RNA helicase DDX5 modulates sorafenib sensitivity in hepatocellular carcinoma via the Wnt/ß-catenin-ferroptosis axis.

Reduced expression of the RNA helicase DDX5 associated with increased hepatocellular carcinoma (HCC) tumor grade and poor patient survival following treatment with sorafenib. While immunotherapy is the first-line treatment for HCC, sorafenib and other multi-tyrosine kinase inhibitors (mTKIs) are widely used when immunotherapy is contra-indicated or fails. Herein, we elucidate the role of DDX5 in sensitizing HCC to sorafenib, offering new therapeutic strategies. Treatment of various human HCC cell lines with sorafenib/mTKIs downregulated DDX5 in vitro and in preclinical HCC models. Conversely, DDX5 overexpression reduced the viability of sorafenib-treated cells via ferroptosis, suggesting a role for DDX5 in sorafenib sensitivity. RNAseq of wild-type vs. DDX5-knockdown cells treated with or without sorafenib identified a set of common genes repressed by DDX5 and upregulated by sorafenib. This set significantly overlaps with Wnt signaling genes, including Disheveled-1 (DVL1), an indispensable Wnt activator and prognostic indicator of poor survival for sorafenib-treated patients. DDX5-knockout (DDX5KO) HCC cells exhibited DVL1 induction, Wnt/ß-catenin pathway activation, and ferroptosis upon inhibition of canonical Wnt signaling. Consistently, xenograft HCC tumors exhibited reduced growth by inhibition of Wnt/ß-catenin signaling via induction of ferroptosis. Significantly, overexpression of DDX5 in HCC xenografts repressed DVL1 expression and increased ferroptosis, resulting in reduced tumor growth by sorafenib. We conclude that DDX5 downregulation by sorafenib mediates adaptive resistance by activating Wnt/ß-catenin signaling, leading to ferroptosis escape. Conversely, overexpression of DDX5 in vivo enhances the anti-tumor efficacy of sorafenib by suppressing Wnt/ß-catenin activation and induction of ferroptosis. Thus, DDX5 overexpression in combination with mTKIs is a promising therapeutic strategy for HCC.

GNS561, a New Autophagy Inhibitor Active against Cancer Stem Cells in Hepatocellular Carcinoma and Hepatic Metastasis from Colorectal Cancer.

Patients with advanced hepatocellular carcinoma (HCC) or metastatic colorectal cancer (mCRC) have a very poor prognosis due to the lack of efficient treatments. As observed in several other tumors, the effectiveness of treatments is mainly hampered by the presence of a highly tumorigenic sub-population of cancer cells called cancer stem cells (CSCs). Indeed, CSCs are resistant to chemotherapy and radiotherapy and can regenerate the tumor bulk. Hence, innovative drugs that are efficient against both bulk tumor cells and CSCs would likely improve cancer treatment. In this study, we demonstrated that GNS561, a new autophagy inhibitor that induces lysosomal cell death, showed significant activity against not only the whole tumor population but also a sub-population displaying CSC features (high ALDH activity and tumorsphere formation ability) in HCC and in liver mCRC cell lines. These results were confirmed in vivo in HCC from a DEN-induced cirrhotic rat model in which GNS561 decreased tumor growth and reduced the frequency of CSCs (CD90+CD45-). Thus, GNS561 offers great promise for cancer therapy by exterminating both the tumor bulk and the CSC sub-population. Accordingly, a global phase 1b clinical trial in liver cancers was recently completed.

In vitro transformation of primary human hepatocytes: Epigenetic changes and stemness properties.

Human hepatocarcinogenesis is a complex process with many unresolved issues, including the cell of origin (differentiated and/or progenitor/stem cells) and the initial steps leading to tumor development. With the aim of providing new tools for studying hepatocellular carcinoma initiation and progression, we developed an innovative model based on primary human hepatocytes (PHHs) lentivirus-transduced with SV40LT+ST, HRASV12 with or without hTERT. The differentiation status of these transduced-PHHs was characterized by RNA sequencing (including lncRNAs), and the expression of some differentiation markers confirmed by RT-qPCR and immunofluorescence. In addition, their transformation capacity was assessed by colony formation in soft agar and tumorigenicity evaluated in immune-deficient mice. The co-expression of SV40LT+ST and HRASV12 in PHHs, in association or not with hTERT, led to the emergence of transformed clones. These clones exhibited a poorly differentiated cell phenotype with expression of stemness and mesenchymal-epithelial transition markers and gave rise to cancer stem cell subpopulations. In vivo, they resulted in poorly differentiated hepatocellular carcinomas with a reactivation of endogenous hTERT. These experiments demonstrate for the first time that non-cycling human mature hepatocytes can be permissive to in vitro transformation. This cellular tool provides the first comprehensive in vitro model for identifying genetic/epigenetic changes driving human hepatocarcinogenesis.

A new signaling cascade linking BMP4, BMPR1A, ΔNp73 and NANOG impacts on stem-like human cell properties and patient outcome.

In a significant number of cases cancer therapy is followed by a resurgence of more aggressive tumors derived from immature cells. One example is acute myeloid leukemia (AML), where an accumulation of immature cells is responsible for relapse following treatment. We previously demonstrated in chronic myeloid leukemia that the bone morphogenetic proteins (BMP) pathway is involved in stem cell fate and contributes to transformation, expansion, and persistence of leukemic stem cells. Here, we have identified intrinsic and extrinsic dysregulations of the BMP pathway in AML patients at diagnosis. BMP2 and BMP4 protein concentrations are elevated within patients' bone marrow with a BMP4-dominant availability. This overproduction likely depends on the bone marrow microenvironment, since MNCs do not overexpress BMP4 transcripts. Intrinsically, the receptor BMPR1A transcript is increased in leukemic samples with more cells presenting this receptor at the membrane. This high expression of BMPR1A is further increased upon BMP4 exposure, specifically in AML cells. Downstream analysis demonstrated that BMP4 controls the expression of the survival factor ΔNp73 through its binding to BMPR1A. At the functional level, this results in the direct induction of NANOG expression and an increase of stem-like features in leukemic cells, as shown by ALDH and functional assays. In addition, we identified for the first time a strong correlation between ΔNp73, BMPR1A and NANOG expression with patient outcome. These results highlight a new signaling cascade initiated by tumor environment alterations leading to stem-cell features and poor patients' outcome.

Disequilibrium of BMP2 levels in the breast stem cell niche launches epithelial transformation by overamplifying BMPR1B cell response.

Understanding the mechanisms of cancer initiation will help to prevent and manage the disease. At present, the role of the breast microenvironment in transformation remains unknown. As BMP2 and BMP4 are important regulators of stem cells and their niches in many tissues, we investigated their function in early phases of breast cancer. BMP2 production by tumor microenvironment appeared to be specifically upregulated in luminal tumors. Chronic exposure of immature human mammary epithelial cells to high BMP2 levels initiated transformation toward a luminal tumor-like phenotype, mediated by the receptor BMPR1B. Under physiological conditions, BMP2 controlled the maintenance and differentiation of early luminal progenitors, while BMP4 acted on stem cells/myoepithelial progenitors. Our data also suggest that microenvironment-induced overexpression of BMP2 may result from carcinogenic exposure. We reveal a role for BMP2 and the breast microenvironment in the initiation of stem cell transformation, thus providing insight into the etiology of luminal breast cancer.

Wnt signaling and hepatocarcinogenesis: molecular targets for the development of innovative anticancer drugs.

Hepatocellular carcinoma (HCC) is one of the most common causes of cancer death worldwide. HCC can be cured by radical therapies if early diagnosis is done while the tumor has remained of small size. Unfortunately, diagnosis is commonly late when the tumor has grown and spread. Thus, palliative approaches are usually applied such as transarterial intrahepatic chemoembolization and sorafenib, an anti-angiogenic agent and MAP kinase inhibitor. This latter is the only targeted therapy that has shown significant, although moderate, efficiency in some individuals with advanced HCC. This highlights the need to develop other targeted therapies, and to this goal, to identify more and more pathways as potential targets. The Wnt pathway is a key component of a physiological process involved in embryonic development and tissue homeostasis. Activation of this pathway occurs when a Wnt ligand binds to a Frizzled (FZD) receptor at the cell membrane. Two different Wnt signaling cascades have been identified, called non-canonical and canonical pathways, the latter involving the ß-catenin protein. Deregulation of the Wnt pathway is an early event in hepatocarcinogenesis and has been associated with an aggressive HCC phenotype, since it is implicated both in cell survival, proliferation, migration and invasion. Thus, component proteins identified in this pathway are potential candidates of pharmacological intervention. This review focuses on the characteristics and functions of the molecular targets of the Wnt signaling cascade and how they may be manipulated to achieve anti-tumor effects.

[The two faces of p63, Janus of the p53 gene family]. / Les deux visages de p63, Janus de la famille p53.

The TP53 family member TP63 encodes two main isoforms TAp63 and ΔNp63 with distinct, often opposite functions during development and in the adult. ΔNp63 is crucial for the formation of the ectodermal derivatives and epidermis, while TAp63 is essential for heart development. In the adult, ΔNp63 behaves as a cell survival factor, controlling cell proliferation, adhesion and cell differentiation. In contrast, TAp63 is a proapoptotic factor that protects oocytes from genotoxic insults and prevents premature aging of dermal stem cells. In agreement with these activities, TAp63 is often lost and ΔNp63 overexpressed in cancer cells. Because of their opposite and competitive effects, p63 isoforms could be viewed as Janus two faces. The review focuses on the accumulating data on the p63 functions and regulation in the last decade.

Effects of the TP53 p.R249S mutant on proliferation and clonogenic properties in human hepatocellular carcinoma cell lines: interaction with hepatitis B virus X protein.

Aflatoxin B(1) (AFB(1)) is a risk factor for hepatocellular carcinoma (HCC) in many low-resource countries. Although its metabolites bind at several positions in TP53, a mutation at codon 249 (AGG to AGT, arginine to serine, p.R249S) accounts for 90% of TP53 mutations in AFB(1)-related HCC. This specificity suggests that p.R249S confers a selective advantage during hepatocarcinogenesis. Using HCC cell lines, we show that p.R249S has lost the capacity to bind to p53 response elements and to transactivate p53 target genes. In p53-null Hep3B cells, stable transfection of p.R249S or of another mutant, p.R248Q, did not induce significant changes in cell proliferation and survival after cytotoxic stress. In contrast, in a cell line that constitutively expresses both p.R249S and the hepatitis B virus antigen HBx (PLC/PRF/5), silencing of either p.R249S or HBx by RNA interference slowed down proliferation, with no additive effects when both factors were silenced. Furthermore, the two proteins appear to form a complex. In human HCC samples, mutation at codon 249 did not correlate with p.R249S protein accumulation or HBx truncation status. We suggest that p.R249S may contribute to hepatocarcinogenesis through interaction with HBx, conferring a subtle growth advantage at early steps of the transformation process, but that this interaction is not required for progression to advanced HCC.

The CD10 enzyme is a key player to identify and regulate human mammary stem cells.

The major components of the mammary ductal tree are an inner layer of luminal cells, an outer layer of myoepithelial cells, and a basement membrane that separates the ducts from the underlying stroma. Cells in the outer layer express CD10, a zinc-dependent metalloprotease that regulates the growth of the ductal tree during mammary gland development. To define the steps in the human mammary lineage at which CD10 acts, we have developed an in vitro assay for human mammary lineage progression. We show that sorting for CD10 and EpCAM cleanly separates progenitors from differentiated luminal cells and that the CD10-high EpCAM-low population is enriched for early common progenitor and mammosphere-forming cells. We also show that sorting for CD10 enriches sphere-forming cells from other tissue types, suggesting that it may provide a simple tool to identify stem or progenitor populations in tissues for which lineage studies are not currently possible. We demonstrate that the protease activity of CD10 and the adhesion function of beta1-integrin are required to prevent differentiation of mammary progenitors. Taken together, our data suggest that integrin-mediated contact with the basement membrane and cleavage of signaling factors by CD10 are key elements in the niche that maintains the progenitor and stem cell pools in the mammary lineage.

Cancer stem cells: the emerging challenge of drug targeting.

Stem cells are defined by their unique property to self-renew and starting from one single cell to generate all the different progenies required for tissue regeneration. In adults, stem cells are still present in the majority of tissues and organs where they are responsible for continuous organs and tissues homeostasis. Adult stem cells have been isolated in various tissues and all share common specific characteristics (localization in stem cell niches, drug transporter expression, adhesion, levels of apoptosis inhibitors, DNA methylation, -) involved in high levels of drug resistance of this specific cell subtype. Several studies have identified different populations of cancer cells, within the same tumor, some of them which present properties closely related to normal stem cells and raised the concept of cancer stem cells. Interestingly, the cell surface markers expressed by these particular cancer cells are the same as those expressed by normal stem cells, suggesting that cancer can arise in some cases from the malignant transformation of stem cells. The cancer stem cell (CaSC) model predicts that, even if "conventional" cancer cells can be killed by chemotherapy or radiotherapy, only the destruction of CaSC, considered responsible for relapse, will allow full recovery, thus demonstrating the importance of CaSC-targeting for patient outcome. Therapeutic innovations will emerge from a better understanding of the biology and environment of cancer stem cells. The tumor environment can create a niche favoring the survival and proliferation of CaSC. It also protects CaSC from chemotherapy-induced apoptosis. Clinically, it is crucial to get rid of quiescent and resistant cells and to adapt the therapeutic strategy to cancer stem cells sheltered in niches. Here, we review the major characteristics of cancer stem cells and their behavior in response to chemotherapy; we also highlight the main issues to be considered for efficient and specific cancer stem cell targeting.

Induction of EMT by twist proteins as a collateral effect of tumor-promoting inactivation of premature senescence.

Twist1 and Twist2 are major regulators of embryogenesis. Twist1 has been shown to favor the metastatic dissemination of cancer cells through its ability to induce an epithelial-mesenchymal transition (EMT). Here, we show that a large fraction of human cancers overexpress Twist1 and/or Twist2. Both proteins override oncogene-induced premature senescence by abrogating key regulators of the p53- and Rb-dependent pathways. Twist1 and Twist2 cooperate with Ras to transform mouse embryonic fibroblasts. Interestingly, in epithelial cells, the oncogenic cooperation between Twist proteins and activated mitogenic oncoproteins, such as Ras or ErbB2, leads to complete EMT. These findings suggest an unanticipated direct link between early escape from failsafe programs and the acquisition of invasive features by cancer cells.

In vitro and in vivo cytotoxic effects of PRIMA-1 on hepatocellular carcinoma cells expressing mutant p53ser249.

Hepatocellular carcinoma (HCC) is highly lethal due to limited curative options. In high-incidence regions, such as parts of Africa and Southeastern Asia, >50% of cases carry an AGG to AGT mutation at codon 249 of the TP53 gene, considered as a 'signature' of mutagenesis by aflatoxins. The protein product, p53ser249, may represent a therapeutic target for HCC. The small molecule p53 reactivation and induction of massive apoptosis (PRIMA)-1 has been shown to induce apoptosis in tumour cells by reactivating the transactivation capacity of some p53 mutants. In this study, we have investigated the cytotoxic effects of PRIMA-1 on HCC cells expressing p53ser249. In p53-null Hep3B cells, over-expression of p53ser249 or p53gln248 by stable transfection increased the cytotoxicity of PRIMA-1 at 50 muM. Furthermore, PRIMA-1 treatment delayed the growth of p53ser249-expressing Hep3B cells xenografted in severe combined immunodeficiency mice. However, PRIMA-1 did not restore wild-type DNA binding and transactivation activities to p53ser249 or to p53gln248 in Hep3B cells. Moreover, in PLC/PRF/5, a HCC cell line constitutively expressing p53ser249, small interfering RNA (siRNA) silencing of the mutant increased the cytotoxic effect of PRIMA-1. These apparently contradictory effects can be reconciled by proposing that p53ser249 exerts a gain-of-function effect, which favours the survival of HCC cells. Thus, both inhibition of this effect by PRIMA-1 and removal of the mutant by siRNA can lead to the decrease of survival capacity of HCC cells.

Gadd45a activation protects melanoma cells from ultraviolet B-induced apoptosis.

Epidemiological and biological studies indicate that solar UVB radiation is involved in cutaneous malignant melanoma etiology. Indeed, melanocytes are very frequently exposed to solar UV radiation, which induces cell damage and may promote cell transformation. We previously showed that melanocytes and melanoma cells exposed to UVB radiation activates a p53-independent pathway involving Gadd45a and, more recently, that Gadd45a plays a critical role in UVB-induced G2 cell cycle arrest of melanoma cells. In this study, we demonstrate that the inhibition of UV-induced Gadd45a overexpression by RNA interference results in a dramatic increase of cell death. We identify this cell death as apoptosis, with activation of Caspase-3 and a decrease in Bcl-x(L) expression. Furthermore, we show that inhibition of UV-induced Gadd45a overexpression also leads to increased sensitivity of melanoma cells to therapeutic agents such as DTIC and Cisplatin. We conclude that UVB-induced Gadd45a overexpression protects melanoma cells from apoptosis, both by causing a G2 cell cycle arrest and by inhibiting the mitochondrial apoptotic pathway. These observations suggest that Gadd45a inactivation could be a useful way to sensitize melanoma cells to chemotherapy. JID journal club article: For questions, answers, and open discussion about this article please go to http://network.nature.com/group/jidclub

TP63 gene in stress response and carcinogenesis: a broader role than expected.

The TP63 gene is a member of the TP53 gene family. In contrast with TP53, this gene is not frequently inactivated by mutation in cancer. Initial experiments with disrupted TP63 have allowed specifying p63 protein a role in the regulation of differentiation and morphogenesis in epithelial and mesenchymal tissues. Nevertheless, there is growing evidence that p63 is also involved in oncogenesis through several mechanisms. Indeed, amplification of TP63 is detected in about 25% of squamous cell carcinomas of lung, head and neck and oesophagus. This results in overexpression of a truncated form of p63 (DeltaNp63) that may counteract growth suppression induced by full length p63 (TAp63), as well as by the other family members, p53 and TAp73. Moreover, mice heterozygous for TP63 develop spontaneous tumours. Whereas p53 plays a major role in response to numerous DNA-damaging agents, the involvement of p63 in this process is not well documented. Nevertheless, several groups recently reported that TAp63 can induce cell cycle arrest and apoptosis in DNA-damaged cells, alone or in synergy with chemotherapeutic agents, and thus appears as a chemosensitivity factor. Overall, in addition to non-redundant, specific functions in differentiation and morphogenesis, p63 appears to exert biological functions similar to those of p53 and to take a growing place in oncogenesis and modulation of responses to anti-cancer therapy.

UVB-induced G2 arrest of human melanocytes involves Cdc2 sequestration by Gadd45a in nuclear speckles.

Exposure to solar UVB radiation is involved in the development of cutaneous melanoma. We previously showed that human melanocytes and melanoma cells respond to UVB radiation via a p53-independent pathway involving GADD45A activation. Here, we determined that UVB-induction of Gadd45a is necessary for G(2) arrest and that Gadd45a and its partner p21(Waf1) colocalize in nuclear bodies called Nuclear Speckles. We further observed that UVB-induced G(2) arrest is associated with Cdc2 accumulation in these Nuclear Speckles. Knock-down of Gadd45a expression by RNA interference prevents both UVB-induced Cdc2 accumulation in Nuclear Speckles and G(2) arrest. Our results demonstrate that UVB-induced G(2) arrest of melanoma cells is Gadd45a-dependent. Furthermore, we show that Cdc2 sequestration by Gadd45a occurs in Nuclear Speckles, suggesting a new role for these nuclear bodies, so far only linked to RNA maturation.

p53 as a target for anti-cancer drug development.

Loss of p53 function compromises genetic homeostasis in cells exhibiting deregulated DNA replication and/or DNA damage, and prevents normal cytotoxic responses to cancer therapies. Genetic and pharmacological approaches are being developed with the ultimate goal of restoring or controlling p53 functions in cancer patients. Progress has recently been made in the clinical use of replication-deficient virus carrying wt-TP53 (Ad5CMV-p53) and/or cancer-selective oncolytic adenoviruses (ONYX-015). These strategies demonstrated clinical activity as monotherapy and were synergistic with traditional chemotherapy agents in the treatment of some types of cancer. In addition, pharmacological methods are under development to either stimulate wild-type p53 protein function, or induce p53 mutant proteins to resume wild-type functions. These methods are based on small chemicals (CP-31388, PRIMA-1), peptides (CDB3) or single-chain Fv antibody fragments corresponding to defined p53 domains. Here, we discuss the mechanisms underlying these approaches and their perspectives for cancer therapy.

The expression of TA and DeltaNp63 are regulated by different mechanisms in liver cells.

The TP63 gene, a member of the TP53 gene family, encodes several isoforms with (TAp63) or without (DeltaNp63) transactivating properties. Whereas the role of p63 in the normal development of squamous epithelia is well established, its function in other cell types remains to be elucidated. Here, we have analysed the expression of TA and DeltaNp63 isoforms in liver cells, by using both primary hepatocytes from wild type and p53-null mice and three human hepatocellular carcinoma (HCC) cell lines, according to the transformation state and the TP53 status of the cells. We observed the expression of DeltaNp63 isoforms only in a p53-null context. On the other hand, the expression of TAp63 isoforms was restricted to the HCC cell lines, whatever the TP53 status. We then studied the expression of TP63 upon genotoxic treatment. When treated with UVB or H(2)O(2), hepatocytes did not exhibit any change in p63 mRNA level. At the opposite, upon treatment with topoisomerase II inhibitors (doxorubicin or etoposide), the expression of TAp63 isoforms was clearly induced, independently of the TP53 status of cells. The same treatment did not induce any variation in the expression of DeltaNp63 isoforms, both at mRNA and protein levels. In HCC cell lines, doxorubicin or etoposide treatment also resulted in an increase of TAp63 transcripts only. This increase was accompanied by an increase in the intracellular level of TAp63 alpha protein. In parallel, we observed an upregulation of some p53-target genes related to cell cycle regulation, such as WAF1/CIP1, PIG3, 14-3-3sigma or GADD45, independently of the TP53 status of cells. In conclusion, we report for the first time that TA and DeltaNp63 alpha proteins are present in liver cells. Furthermore, our results suggest that p63 may partially substitute for wild-type p53, in counteracting uncontrolled liver cell proliferation in response to certain forms of DNA-damage.

The tumor suppressor gene TP53: implications for cancer management and therapy.

The p53 protein is an inducible transcription factor with multiple anti-proliferative roles in response to genotoxic damage; unprogrammed proliferative stimuli; and deprivation of oxygen, nutrients, or ribonucleotides. Inactivation of the TP53 gene by mutation or deletion is the most common event in human cancer. Loss of p53 function compromises genetic homeostasis in cells exposed to mutagens and prevents normal cytotoxic responses to cancer therapies. Genetic and pharmacological approaches are being developed with the ultimate goal of restoring or controlling p53 functions in cancer patients. Genetic interventions aiming at expressing wild-type TP53 in cancer cells, either by retroviral or adenoviral transfer, have met limited clinical success. However, recently, the use of a defective adenovirus (ONYX-015) that selectively kills p53-incompetent cells has shown promising effects in pre-clinical and clinical studies. Pharmacological methods are under development to either stimulate wild-type p53 protein function or induce p53 mutant proteins to resume wild-type functions. These methods are based on small chemicals (CP-31388, PRIMA-1), peptides (CDB3), or single-chain Fv antibody fragments corresponding to defined p53 domains. In addition, detection of mutant TP53 may also serve as a marker for early cancer detection, prediction, and prognosis. In this review, we discuss the mechanisms underlying these approaches and their perspectives for cancer therapy.

TP53 mutation spectra and load: a tool for generating hypotheses on the etiology of cancer.

Among genetic alterations, the activation of proto-oncogenes and inactivation of tumour suppressor genes in affected cells are considered to be the core molecular events that provide a selective growth advantage and clonal expansion during the multistep process of carcinogenesis. The TP53 tumour suppressor gene is mutated in about half of all human cancer cases. The p53 protein modulates multiple cellular functions, such as gene transcription, DNA synthesis and repair, cell cycle arrest, senescence and apoptosis. Mutations in the TP53 gene can abrogate these functions, leading to genetic instability and progression to cancer. The molecular archaeology of the TP53 mutation spectrum generates hypotheses concerning the etiology and molecular pathogenesis of each type of cancer. The spectrum of somatic mutations in the TP53 gene, of which 75% are missense mutations, implicates environmental carcinogens and endogenous processes in the etiology of human cancer. The presence of a characteristic TP53 mutation can also manifest a molecular link between exposure to a particular carcinogen and a specific type of human cancer, e.g. exposure to aflatoxin B1 (AFB1) and codon 249 mutations in hepatocellular carcinoma; exposure to ultraviolet (UV) light and C:C-->T:T tandem mutations in skin cancer; and cigarette smoking and the prevalence of G-->T transversions in lung cancer. Although exogenous carcinogens have been shown to target p53 selectively, evidence supporting the endogenous insult of TP53 from oxyradicals and nitrogen-oxyradicals is also accumulating. TP53 mutations can be a biomarker of carcinogen effect. Determining the characteristic TP53 mutation load in non-tumorous tissue, using a highly sensitive mutation assay, can indicate exposure to a specific carcinogen and may also help in identifying individuals at an increased risk of cancer.