ubiquitination of p53 is generally associated with the loss of its tumor suppressor function.
- PTEN Ubiquitination: PTEN is a critical tumor suppressor protein, and its ubiquitination and degradation are linked to the development of various cancers, such as prostate cancer and glioblastoma.
Detection Methods for Ubiquitination Biomarkers
Several technologies have been developed to detect ubiquitination-related biomarkers, each with its own advantages and limitations. Traditional methods like Western blotting and immunohistochemistry can be used to assess the presence of ubiquitin-conjugated proteins.
Mass spectrometry (MS) has become a powerful technique for detecting ubiquitination biomarkers in cancer due to its high sensitivity and specificity. By using selective enrichment strategies, such as ubiquitin-binding domain-based affinity capture, MS can identify ubiquitin-modified peptides in complex samples. Once isolated, the peptides are analyzed by MS, which measures their mass-to-charge ratio to detect ubiquitination sites. Fragmentation techniques like MS/MS allow for precise identification of ubiquitin modifications on specific lysine residues, offering insights into the role of ubiquitination in cancer-related processes such as tumorigenesis and metastasis. This method provides not only the identification of ubiquitination sites but also quantification, enabling the study of differential ubiquitination patterns between cancerous and normal tissues.
Ubiquitination and Cancer Therapy
Targeting Ubiquitination Pathways for Cancer Treatment
Given the central role of ubiquitination in cancer biology, targeting the UPS represents an exciting therapeutic strategy. Researchers are exploring drugs that can modulate the activity of specific E3 ligases, DUBs, or the proteasome itself to treat cancer. For example, small molecules that inhibit MDM2, thereby stabilizing p53, are currently being evaluated in clinical trials as potential cancer treatments.
The potential of targeting the UPS in cancer therapy lies in its ability to selectively regulate the degradation of oncogenic proteins while stabilizing tumor suppressors, offering a more precise approach than traditional chemotherapy.
Proteasome Inhibitors
Proteasome inhibitors, such as Bortezomib, have already been approved for the treatment of certain cancers, including multiple myeloma. These drugs work by blocking the proteasome's ability to degrade proteins, leading to the accumulation of misfolded or damaged proteins, which triggers apoptosis in cancer cells. While effective, proteasome inhibitors come with side effects, including neurotoxicity and immune suppression, limiting their broader application.
Deubiquitinase (DUB) Inhibitors
DUB inhibitors are an emerging class of drugs that target the enzymes responsible for removing ubiquitin from proteins. By inhibiting DUBs, these drugs can prevent the deubiquitination and stabilization of oncoproteins, thus promoting their degradation. Research into DUB inhibitors is still in its early stages, but they hold promise as a novel therapeutic approach in cancer treatment.
Ubiquitination and Protein Degradation in Cancer
The Ubiquitin-Proteasome System (UPS) in Cancer
The UPS is responsible for the degradation of most intracellular proteins, ensuring cellular homeostasis. This system involves a cascade of events beginning with the attachment of ubiquitin molecules to target proteins, followed by their recognition and degradation by the 26S proteasome. The E3 ligases are key regulators in this process, determining the specificity of substrate recognition, while the proteasome is responsible for executing the degradation.
In cancer, several key components of the UPS are frequently altered. E3 ligases can become overactive, leading to the excessive degradation of tumor suppressors, or they may fail to degrade oncoproteins, leading to their accumulation. Similarly, the proteasome itself may become hyperactivated, removing regulatory proteins that would otherwise inhibit cancer progression.
Ubiquitination and Tumor Suppressors
Many tumor suppressor proteins are tightly regulated by ubiquitination, and their degradation is often critical for maintaining normal cell function. However, in cancer, the degradation of tumor suppressors is frequently deregulated, contributing to the loss of tumor suppressor function and promoting tumorigenesis.
- p53: p53, a key tumor suppressor, regulates cell cycle arrest and apoptosis. Overexpression of MDM2 in cancer leads to excessive degradation of p53, promoting tumor growth.
- PTEN: PTEN inhibits the PI3K/AKT pathway. Its degradation by ligases like HECTD1 or MDM2 in cancer contributes to tumorigenesis, especially in prostate and glioblastoma cancers.
- RB: RB controls the G1 to S phase transition. Dysregulated degradation in cancer leads to uncontrolled cell cycle progression, driving tumor growth.
Ubiquitination and Oncoproteins
In contrast to tumor suppressors, oncoproteins are typically stabilized in cancer cells through alterations in the ubiquitination machinery. These proteins drive processes such as cell cycle progression, survival, and angiogenesis. By escaping normal degradation pathways, oncoproteins accumulate to levels that promote cancer cell proliferation and metastasis.
- c-Myc: c-Myc is a transcription factor that promotes cell proliferation by driving the expression of genes involved in cell cycle progression and metabolism. The stability of c-Myc is regulated by ubiquitination, with E3 ligases like FBW7 playing a major role in targeting it for degradation. In many cancers, mutations in FBW7 or other components of the ubiquitination machinery lead to the stabilization of c-Myc, allowing it to drive tumorigenesis in leukemia, lymphoma, and solid tumors like breast cancer.
- Ras: Ras proteins are small GTPases that regulate several signaling pathways critical for cell survival and proliferation. In their mutated, constitutively active forms, Ras proteins are often resistant to ubiquitination-mediated degradation, leading to prolonged activation of downstream signaling pathways such as MAPK and PI3K. Mutations in Ras are common in cancers like pancreatic, lung, and colorectal cancers. The dysregulation of Ras degradation through impaired ubiquitination contributes to the aberrant signaling that drives tumorigenesis.
Ubiquitination and Cell Cycle Control
The cell cycle is tightly regulated by a network of cyclins, cyclin-dependent kinases (CDKs), and their inhibitors, many of which are subjected to ubiquitination-dependent degradation. Abnormalities in the regulation of these proteins can lead to uncontrolled cell division, a hallmark of cancer.
- Cyclins and CDKs: Cyclins are regulatory subunits that activate CDKs, driving progression through different phases of the cell cycle. The degradation of cyclins at appropriate points in the cell cycle ensures proper transition between phases. Ubiquitination is a key mechanism for regulating cyclin degradation. The APC/C complex, for example, targets cyclins like cyclin A and cyclin B for degradation at the metaphase-anaphase transition. In many cancers, cyclins are overexpressed or stabilized due to defects in their ubiquitination pathways, resulting in unregulated cell cycle progression.
- CDK Inhibitors: CDK inhibitors like p21 and p27 act to block CDK activity and prevent unscheduled cell cycle progression. Ubiquitination regulates the levels of these inhibitors. For example, the SCF complex targets p27 for degradation to allow cell cycle progression. Loss of p27 expression, often through deregulated ubiquitination, is a frequent finding in aggressive cancers, such as breast and prostate cancers.
Autophagy and Ubiquitination
While the proteasome system is the primary pathway for degrading most cellular proteins, autophagy is another important protein degradation mechanism. Autophagy allows the degradation of long-lived proteins, organelles, and other cellular debris through the lysosome. In cancer, the regulation of both proteasomal degradation and autophagy is often disrupted, leading to altered cellular homeostasis. Ubiquitination plays a critical role in directing substrates to autophagic degradation. For example, the p62 protein, which is involved in autophagy, binds to polyubiquitinated proteins and helps shuttle them to the autophagosome for degradation.
In cancer, autophagy can act as a double-edged sword. On one hand, autophagy supports tumor growth by helping cancer cells cope with stress, such as nutrient deprivation. On the other hand, inhibiting autophagy can lead to the accumulation of damaged proteins and organelles, contributing to cancer cell death. Therefore, strategies to modulate both the proteasome and autophagy systems are being explored for cancer therapy.
Reference
- Liu, Fangfang, et al. "Ubiquitination and deubiquitination in cancer: from mechanisms to novel therapeutic approaches." Molecular Cancer 23.1 (2024): 148. https://doi.org/10.1186/s12943-024-02046-3
