Lung-enriched Mutations in the p53 Tumor Suppressor: A Paradigm for Tissue-specific Gain of Oncogenic Function

Abstract

Lung cancer, the leading cause of cancer-related mortality in the US, occurs primarily due to prolonged exposure to an array of carcinogenic compounds in cigarette smoke. These carcinogens create bulky DNA adducts, inducing alterations including missense mutations in the tumor suppressor gene TP53. TP53 is the most commonly mutated gene in many human cancers, and a specific set of these variants are enriched in lung cancer (at amino acid residues V157, R158, and A159). This perspective postulates that lung-enriched mutations can be explained, in part, by biological selection for oncogenic gain-of-function (GOF) mutant p53 alleles at V157, R158, and A159. This hypothesis explaining tissue-specific TP53 mutations, is further supported by mouse model studies of the canonical TP53 hotspots showing that tumor spectra and GOF activities are altered with mutation type. Therefore, while smoking-related lung cancer unequivocally arises due to the mutagenic environment induced by tobacco carcinogens, this perspective provides a rationale for the preferential selection of lung-enriched V157, R158, and A159 mutant p53.

Introduction

Although the p53 tumor suppressor, as the “guardian of the genome,” has been studied extensively, the availability of more complete sequencing data across the entire genome has allowed the current era of human genetic studies to be more comprehensive than ever.(1,2) One of the most active areas of research is in mutant p53 gain of oncogenic function (GOF), whereby single nucleotide missense mutations exhibit not only loss of tumor suppressor function but also acquisition of novel activities such as increased invasion, chemoresistance, and metabolic reprogramming.(3,4) Identification of novel tumorigenic functions may open new avenues for p53-targeted therapies.

Somatic alterations in the TP53 gene occur primarily at one of six major canonical hotspots in the DNA-binding domain: R175, G245, R248, R249, R273, and R282. Specifically, the eight most common mutants individually range in frequency from 2.9 to 7% and together account for 27.7% of all TP53 mutations in a database of 11,145 sequenced tumors.(5) Notably, when lung cancer cases are reviewed in isolation, the frequency of mutations at amino acid residues V157 and R158 increases 5- to 6-fold (0.9% to 4.6% and 1.1% to 5.9%, respectively) and surpasses that of many of the traditional hotspots. This phenomenon has been reported extensively and attributed to preferential DNA adduct formation at these codons by carcinogenic polycyclic aromatic hydrocarbons (PAH) found in cigarette smoke.(6–10)

The PAH benzo[a]pyrene, which is metabolically activated to benzo[a]pyrene-diol-epoxide (BPDE), preferentially binds the N2 position of guanine at TP53 codons 157, 248, and 273 in normal human bronchial epithelial cells and normal human fibroblasts.(6) These bulky DNA adducts generate a COSMIC smoking signature described as G:C > T:A transversion mutations with a bias for the nontranscribed DNA strand, where DNA is less efficiently repaired.(11) Moreover, DNA adduct formation by BPDE is enhanced at methylated CpG sites in both lung-enriched V157F and R158 codons and the canonical hotspots G245, R248, and R273.(6) While the precise mechanism is not yet understood, it has been proposed that cytosine methylation increases the electron density of the amine group of the paired guanine. This enhances its affinity with BPDE and acrolein, a mutagenic aldehyde also found in cigarette smoke.(12–14) Alternative hypotheses have been put forth as well, with the methyl group of 5-methylcytosine allowing increased intercalation of BPDE with subsequent increase in covalent interaction; or increased probability of base flipping of the paired guanine into the major groove, allowing it to interact with BPDE. (12,13) A variety of in vitro and in vivo studies to examine mutations induced by exposure of p53 cDNA, bronchial epithelial cells, lung fibroblasts, and human TP53 knock-in mouse embryo fibroblasts to BPDE have confirmed a high frequency of G>T transversions at codons V157 and R158 in addition to the canonical p53 hotspots. (12–18)

TP53 mutational spectra in lung tumors

To investigate the clinical significance of V157, R158, and A159 mutations in lung cancer, we evaluated lung adenocarcinoma and lung squamous cell lung cancer sequencing data from the Pan-Lung Cancer Study of The Cancer Genome Atlas (TCGA). (19) As noted above, the frequency of R158 and V157 mutations in lung cancers is markedly elevated. This finding is not observed in head and neck squamous cell carcinomas (HNSCC), for which cigarette smoking is also the primary risk factor (Figure 1). In the TCGA database which includes 528 patient samples, TP53 is altered in 367 cases of HNSCC (69.5%), and 253 (68.9%) of these are missense mutations.(20) These frequencies are nearly identical to the TCGA Pan-Lung Cancer Study, where 1,144 patient samples exhibit TP53 alterations in 776 cases (67.8%), and 505 (65.1%) of these are missense mutations.(19) However, in sharp contrast to lung cancer, only five samples harbor V157 mutations (5/5 V157F, 2.0% of total), three have R158 mutations (2/3 R158L, 1.2% of total), and two have A159 mutations (1/2 A159P, 0.8%). In bladder urothelial carcinoma where cigarette smoking is also a major risk factor, there is a much lower rate of TP53 mutations.(21) Among 412 patient samples, 74 (18.0%) exhibit alterations in TP53, and 57 (77.0%) of these are missense mutations. No V157 mutations and only one R158 mutations (R158H) exist in this cohort, and although there are four A159 mutations, the overall lower frequency of TP53 mutations suggests that changes in the p53 tumor suppressor do not play a major role in bladder tumorigenesis. These data show that the V157, R158, and A159 TP53 mutations occur preferentially in lung cancer over HNSCC and bladder cancer despite the critical role that cigarette smoke exposure plays in all three tumor types. Hence, it is possible that these TP53 mutations are not only induced by tobacco carcinogens but exhibit a relative abundance in the lung driven by selective biological pressure which may be tissue-specific.

Figure 1. Patterns in TP53 mutations vary by tumor type.

Number of somatic missense mutations are plotted for the most frequently mutated amino acid residues in the TP53 gene, by solid tumor type. Data are from The Cancer Genome Atlas (TCGA). (19) Total numbers of samples with TP53 missense mutations are: Lung adenocarcinoma and squamous cell carcinoma, 505; Breast adenocarcinoma, 179; Head and neck squamous cell carcinoma (HNSCC), 253; Gastric adenocarcinoma, 131; Ovarian serous cystadenocarcinoma, 179; Esophageal adenocarcinoma, 110; Hepatocellular carcinoma (HCC), 71; Colorectal adenocarcinoma, 83; Pancreatic adenocarcinoma, 72.

Our analysis of the larger and more recently curated AACR Project GENIE cohort revealed a similar enrichment for mutations at V157, R158, and A159 among non-small cell lung cancer samples (Table 1). Specifically, in lung adenocarcinoma (n = 5,060 samples), the overall prevalence of TP53 alterations was 46.1%, or 2,333 samples, and R158 was the third most frequent amino acid residue at which mutations occurred (increasing 4-fold in frequency to 4.2%, from 1.0% when compared to non-lung cancers). Alterations in TP53 in general were more common in squamous cell lung cancer (n = 559 samples) than in adenocarcinomas, with an overall prevalence of 78.4%, or 438 samples. Mutations at the traditional hotspot R248 and the lung-enriched hotspot R158 occurred most commonly, each with a frequency of 7.4%, or 22 of 297 missense mutations. The lung-enriched hotspots V157 and A159 also occurred with higher frequency than in non-lung cancers. In small cell lung cancer (SCLC, n = 236 samples) TP53 alterations occurred in 78.0%, or 184 samples. This is lower than in other published cohorts, where alterations were seen in 98% of samples.(22) While the general distribution of mutations is starkly different from that in lung adenocarcinoma and squamous cell lung cancers, with mutations seen at a variety of amino acid residues including H179, Y220, and P278, the lung-enriched loci V157 and R158 continue to be hotspots in SCLC. Together, these findings show that while minor differences exist among histologic groups – primarily, high R158 frequencies in squamous cell lung cancer in particular – mutations at the lung-enriched hotspots have a high relative abundance in all subtypes of lung cancer.

Table 1.

TP53 mutations in AACR Project GENIE registry v3.0.0.

	Non-lung cancers n = 33,011 samples	Adenocarcinoma n = 5,060 samples	Squamous cell lung cancer n = 559 samples	Small cell lung cancer (SCLC) n = 236 samples
TP53 mutations#	13,080 mutations in11,983 samples (36.3%)	2,513 mutations in 2,333 samples (46.1%)	479 mutations in 438 samples (78.4%)	190 mutations in 184 samples (78.0%)
TP53 missense mutations	8,813	1,750	297	112
Traditional TP53 hotspot missense mutations*
R273	951 (10.8%)	117 (6.7%)	16 (5.4%)	8 (7.1%)
R248	847 (9.6%)	106 (6.1%)	22 (7.4%)	4 (3.6%)
R175	736 (8.4%)	44 (2.5%)	7 (2.4%)	4 (3.6%)
G245	324 (3.7%)	68 (3.9%)	10 (3.4%)	3 (2.7%)
Lung-enriched TP53 hotspot missense mutations*
R158	87 (1.0%)	73** (4.2%)	22 (7.4%)	6 (5.4%)
V157	67 (0.8%)	49** (2.8%)	9 (3.0%)	5 (4.5%)
A159	48 (0.5%)	25** (1.4%)	4 (1.3%)	0 (0%)

^#Frequency is represented in percent of samples with TP53 mutations (including missense, truncating, inframe, and other mutations) out of total number of samples for each histologic subtype.

^*Frequency is represented in percent of missense mutations out of total number of missense mutations for each histologic subtype.

^**One case had V157F and A159 mutations (counted separately), and one case had R158L and R158C mutations (counted separately).

Recent studies have defined subsets of lung adenocarcinoma based upon frequently co-occurring mutations, namely KRAS and TP53 or STK11, with impacts on treatment response and survival.(23–25) We sought to determine whether co-mutational events differ among lung cancers harboring the lung-enriched mutations in the Project GENIE cohort. Similar to all TP53-altered lung adenocarcinomas, the most frequently co-occurring mutations in V157-, R158-, and A159-mutated lung adenocarcinomas were found in KRAS and EGFR (which are mutually exclusive). Co-mutated TP53 and KRAS were slightly more common among lung-enriched p53-mutated samples (35.6%, or 52 of 146 samples vs. 26.0%, or 434 of 1,667 samples; Figure 2A and Table 2A). Co-occurrence of KRAS and STK11 in p53-mutant tumors was rare in both groups (2.7%, or 4 of 146 samples, vs 3.1%, or 52 of 1,667 samples), consistent with existing literature.(26) Among lung squamous cell cancers, the most commonly co-mutated genes with TP53 were KMT2D and CDKN2A. (Table 2B) In the lung-enriched mutant p53 cohort, the frequency of co-occurring CDKN2A mutations was higher (31.4%, or 11 of 35 samples) than among squamous cell lung cancers with any TP53 mutation (20.6%, or 59 of 287 samples; Figure 2B). Together, these findings show that common co-occurring mutations (KRAS in lung adenocarcinoma and CDKN2A in squamous cell lung cancer) are even more frequent in tumors harboring lung-enriched p53 mutations.

Figure 2. Co-occurring mutations with TP53 in lung adenocarcinoma and lung squamous cell samples from AACR Project GENIE v3.0.0.

Prevalence of common co-occurring mutations in A. lung adenocarcinoma, and B. squamous cell lung cancers is shown for tumors harboring any mutation in TP53 vs. the cohort of lung-enriched mutations.

Table 2A.

AACR GENIE Lung Adenocarcinoma Co-Occurring Mutations

	All lung adenocarcinomas (n = 5,060 samples)
TP53 mutation*	2,333 (46.1%)
KRAS mutation**	1,649 (32.6%)
STK11 mutation***	665 (13.4%)
Mutations#	All TP53 mutations n = 1,667 samples (1,750 missense mutations)	Lung-enriched TP53 mutations (V157, R158, A159) n = 146 samples (147 missense mutations)
TP53 without KRAS or STK11	1,100 (66.0%)	86 (58.9%)
TP53 and KRAS	434 (26.0%)	52 (35.6%)
TP53 and STK11	185 (11.1%)	12 (8.2%)
TP53, KRAS, and STK11	52 (3.1%)	4 (2.7%)

^*TP53 mutations include 2,325 samples with mutations, 3 samples with fusions, and 5 samples with multiple alterations.

^**KRAS mutations include 1,625 samples with mutations and 24 samples with multiple alterations.

^***STK11 mutations include 652 samples with mutations, 12 samples with fusions, and 1 sample with multiple alterations.

Amplifications and deep deletions are excluded.

^#Frequency is represented in percent of samples with missense mutations out of total number of samples with TP53 missense mutations.

Table 2B.

AACR GENIE Squamous Cell Lung Cancer Co-Occurring Mutations

	All squamous cell lung cancer (n = 559 samples)
TP53 mutation*	438 (78.4%)
KMT2D mutation**	96 (17.2%)
CDKN2A mutation***	88 (15.7%)
Mutations#	All TP53 mutations n = 287 samples (297 missense mutations)	Lung-enriched TP53 mutations (V157, R158, A159) n = 35 samples (35 missense mutations)
TP53 without KMT2D or CDKN2A	182 (63.4%)	17 (48.6%)
TP53 and KMT2D	59 (20.6%)	9 (25.7%)
TP53 and CDKN2A	59 (20.6%)	11 (31.4%)
TP53, CDKN2A, and KMT2D	13 (4.5%)	2 (5.7%)

^*TP53 mutations include 435 samples with mutations, 2 samples with fusions, and 1 sample with multiple alterations.

^**KMT2D mutations include 94 samples with mutations, 1 sample with fusions, and 1 sample with multiple alterations.

^***CDKN2A mutations include 86 samples with mutations and 2 samples with fusions.

Amplifications and deep deletions are excluded.

^#Frequency is represented in percent of samples with missense mutations out of total number of samples with TP53 missense mutations.

While a comparison of lung tumors occurring in current or former smokers vs. never smokers would better elucidate the relationship between tobacco carcinogens and the lung-enriched p53 mutations, 64 of the 65 samples with missense mutations at V157, R158, or A159 in the TCGA Pan-Lung Cancer study are from patients with a history of cigarette smoke exposure. (19) However, this delineation has been addressed in older datasets, with the conclusion that the p53 mutation spectra are highly different between smokers and non-smokers. (7,9) Newer datasets such as the AACR Project GENIE, which is not yet fully clinically annotated, may provide more detailed knowledge as smoking rates continue to decline in the US. (27)

In addition to the enriched loci at which TP53 mutations occur in lung cancer, the frequency of specific nucleotide changes is also altered in these tumors. Smoking-related lung cancer arises in a highly mutagenic environment and is associated with a specific G>T mutational signature. This G>T signature is found frequently in p53 mutations in lung cancer – in contrast to the G>A transitions that predominate p53 mutations in non-lung cancers – and supports the important role of smoking in oncogenic p53 mutations in the pathogenesis of lung cancer. Given this, one would expect that if G>T transversions at the canonical hotspots are possible and sufficient for lung tumorigenesis, the presence of non-classical nucleotide changes at non-canonical codons might be unnecessary. Notably, while G>T transversions are also the most common nucleotide substitution in V157 and R158 mutations, a minority of tumors show a G>C transversion.(19) Furthermore, TCGA data reveals that A159, despite including a guanine, exclusively participates in G>C transversions (A159P) or C>T transitions (A159V) rather than the canonical G>T smoking-related signature. Finally, it has been noted that the G>T smoking signature dominates the nucleotide changes at the canonical hotspots R273 (51.6% of R273 mutations in the Pan-Lung Cancer Study), G245 (84%), and R248 (32%) which still occur with moderate frequency, albeit reduced. In contrast, at R175 there are no G>T transversions in the Pan-Lung Cancer Study but rather an increase in C>G transversions Furthermore, there is an overall decrease in frequency of mutations at this traditional hotspot which is usually among the most commonly altered amino acid residues in other solid tumors.(19) This diversity of nucleotide changes suggests that factors beyond cigarette smoke exposure may play a role in selection for p53 mutation and cancer progression in the lung.

Additional support for preferential biological selection of the V157 mutant is illustrated by a case report of a 22-year old patient with Li Fraumeni-like syndrome due to germline V157D p53 and PMS2 mutations who developed lung cancer with loss of heterozygosity (LOH) restricted to lung tumor cells.(28) Notably, the proband’s father also carried a germline p53 V157D mutation with heterozygous mutation noted in a sample of the colon cancer from which he died at age 31. While one interpretation would be that selective pressure exists specifically in the lung for mutations at V157F p53, it is also possible that LOH in the lung adenocarcinoma resulted from its inability to tolerate the presence of even a single allele of wildtype p53 tumor suppressive capacity. Larger scale studies of Li Fraumeni patients harboring different p53 mutations have demonstrated a pattern of variable tumor type and age at onset depending on the mutant allele.(29,30)

Together, this evidence supports our hypothesis that while mutations at codons V157, R158, and A159 may occur as a result of tobacco carcinogen exposure, they may be preferentially selected in lung cancer for additional reasons such as GOF activities with inherent benefit to lung tumors.

Biochemical and molecular rationale for selection of p53 mutations

It is plausible that the lung-enriched mutations are selected for structural alterations resulting in a highly debilitated mutant protein. Fersht and colleagues characterized V157F as a severely destabilizing mutation, with a large number of neighboring side chains affected by the substitution of a larger, hydrophobic phenylalanine for valine, which eliminates function at physiological temperature.(31) Located in the β-sandwich strand S4, V157F mutations cause global effects on the DNA-binding core domain and loop-sheet helix motif, which directly contacts DNA.(32) Additional structural studies, however, have reported that the most common p53 mutant proteins exhibit a wide range of structural debility, suggesting that p53 mutations are selected for more than just a disrupted structure.(5,33)

Early in vivo studies of GOF effects by mutant p53 demonstrated allele-specific differences in tumor type and invasiveness.(34–36) Specifically, Olive and colleagues found, in genetically engineered murine models, that p53^R270H/− and p53^R172H/− mice exhibited similar survival to that of p53^−/− mice, but the heterozygous mutant p53 mice exhibited an increased incidence of epithelial tumors with evidence of invasion and/or metastasis while none of the null mice in the study developed carcinomas.(34) Similarly, p53^R270H/+ mice had an increased incidence of carcinomas, most commonly lung adenocarcinomas. In contrast, p53^R172H/+ mice were found to develop osteosarcomas at a rate twice that of p53^R270H/+ mice. These results together demonstrated GOF effects (invasion and metastasis) due to structural (R175H) and conformational (R273H) mutations in p53 and the emergence of distinct tumor spectra depending on the specific allele present. More recently, Hanel and colleagues demonstrated differential gain of function in tumorigenesis, with accelerated tumor onset of all tumor types and shortened survival in humanized p53 knock-in (HUPKI) p53hupki^R248Q/− mice, in contrast to p53hupki^G245S/− mice which were similar to p53 null mice in tumor latency and survival.(37) To examine the effect of p53 alterations on K-ras-induced lung cancer, Jackson and colleagues generated a compound conditional mouse harboring the LSL-K-ras^G12D allele and combinations of p53^LSL.R270H, p53^LSL.R172H, and p53^Flox alleles.(38) This work demonstrated that mice with endogenous expression of the contact mutant p53^R270H develop significantly greater tumor burden compared with mice expressing the structural mutant p53^R172H. The authors postulated that this difference in tumorigenic potential may explain the relative underrepresentation of mutations at the conventional hotspot codon R175 in human non-small cell lung cancers. Using a similar model, Turrell and colleagues found genotype-specific changes in transcriptional GOF, with increased dependency on mevalonate pathway gene expression in R270H but not R172H lung tumor cells.(39) Furthermore, p53^R270H mutant Kras^G12D-driven lung tumors exhibited a robust decrease in cell proliferation and survival upon mevalonate pathway inhibition by statin treatment, while p53-null and R172H lung tumors displayed no antiproliferative or apoptotic effect with statin therapy. Collectively, these data demonstrate that mutant p53 proteins exhibit GOF activities during tumor progression in vivo, and also that different hotspot mutations in p53 manifest GOF properties of varying magnitudes and tumor spectra. As yet, no murine studies have examined the lung-enriched p53 mutations at V157, R158, and A159.

Conclusions

We have proposed a paradigm for tissue-specificity in the selection of p53 mutations, which translates into varying frequencies of hotspot mutations unique to specific tumor types. Additionally, we hypothesize that while the unique pattern of mutations at V157, R158, and A159 in lung cancer is in part due to the mutagenic effect of tobacco carcinogens, these specific codons may also impart an oncogenic GOF leading to selection for V157, R158, or A159 mutations. Notably, the clustered nature of the lung-enriched loci suggests structurally or functionally related mutant p53 proteins, although the innate hypersensitivity of this genomic region to DNA adducts cannot be ruled out. From a translational standpoint, elucidating the biological activities associated with the lung-enriched p53 mutations will add to the growing understanding of mutant p53 gain of function and may allow for specific therapies targeted against GOF pathways. Existing treatment strategies against mutant p53 include restoration of wildtype p53 function or degradation of mutant p53 proteins. The GOF pathways induced or repressed by lung-enriched mutant p53 may identify actionable targets for precision therapy in certain subsets of lung cancer. As an example, statins hold promise in targeting the mevalonate pathway, which is responsible for disordered and invasive morphologic GOF exhibited by p53 R273H mutant breast cancers.(40) Many additional questions remain, including the impact of tissue- and microenvironment context on GOF activities and whether different mutant p53 alleles function through different molecular mechanisms.