Dysfunction of the TP53 tumor suppressor gene in lymphoid malignancies

Abstract

Mutations of the TP53 gene and dysregulation of the TP53 pathway are important in the pathogenesis of many human cancers, including lymphomas. Tumor suppression by p53 occurs via both transcription-dependent activities in the nucleus by which p53 regulates transcription of genes involved in cell cycle, DNA repair, apoptosis, signaling, transcription, and metabolism; and transcription-independent activities that induces apoptosis and autophagy in the cytoplasm. In lymphoid malignancies, the frequency of TP53 deletions and mutations is lower than in other types of cancer. Nonetheless, the status of TP53 is an independent prognostic factor in most lymphoma types. Dysfunction of TP53 with wild-type coding sequence can result from deregulated gene expression, stability, and activity of p53. To overcome TP53 pathway inactivation, therapeutic delivery of wild-type p53, activation of mutant p53, inhibition of MDM2-mediated degradation of p53, and activation of p53-dependent and -independent apoptotic pathways have been explored experimentally and in clinical trials. We review the mechanisms of TP53 dysfunction, recent advances implicated in lymphomagenesis, and therapeutic approaches to overcoming p53 inactivation.

Figure 1

Schematic structure of TP53 and p53, and numbers of mutations in exons in lymphoid malignancies. (A) TP53 gene structure, p53 functional domains, and posttranslational modifications. Exons are in blue (UTRs) or green (CDS) and are drawn proportionally to their sizes; introns are dark blue and not drawn to scale. Sizes of exons/introns are according to NCBI (reference NC_000017.10 sequence). Domains of p53 include transactivation domain (TAD), proline-rich domain (PRD), DBD, nuclear localization sequence (NLS), oligomerization domain (OD), and basic/repression (BR) of DBD. Both the TAD and OD have a nuclear export signal (NES). Posttranslational modification of p53 can occur by phosphorylation (P), acetylation (A), ubiquitination (U), methylation (M), neddylation (N), or sumoylation (S). (B) Schematic of p53 protein structure. Shown are positions in the p53 primary sequence for 3 loops (L1, L2, L3) involved in DNA binding, 11 β-strands (S1-S10) as components of 2 anti-parallel β-sheets, and 3 α-helices, including 2 in the helix-loop-helix motif. (C) TP53 CDS mutation numbers in lymphoid malignancies. These mutations are not randomly distributed, as indicated by the finding that mutation numbers (shown on right side and illustrated by the length of red bars) in each exon are not proportional to exon sizes (on the left side). Mutation numbers (unique mutation variants and sample/mutation associations) are according to the IARC TP53 database (R15 release, November 2010).

Figure 2

TAs and TIAs of p53 in lymphocytes. TAs are those that p53 activates or represses in nucleus by binding directly or indirectly to target genes. TIAs include regulation of the intrinsic apoptosis pathway and autophagy through protein-protein interactions in the cytoplasm. Ub indicates ubiquitination.

Figure 3

Illustration of p53 TAs in lymphocytes. TAs of p53 transactivate or transrepress hundreds of target genes, whose products are depicted according to their main functions. Downstream events fulfill the tumor suppression function with apoptosis, cell-cycle arrest, DNA repair, senescence, or autophagy as consequences. In the diagram, green hyphenated lines with arrows indicate up-regulation of gene expression; and red hyphenated lines, down-regulation of gene expression. For the downstream events, proteins/effectors are grouped according to their major functions and subcellular locations. Up-regulated effectors are marked in bold and depicted in colors representing functional groups, whereas down-regulated effectors are not in bold and are all depicted in blue.

Figure 4

Illustration of p53 TIAs in lymphocytes. TIAs of p53, via protein-protein interactions with Bcl-2 family members and the mTOR pathway, induce mitochondria-mediated apoptosis and inhibit autophagy. The mechanism for p53-mediated apoptosis is clear, whereas the autophagic role of p53 is not well elucidated. Under stress, PUMA sequestrates Bcl-2 and Bcl-X(L), which associate with cytoplasmic p53. Within 30 minutes, cytoplasmic p53 and proapoptotic proteins translocate to mitochondria. In mitochondria, p53 releases BAX, BAK, BIM, and BID from Bcl-2, Bcl-X(L), BCL2A1, and Mcl-1; BIK, BAD, and Noxa act in similar ways as p53. Their interactions with antiapoptotic Bcl-2, Bcl-X(L) BCL2A1, and Mcl-1 in the cytoplasm are also shown. In addition, transient association of p53 with tBid (BID cleaved by caspase-8 as a result of death-inducing signaling complex activation in extrinsic apoptosis pathway; not shown) or with BAX, BAK, BID, and BAD activates these proapoptotic proteins through structural changes. BAX, BAK, and BID form homo- or hetero-oligomers and trigger mitochondrial outer membrane permeabilization (MOMP) and consequently cytochrome c (cyt c) release. Cyt c then associates with APAF-1, together activating the downstream caspase cascade leading to apoptosis, preceding a second wave of apoptosis triggered by p53 transcription-dependent activities. The effectors in the mitochondrial pathway of apoptosis might vary in different cells by different stimuli. Green arrows indicate positive protein-protein interactions, and red lines, negative protein-protein interactions. Ub indicates ubiquitination; and VDAC1, voltage-dependent anion-selective channel protein 1.

Figure 5

Regulation and dysregulation of TP53 function implicated in lymphomagenesis. (1) At the DNA level, dysregulation of transcription factors and hypermethylation can silence gene expression. (2) At the RNA level, posttranscriptional regulation events include alternative splicing that produces p53 isoforms with altered function, mRNA stability/degradation, and translational regulation. (3) At the protein level, posttranslational modification, redox regulation, and p53 regulators affect p53 stability and function in the nucleus and cytoplasm. (4) p53-independent pathways, including PI3K/Akt affects p53-dependent apoptotic pathway. (5) Autophagy caused by ER stress, starvation, and other forms of stress inhibits p53-dependent apoptosis in most cases. Conversely, cytoplasmic p53 inhibits autophagy by promoting the mTOR pathway, whereas nuclear p53 stimulates autophagy by transactivating genes involved in autophagy. MOMP indicates mitochondrial outer membrane permeabilization; and U or Ub, ubiquitination.

Figure 6

The prognostic significance of TP53 mutations in patients with DLCBL. (A) OS (in years) after treatment with the cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) regimen among patients with a p53 mutation versus those with WT-p53. (B) OS (in months) after treatment with the rituximab-CHOP regimen among patients with p53 mutations versus those with WT-p53. (C) OS (in years) after CHOP treatment among patients with a TP53 DNA-binding domain mutation versus those with WT-p53. (D) Location of critical p53 residues in the p53 domain model designed from the published crystal structure. The mutations depicted are associated with poor outcome identified from our group study in 1187 DLBCL cases. The residues are color-coded as follows: green represents mutation from or to proline; deep blue, residues close to zinc sites; light blue, residues close to DNA-binding sites; brown, residues far from both zinc and DNA; and orange, cysteine residues implicated in the oxidation-reduction activity of p53.

Figure 7

Posttranslational regulation of p53 and regulation of p53-effectors. (A) Regulation of p53 transcriptional activities and stability. Under stress conditions, p53 protein is stabilized and activated. Various proteins (mostly shown on the left) and posttranslational modifications (mostly shown on the right) regulate p53 degradation and p53 TAs. Generally, acetylation (eg, by p300, CBP) and phosphorylation (eg, by CK2, Chk2) inhibit ubiquitination, stabilize and enhance p53 activity, whereas neddylation (eg, by FBXO11, NEDD) increases p53 stability but suppresses p53 function. However, association with p300 is required for MDM2-mediated polyubiquitination and degradation of p53. Methylation on different residues has different effects on p53. The methyltransferase SETD7 increases p53 stability; SETD8 inhibits p53 activity. Controversially, Setd7 is dispensable for the p53 function (cell-cycle arrest or apoptosis) in vivo. Effect of sumoylation is also controversial. Acetylation state of SUMO-1 affects its activity toward p53 stability. Ubiquitination decreases p53 stability and activity. MDM2 mediates p53 degradation and inhibits p53 acetylation and activity. NUB1 decreases neddylation by NEDD8 and stimulates p53 ubiquitination, promotes cytoplasmic localization of p53, and inhibits p53 TA. The atypical ubiquitin ligase E4F1 has no effect on p53 degradation or localization but modifies p53 transcriptional program by enhancing cell-cycle arrest and not apoptosis. In the diagram, solid green arrows indicate positive regulation; solid red lines, negative regulation; and hyphenated green arrows, up-regulation of gene expression. (B) Downstream dysregulation of the TP53 pathway by p53-independent pathways, including the PI3K/Akt and NF-κB pathways. The PI3K/Akt pathway counters both the p53-mediated extrinsic (designated as 1: by inhibiting Fas/CD95 death-inducing signaling complex activation without affecting Fas expression) and intrinsic (designated as 2: by increasing antiapoptotic gene expression and decreasing PUMA expression; designated as 4: by suppressing the metabolic up-regulation of PUMA, decreasing PUMA stability, and inhibiting BIM cytotoxicity) apoptotic pathways, and activates the NF-κB pro-survival function (designated as 3: by phosphorylation of NF-κB inhibitor IκBα). DISC indicates death-inducing signaling complex.

Figure 8

Therapeutic modulation of the TP53 pathway. Strategies to activate p53 functions or p53-independent apoptotic pathways have been explored in p53-wt or p53-mut cancer cells. (1) Induction and activation of p53 by stress within the nucleus, including DNA damage caused by alkylating agents, DNA-intercalating agents, base analogs, irradiation, and ROS. Mitotic inhibitors and cell cycle-mediated drugs also effectively activate p53. (2) Therapeutic gene delivery of p53 and pharmacologic activation of p53 mutant. (3) Antagonism of MDM2-mediated degradation by MDM2 inhibitors and proteasome inhibitors. (4) Activation of the extrinsic apoptotic pathway by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and agonistic anti-TNF antibodies, or CFLAR/c-Flip inhibitors. (5) Enhancement of the intrinsic mitochondrial pathway of apoptosis by targeting Bcl-2 and IAP family members, or by directly activating caspases or BAX/BAK. (6) Induction of p53-independent apoptosis by various compounds and agents, mostly via the mitochondrial pathway and ROS generation. (7) Inhibition of survival pathways, including NF-κB, PI3K/Akt, and autophagy. (8) Induction of apoptosis by Cr(VI) through the calcium/Ca²⁺-calpain pathway and mitochondrial pathway induced by oxidative stress. (9) Increased unfolded/misfolded proteins that can be induced by proteasome inhibitors (Syrbactin, bortezomib), and increased intracellular Ca²⁺ concentration that can be induced by TG, can induce ER stress and autophagic cell death in several cancer cells.