It is not all about the alpha: elevated expression of p53ß variants is associated with lower probability of survival in a retrospective melanoma cohort.

BACKGROUND: Melanoma is the deadliest type of skin cancer and despite improvements in treatment outcomes, melanoma claimed 57,043 lives in 2020. In most malignancies, p53 mutation rates are above 50% and provide prognostic indications. However, in melanoma where less than a quarter of cases harbour a p53 mutation, the significance of the tumour suppressor may be questioned. Instead, p53 isoforms, which modulate p53's canonical function, may be of greater clinical importance. METHODS: The expression of p53 isoforms was evaluated in 123 melanoma specimens by immunohistochemistry using p53 isoform-specific antibodies (DO-1, KJC8, KJC40, and KJC133). To determine whether TP53 mutations may be driving p53 isoform expression, TP53 was sequenced in 30 FFPE melanoma samples. RESULTS: The C-terminally truncated p53ß isoforms (KJC8) were found to be the most highly expressed p53 isoforms compared to all other isoforms. Further, elevated KJC8 staining was found to correlate with reduced probability of melanoma-specific survival, while KJC40 staining (Δ40p53) positively correlated with reduced melanoma thickness. TAp53 isoforms (p53 retaining both transactivation domains, DO-1), were the second highest p53 isoforms expressed across all samples. Elevated DO-1 staining was also associated with worse survival outcomes and more advanced stages of cancer. Given that the isoforms are likely to work in concert, composite isoform profiles were generated. Composite biomarker profiles revealed that elevated TAp53 (DO-1) and p53ß (KJC8) expression, accompanied by low Δ40p53 (KJC40) and Δ133p53 (KJC133) expression was associated with the worst survival outcomes. Supporting the lack of predictive biomarker potential of TP53 in melanoma, no clinicopathological or p53 isoform expression associations could be linked to TP53 status. CONCLUSIONS: Given the lack of prognostic biomarker potential derived from TP53 status, this study highlights how p53 isoform expression might progress this field and, pending further validation, may provide additional information to treating oncologists that might be factored into treatment decisions.

Adaptive homeostasis and the p53 isoform network.

All living organisms have developed processes to sense and address environmental changes to maintain a stable internal state (homeostasis). When activated, the p53 tumour suppressor maintains cell and organ integrity and functions in response to homeostasis disruptors (stresses) such as infection, metabolic alterations and cellular damage. Thus, p53 plays a fundamental physiological role in maintaining organismal homeostasis. The TP53 gene encodes a network of proteins (p53 isoforms) with similar and distinct biochemical functions. The p53 network carries out multiple biological activities enabling cooperation between individual cells required for long-term survival of multicellular organisms (animals) in response to an ever-changing environment caused by mutation, infection, metabolic alteration or damage. In this review, we suggest that the p53 network has evolved as an adaptive response to pathogen infections and other environmental selection pressures.

Cytoplasmic p53ß Isoforms Are Associated with Worse Disease-Free Survival in Breast Cancer.

TP53 mutations are associated with tumour progression, resistance to therapy and poor prognosis. However, in breast cancer, TP53's overall mutation frequency is lower than expected (~25%), suggesting that other mechanisms may be responsible for the disruption of this critical tumour suppressor. p53 isoforms are known to enhance or disrupt p53 pathway activity in cell- and context-specific manners. Our previous study revealed that p53 isoform mRNA expression correlates with clinicopathological features and survival in breast cancer and may account for the dysregulation of the p53 pathway in the absence of TP53 mutations. Hence, in this study, the protein expression of p53 isoforms, transactivation domain p53 (TAp53), p53ß, Δ40p53, Δ133p53 and Δ160p53 was analysed using immunohistochemistry in a cohort of invasive ductal carcinomas (n = 108). p53 isoforms presented distinct cellular localisation, with some isoforms being expressed in tumour cells and others in infiltrating immune cells. Moreover, high levels of p53ß, most likely to be N-terminally truncated ß variants, were significantly associated with worse disease-free survival, especially in tumours with wild-type TP53. To the best of our knowledge, this is the first study that analysed the endogenous protein levels of p53 isoforms in a breast cancer cohort. Our findings suggest that p53ß may be a useful prognostic marker.

Loss of the p53 transactivation domain results in high amyloid aggregation of the Δ40p53 isoform in endometrial carcinoma cells.

Dysfunctional p53 formation and activity can result from aberrant expression and subcellular localization of distinct p53 isoforms or aggregates. Endometrial carcinoma (EC) is a cancer type in which p53 status is correlated with prognosis, and TP53 mutations are a frequent genetic modification. Here we aimed to evaluate the expression patterns of different p53 isoforms and their contributions to the formation and subcellular localization of p53 amyloid aggregates in both EC and endometrial nontumor cell lines. We found that full-length (fl) p53 and a truncated p53 isoform, Δ40p53, resulting from alternative splicing of exon 2 or alternative initiation of translation at ATG-40, are the predominantly expressed p53 variants in EC cells. However, Δ40p53 was the major p53 isoform in endometrial nontumor cells. Immunofluorescence assays revealed that Δ40p53 is mainly localized to cytoplasmic punctate structures of EC cells, resembling solid-phase structures similar to those found in neurodegenerative pathologies. Using light-scattering kinetics, CD, and transmission EM, we noted that the p53 N-terminal transactivation domain significantly reduces aggregation of the WT p53 DNA-binding domain, confirming the higher aggregation tendency of Δ40p53, which lacks this domain. This is the first report of cytoplasmic Δ40p53 in EC cells being a major component of amyloid aggregates. The differential aggregation properties of p53 isoforms in EC cells may open up new avenues in the development of therapeutic strategies that preferentially target specific p53 isoforms to prevent p53 amyloid aggregate formation.

High expression of the p53 isoform γ is associated with reduced progression-free survival in uterine serous carcinoma.

BACKGROUND: Uterine serous carcinoma (USC) is a rare but aggressive subtype of endometrial carcinoma. Large-scale comprehensive efforts have resulted in an improved molecular understanding of its pathogenesis, and the p53 pathway has been proposed as a key player and is potentially targetable. Here we attempt to further portray the p53 pathway in USC by assessing p53 isoform expression. METHODS: We applied quantitative Real-Time PCRs (RT-qPCR) for expression analyses of total p53 mRNA as well as quantitative distinction of p53ß, p53γ, and the total mRNA of amino-terminal truncated Δ40p53 and Δ133p53 in a retrospective cohort of 37 patients with USC. TP53 mutation status was assessed by targeted massive parallel sequencing. Findings were correlated with clinical data. RESULTS: The p53 isoform expression landscape in USCs was heterogeneous and dominated by total Δ133p53, while the distinct p53ß and p53γ variants were found at much lower levels. The isoform expression profiles varied between samples, while their expression was independent of TP53 mutation status. We found high relative p53γ expression to be associated with reduced progression-free survival (PFS). CONCLUSIONS: This is the first indication that elevated p53γ expression is associated with reduced PFS in USC. This single-center study may offer some insight in the landscape of p53 isoform expression in USC, but further validation studies are crucial to understand the context-dependent and tissue-specific role of the p53 isoform network in gynecological cancer.

Cancer-specific mutations in p53 induce the translation of Δ160p53 promoting tumorigenesis.

Wild-type p53 functions as a tumour suppressor while mutant p53 possesses oncogenic potential. Until now it remains unclear how a single mutation can transform p53 into a functionally distinct gene harbouring a new set of original cellular roles. Here we show that the most common p53 cancer mutants express a larger number and higher levels of shorter p53 protein isoforms that are translated from the mutated full-length p53 mRNA. Cells expressing mutant p53 exhibit "gain-of-function" cancer phenotypes, such as enhanced cell survival, proliferation, invasion and adhesion, altered mammary tissue architecture and invasive cell structures. Interestingly, Δ160p53-overexpressing cells behave in a similar manner. In contrast, an exogenous or endogenous mutant p53 that fails to express Δ160p53 due to specific mutations or antisense knock-down loses pro-oncogenic potential. Our data support a model in which "gain-of-function" phenotypes induced by p53 mutations depend on the shorter p53 isoforms. As a conserved wild-type isoform, Δ160p53 has evolved during millions of years. We thus provide a rational explanation for the origin of the tumour-promoting functions of p53 mutations.

Activated CD90/Thy-1 fibroblasts co-express the Δ133p53ß isoform and are associated with highly inflamed rheumatoid arthritis.

BACKGROUND: The p53 isoform Δ133p53ß is known to be associated with cancers driven by inflammation. Many of the features associated with the development of inflammation in rheumatoid arthritis (RA) parallel those evident in cancer progression. However, the role of this isoform in RA has not yet been explored. The aim of this study was to determine whether Δ133p53ß is driving aggressive disease in RA. METHODS: Using RA patient synovia, we carried out RT-qPCR and RNAScope-ISH to determine both protein and mRNA levels of Δ133p53 and p53. We also used IHC to determine the location and type of cells with elevated levels of Δ133p53ß. Plasma cytokines were also measured using a BioPlex cytokine panel and data analysed by the Milliplex Analyst software. RESULTS: Elevated levels of pro-inflammatory plasma cytokines were associated with synovia from RA patients displaying extensive tissue inflammation, increased immune cell infiltration and the highest levels of Δ133TP53 and TP53ß mRNA. Located in perivascular regions of synovial sub-lining and surrounding ectopic lymphoid structures (ELS) were a subset of cells with high levels of CD90, a marker of 'activated fibroblasts' together with elevated levels of Δ133p53ß. CONCLUSIONS: Induction of Δ133p53ß in CD90+ synovial fibroblasts leads to an increase in cytokine and chemokine expression and the recruitment of proinflammatory cells into the synovial joint, creating a persistently inflamed environment. Our results show that dysregulated expression of Δ133p53ß could represent one of the early triggers in the immunopathogenesis of RA and actively perpetuates chronic synovial inflammation. Therefore, Δ133p53ß could be used as a biomarker to identify RA patients more likely to develop aggressive disease who might benefit from targeted therapy to cytokines such as IL-6.

Uncoupling elephant TP53 and cancer.

Elephant testicles do not descend, with implications for sperm production being hot enough to compromise germline DNA replication/repair. Uniquely, elephants also possess 20 copies of a gene encoding for the p53 protein. Did elephants evolve multiplication of the TP53 gene complex to protect their germline rather than to fight cancer?

Interaction of C-terminal p53 isoforms depends strongly upon DNA sequence and topology.

The p53 protein is a key tumor suppressor and the most commonly mutated and down-regulated protein in human tumors. It functions mainly through interaction with DNA, and p53 acts as a transcription factor that recognizes the so-called p53 target sites on the promoters of various genes. P53 has been shown to exist as many isoforms, including three C-terminal isoforms that are produced by alternative splicing. Because the C-terminal domain is responsible for sequence-nonspecific binding and regulation of p53 binding, we have analyzed DNA recognition by these C-terminal isoforms. Using atomic force microscopy, we show for the first time that all C-terminal isoforms recognize superhelical DNA. It is particularly noteworthy that a sequence-specific p53 consensus binding site is bound by p53α and ß isoforms with similar affinities, whilst p53α shows higher binding to a quadruplex sequence than both p53ß and p53γ, and p53γ loses preferential binding to both the consensus binding sequence and the quadruplex-forming sequence. These results show the important role of the variable p53 C-terminal amino acid sequences for DNA recognition.

p53 Isoforms as Cancer Biomarkers and Therapeutic Targets.

This review aims to summarize the implications of the major isoforms of the tumor suppressor protein p53 in aggressive cancer development. The current knowledge of p53 isoforms, their involvement in cell-signaling pathways, and their interactions with other cellular proteins or factors suggests the existence of an intricate molecular network that regulates their oncogenic function. Moreover, existing literature about the involvement of the p53 isoforms in various cancers leads to the proposition of therapeutic solutions by altering the cellular levels of the p53 isoforms. This review thus summarizes how the major p53 isoforms Δ40p53α/ß/γ, Δ133p53α/ß/γ, and Δ160p53α/ß/γ might have clinical relevance in the diagnosis and effective treatments of cancer.

Δ133p53α Protects Human Astrocytes from Amyloid-beta Induced Senescence and Neurotoxicity.

Cellular senescence is an important contributor to aging and age-related diseases such as Alzheimer's disease (AD). Senescent cells are characterized by a durable cell proliferation arrest and the acquisition of a proinflammatory senescence-associated secretory phenotype (SASP), which participates in the progression of neurodegenerative disorders. Clearance of senescent glial cells in an AD mouse model prevented cognitive decline suggesting pharmacological agents targeting cellular senescence might provide novel therapeutic approaches for AD. Δ133p53α, a natural protein isoform of p53, was previously shown to be a negative regulator of cellular senescence in primary human astrocytes, with clinical implications from its diminished expression in brain tissues from AD patients. Here we show that treatment of proliferating human astrocytes in culture with amyloid-beta oligomers (Aß), an endogenous pathogenic agent of AD, results in reduced expression of Δ133p53α, as well as induces the cells to become senescent and express proinflammatory SASP cytokines such as IL-6, IL-1ß and TNFα. Our data suggest that Aß-induced astrocyte cellular senescence is associated with accelerated DNA damage, and upregulation of full-length p53 and its senescence-inducing target gene p21WAF1. We also show that exogenously enhanced expression of Δ133p53α rescues human astrocytes from Aß-induced cellular senescence and SASP through both protection from DNA damage and dominant-negative inhibition of full-length p53, leading to inhibition of Aß-induced, astrocyte-mediated neurotoxicity. The results presented here demonstrate that Δ133p53α manipulation could modulate cellular senescence in the context of AD, possibly opening new therapeutic avenues.

p53 and Its Isoforms in Renal Cell Carcinoma-Do They Matter?

p53 is a transcription al factor responsible for the maintenance of cellular homeostasis. It has been shown that more than 50% of tumors are connected with mutations in the Tp53 gene. These mutations cause a disturbance in cellular response to stress, and eventually, cancer development. Apart from the full-length p53, at least twelve isoforms of p53 have been characterized. They are able to modulate p53 activity under stress conditions. In 2020, almost a half of million people around the world were diagnosed with renal cancer. One genetic disturbance which is linked to the most common type of kidney cancer, renal cell carcinoma, RCC, occurs from mutations in the VHL gene. Recent data has revealed that the VHL protein is needed to fully activate p53. Disturbance of the interplay between p53 and VHL seems to explain the lack of efficient response to chemotherapy in RCC. Moreover, it has been observed that changes in the expression of p53 isoforms are associated with different stages of RCC and overall survival. Thus, herein, an attempt was made to answer the question whether p53 and its isoforms are important factors in the development of RCC on the one hand, and in positive response to anti-RCC therapy on the other hand.

Distinct p53 isoforms code for opposing transcriptional outcomes.

p53 genes are conserved transcriptional activators that respond to stress. These proteins can also downregulate genes, but the mechanisms are not understood and are generally assumed to be indirect. Here, we investigate synthetic and native cis-regulatory elements in Drosophila to examine opposing features of p53-mediated transcriptional control in vivo. We show that transcriptional repression by p53 operates continuously through canonical DNA binding sites that confer p53-dependent transactivation at earlier developmental stages. p53 transrepression is correlated with local H3K9me3 chromatin marks and occurs without the need for stress or Chk2. In sufficiency tests, two p53 isoforms qualify as transrepressors and a third qualifies as a transcriptional activator. Targeted isoform-specific knockouts dissociate these opposing transcriptional activities, highlighting features that are dispensable for transactivation but critical for repression and for proper germ cell formation. Together, these results demonstrate that certain p53 isoforms function as constitutive tissue-specific repressors, raising important implications for tumor suppression by the human counterpart.

Astrocyte senescence and SASP in neurodegeneration: tau joins the loop.

Tau accumulation is a core component of Alzheimer's disease and other neurodegenerative tauopathies. While tau's impact on neurons is a major area of research, the effect of extracellular tau on astrocytes is largely unknown. This article summarizes our recent studies showing that astrocyte senescence plays a critical role in neurodegenerative diseases and integrates extracellular tau into the regulatory loop of senescent astrocyte-mediated neurotoxicity. Human astrocytes in vitro undergoing senescence were shown to acquire the inflammatory senescence-associated secretory phenotype (SASP) and toxicity to neurons, which may recapitulate aging- and disease-associated neurodegeneration. Here, we show that human astrocytes exposed to extracellular tau in vitro also undergo cellular senescence and acquire a neurotoxic SASP (e.g. IL-6 secretion), with oxidative stress response (indicated by upregulated NRF2 target genes) and a possible activation of inflammasome (indicated by upregulated ASC and IL-1ß). These findings suggest that senescent astrocytes induced by various conditions and insults, including tau exposure, may represent a therapeutic target to inhibit or delay the progression of neurodegenerative diseases. We also discuss the pathological activity of extracellular tau in microglia and astrocytes, the disease relevance and diversity of tau forms, therapeutics targeting senescence in neurodegeneration, and the roles of p53 and its isoforms in astrocyte-mediated neurotoxicity and neuroprotection.

p53/p73 Protein Network in Colorectal Cancer and Other Human Malignancies.

The p53 tumor suppressor protein is crucial for cell growth control and the maintenance of genomic stability. Later discovered, p63 and p73 share structural and functional similarity with p53. To understand the p53 pathways more profoundly, all family members should be considered. Each family member possesses two promoters and alternative translation initiation sites, and they undergo alternative splicing, generating multiple isoforms. The resulting isoforms have important roles in carcinogenesis, while their expression is dysregulated in several human tumors including colorectal carcinoma, which makes them potential targets in cancer treatment. Their activities arise, at least in part, from the ability to form tetramers that bind to specific DNA sequences and activate the transcription of target genes. In this review, we summarize the current understanding of the biological activities and regulation of the p53/p73 isoforms, highlighting their role in colorectal tumorigenesis. The analysis of the expression patterns of the p53/p73 isoforms in human cancers provides an important step in the improvement of cancer therapy. Furthermore, the interactions among the p53 family members which could modulate normal functions of the canonical p53 in tumor tissue are described. Lastly, we emphasize the importance of clinical studies to assess the significance of combining the deregulation of different members of the p53 family to define the outcome of the disease.

A Comparison of p53 Isoform Profiles and Apoptosis Induced by Camptothecin or a Herbal Khat Extract (Catha Edulis (Vahl) Forssk. ex Endl.) in Leukemic Cell Lines: Exploring Cellular Responses in Therapy Development.

Khat (Catha edulis (Vahl) Forssk. ex Endl.) is habitually used as a natural stimulant by millions of people, but is associated with adverse effects on gastrointestinal, cardiovascular and central neural systems. At the cellular level khat toxicity involves p53 induction and cell cycle arrest, decreased mitochondrial function and activation of receptor- and mitochondria-mediated cell death pathways. In this study we have examined an extract of khat for induction of p53 post-translational modifications (PTMs) and the functional role of p53 in khat-mediated cell death. Khat was shown to induce phosphorylation and acetylation of p53 in both the khat-sensitive MOLM-13 and the khat-resistant MV-4-11 cell line, but accumulation of the full-length p53 isoform was only observed in the khat sensitive cell line. Small molecule inhibitors of p38 MAP kinase sensitized MV-4-11 cells for khat-treatment without concomitant stabilization of p53. Experiments using a p53 knock-down cell line and murine p53 knock-out bone marrow cells indicated that p53 was redundant in khat-mediated cell death in vitro. We suggest that analysis of isoform patterns and p53 PTMs are useful for elucidation of biological effects of complex plant extracts, and that p53 protein analysis is particularly useful in the search for new chemical probes and experimental cancer therapeutics.

The Δ133p53 Isoforms, Tuners of the p53 Pathway.

The TP53 gene is a critical tumor suppressor and key determinant of cell fate which regulates numerous cellular functions including DNA repair, cell cycle arrest, cellular senescence, apoptosis, autophagy and metabolism. In the last 15 years, the p53 pathway has grown in complexity through the discovery that TP53 differentially expresses twelve p53 protein isoforms in human cells with both overlapping and unique biologic activities. Here, we summarize the current knowledge on the Δ133p53 isoforms (Δ133p53α, Δ133p53ß and Δ133p53γ), which are evolutionary derived and found only in human and higher order primates. All three isoforms lack both of the transactivation domains and the beginning of the DNA-binding domain. Despite the absence of these canonical domains, the Δ133p53 isoforms maintain critical functions in cancer, physiological and premature aging, neurodegenerative diseases, immunity and inflammation, and tissue repair. The ability of the Δ133p53 isoforms to modulate the p53 pathway functions underscores the need to include these p53 isoforms in our understanding of how the p53 pathway contributes to multiple physiological and pathological mechanisms. Critically, further characterization of p53 isoforms may identify novel regulatory modes of p53 pathway functions that contribute to disease progression and facilitate the development of new therapeutic strategies.

Good Cop, Bad Cop: Defining the Roles of Δ40p53 in Cancer and Aging.

The tumour suppressor p53 is essential for maintaining DNA integrity, and plays a major role in cellular senescence and aging. Understanding the mechanisms that contribute to p53 dysfunction can uncover novel possibilities for improving cancer therapies and diagnosis, as well as cognitive decline associated with aging. In recent years, the complexity of p53 signalling has become increasingly apparent owing to the discovery of the p53 isoforms. These isoforms play important roles in regulating cell growth and turnover in response to different stressors, depending on the cellular context. In this review, we focus on Δ40p53, an N-terminally truncated p53 isoform. Δ40p53 can alter p53 target gene expression in both a positive and negative manner, modulating the biological outcome of p53 activation; it also functions independently of p53. Therefore, proper control of the Δ40p53: p53 ratio is essential for normal cell growth, aging, and responses to cancer therapy. Defining the contexts and the mechanisms by which Δ40p53 behaves as a "good cop or bad cop" is critical if we are to target this isoform therapeutically.

What is the potential of p53 isoforms as a predictive biomarker in the treatment of cancer?

INTRODUCTION: For decades, p53 was researched as a single protein with alterations described as mutants. The discovery of 12 human p53 isoforms expressed from 9 transcripts changed this perception, eloquently explaining the numerous roles p53 plays, including apoptosis, senescence, and regeneration. Area covered: Here, we summarise the p53 isoforms and their relevance to cancer to establish an understanding and theorise on potential applications of the isoforms in clinical practice. Expert commentary: Pertaining to the different expression of isoforms in different tumors, it is concluded that the clinical use of isoforms as prognostic and predictive biomarkers will be different depending on the cell type, the tissue origin of the tumors, the position of the TP53 mutation and the driver-oncogene.

The role of p53 isoforms' expression and p53 mutation status in renal cell cancer prognosis.

OBJECTIVES: To analyze p53 mutations and gene expression of p53, ∆40p53, and ∆133p53 isoforms in renal cell cancer (RCC) tissues and normal adjacent tissue (NAT) and to associate them to clinical features and outcome. PATIENTS AND METHODS: Forty-one randomly selected patients, with primary, previously untreated RCC, with complete clinicopathohistological data were analyzed. NAT samples were available for 37 cases. Expression of p53, ∆40p53 and ∆133p53 was determined using RT-qPCR. A functional yeast-based assay was performed to analyze p53 mutations. RESULTS: More than half (56.1%) of patients harbored functional p53 mutations, and they were significantly younger than those with wild type (WT) p53 (P = 0.032). Expression of p53, ∆40p53, and ∆133p53 was upregulated in mutant (MT) p53 RCC compared to WT p53 RCC tissues. However, there was no difference in expression of these isoforms between MT p53 RCC tissues and NAT. Expression of ∆133p53 was significantly downregulated in WT p53 tissues compared to NAT (P = 0.006). Patients that harbored functional p53 mutation had better overall survival (hazard ratio 4.32, 95% confidence interval 1.46-18.82, P = 0.006). Multivariate analysis demonstrated that tumor stage and p53 mutation might be used as independent prognostic marker for overall survival in RCC patients. CONCLUSIONS: Our findings support the specific events in the carcinogenesis of RCC. p53 isoforms can be differentially expressed depending on p53 mutational status.