Tobacco Smoking is Positively Associated with Hepatocellular Carcinoma Due to the p53 Mutation at Serine 249


 Background The major risk factors for hepatocellular carcinoma (HCC) are known to be hepatitis b virus (HBV) infection and aflatoxin B1(AFB1) exposure. However, there is still controversy about whether smoking is related to HCC.Methods A binary unconditional logistic regression was used for the data about a total of 300 cases and 612 controls. The approach of functional analysis of separated alleles in yeast (FASAY) was applied to analyze p53 status in HCC group. The relationship between p53 mutation at Serine 249 (p53-RS) and smoking was assessed. Quantitative reverse-transcription PCR (qRT-PCR) was employed for the evaluation to transcriptional activity of p53 and p53-RS. Results Smoking was linked to the risk of HCC with an increased dose-response effect. Moreover, among subjects without alcohol drinker, the risks of HCC were significantly increased for smokers between HCC and controls. Besides, there was an increase in the number of HCC in smokers compared to non-smokers after exclusion of HBV and/or HCV infection. Also, a significant difference was observed about p53-RS between smokers and nonsmokers. Furthermore, the p53-RS transcriptional activity was significantly increased in tumor tissues.Conclusions It demonstrated that tobacco smoking is positively and independently associated with HCC, which may be attributed to p53-RS and its gain of function.


Background
Hepatocellular carcinoma (HCC) accounts for > 80% of primary liver cancers, which is one of the most common and deadly cancers worldwide [1]. HCC causes a heavy disease burden and is the leading cause of cancer-related deaths in many parts of the world. It is estimated that HCC is the fourth most common cause of cancer-related deaths worldwide [2]. Even HCC is not the most common cancer in most parts of the world, and its high mortality rate (the incıdence to mortality rate is 0.95) and short survival (5-year survival rate is only 6.9%) create a serious global health burden [3]. Multiple risk factors leading to HCC have been well identi ed, including chronic HBV and/or HCV infections, carcinogen exposure (e.g. a atoxin B1, AFB1), excessive alcohol consumption, and multiple genetic factors [4,5].
Chronic HBV and HCV infections are the most important causes of liver cancer, accounting for 80% of global HCC cases [6].
Chronic HBV infection is a major cause of HCC in East Asia and most African countries, with the exception of North Africa, where HCV prevalence is highest [7,8]. HBV infections are usually obtained vertically from mothers to children in China and other parts of Asia, while sibling transmission is more common in Africa [9]. Overall, the annual incidence of HCC in patients with cirrhosis caused by chronic HBV or HCV infection is 2-5% [10].
A atoxins are difuranocoumarin derivatives of Aspergillus avus and A. parasiticus. These fungal contamination occurs both during crop growth and due to the improper storage. High levels of exposure to fungal toxin are widely distributed in sub-Saharan Africa and the Asia-Paci c region. AFB1 is the a atoxin most often found in contaminated human foodstuffs and is the most potent hepatocarcinogen [11]. One study con rmed the effectiveness of preventing post-harvest contamination through simple interventions such as drying crops on cloth rather than soil, hand sorting to remove moldy crops and better storage practices [12].
Heavy drinking (> 80 g/day) for more than 10 years increases the risk of HCC by approximately 5 times [13]. Annual incidence of HCC in patients with alcoholic cirrhosis is 1-2% [14]. Alcohol consumption and viral hepatitis have synergistic effects on HCC. Alcoholic cirrhosis is the second most common risk factor for liver cancer in the United States and Europe [15]. The World Cancer Research Fund conducted a meta-analysis of 19 studies [n = 5,650] and found a statistically signi cant increase in risk of 4% per 10 grams of alcohol intake per day, with odds ratios (OR) being 1.04 and 95% con dence intervals (CI) being 1.02 to 1.06, respectively [16].
However, the contribution of smoking to liver cancer risk remains unclear, unlike HBV and HCV infections, a atoxin expression, and excessive drinking. Although smoking is also associated with HCC risk in case-control and cohort studies in different populations, the validity of such results is often questionable as a result of confounding effect of alcohol [17][18][19], which is often too closely correlated smoking. Similarly, in populations where HBV and HCV infection is endemic, it may be also di cult to rule out the infection as a confounding factor [20]. Moreover, HCC only develops in a small part of the exposed population, which means that genetic factors may contribute to the carcinogenic mechanism. In the present study, we also further sought to understand more fully the critical determinants for the carcinogenic activity of tobacco smoking. It showed a signi cant dose-response, positive association between smoking and HCC risk. Moreover, there was a statistically signi cant correlation between p53 mutations in serine 249(p53-RS) and smoking status.

Study population
The study had a hospital-based case-control design. Between June 2015 and December 2018, we enrolled 300 cases HCC, and those who were diagnosed within 1 year prior to admission were also regarded as eligible. HCC diagnosis is based primarily on pathology, cytology or an elevated alpha-fetoprotein levels (400 ng/ml) as well as positive results from at least two diagnostic imaging examinations, including angiography, ultrasound and computed tomography scans. The control group subjects were those who did not suffer from liver disease, such as chronic hepatitis or hepatic cirrhosis, as well as neoplasms and tobacco-or alcohol-related disease, and were genetically unrelated family member (i.e., spouses and in-laws) and matched for age (within the same 5-year age class), gender, residence (prefecture) and time of hospitalization (with 2 months after a case interview).

Laboratory testing
HBV and HCV were tested from the blood samples of the case (n = 300) and control group (n = 612). Hepatitis-B surface antigen (HBsAg) was detected by an immunoassay (Bio-Rad Laboratories, Redmond, WA). Antibody to hepatitis C virus (anti-HCV) was determined by a second-generation enzyme immunoassay (Bio-Rad Laboratories, Redmond, WA). Results were interpreted according to the manufacturer's instructions. Positive results prompted repeated con rmatory enzyme-linked immunosorbent assay testing. The technician who performed the blood test turned a blind eye to the identity of the participant and the disease. Peripheral blood samples, including white blood cells and serum, were collected from patients and controls and frozen at -80℃ until analysis.

Questionnaire
All HCC patients and hospital controls were interviewed in the hospital. Personal general information including age, gender, height, alcohol and smoking status was elicited during questionnaire-based interviews. Also, questionnaires were structured to obtain information on participants' weight before cancer diagnosis (HCC patients) or recruitment (control group). the body mass index (BMI) was estimated based on participants' weight (kg) and height (m). Drinkers are de ned as individuals who drink any alcoholic beverage monthly or more often. Anyone who quit smoking more than three years before HCC onset is considered a former smoker. Those who quit smoking three years before the diagnosis are considered current smokers. Besides, former smokers were classi ed on basis of the number of smoking per day in the past, while current ones were classi ed in line with the daily number of cigarettes smoked before the onset of HCC or a corresponding date in the controls.

p53 determination
All patients underwent complete hepatectomy. In each case, tumor fragments and corresponding normal tissues were obtained and partially frozen in liquid nitrogen and stored at -80 °C, until further testing of p53 transcriptional activity to prevent RNA degradation. The remaining tumor materials were xed in buffered formalin (10%), embedded in para n and sliced, followed by staining with hematoxylin-eosin for pathological evaluation. Pathological classi cation, including tumor grade and staging, was assessed on the basis of the world health organization system and TNM classi cation [21], respectively.
The functional status of p53 gene was determined by the functional analysis of separated alleles in yeast (FASAY) method described previously [22]. Brie y, according to the manufacturer's instructions, use the RNeasy Mini kit to extract total RNA from the tumor material, and then reverse transcribe the RNA sample at 37 °C. PCR ampli ed p53cDNA with the following primers: 5'-CCT TGC CGT CCC AAG CAA TGG ATG AT-3' for P3 and 5'-ACC CTT TTT GGA CTT CAG GTG GCT GGA GT-3' for P4 (Invitrogen, Carlsbad, CA, USA) and Pfu DNA Polymerase (Stratagene). The crude polymerase chain reaction product and linearized p53 expression vector were co-transfected into the yIG397 of yeast reporter strain. The transformed yeast cells were plated on a minimal medium without leucine and incubated with a small amount of adenine (5 µg/ml) at 35 °C for 2-3 days and then at room temperature for 2-3 days. The experiment was repeated at least twice to determine the average percentage of red colonies. p53 status was considered mutated on the basis of the following criteria from Lehmann-Che et al [23]: (a) more than 10% of the yeast colonies were red, (b) defects in the 5' or 3' section of the gene can be identi ed through analysis of the split versions of the test, and (c) sequence analysis of mutant yeast colonies can identify clear genetic defects. Sequencing the presence of more than four clonal mutations in yeast colonies was the nal criterion for de ning mutations.

RNA isolation and quantitative reverse-transcription PCR (qRT-PCR) analysis
Following the manufacturer's instructions, RNA kit was used for the extraction of samples from freshly frozen tumor tissue and normal non-tumor mucosa (RNeasy Lipid Tissue Qiagen kit, 74804; Qiagen, Valencia, CA). qRT-PCR was done using IQ SYBR green (Bio-Rad, USA) and GAPDH as reference gene. The relative expression of mRNA was determined by the comparative Ct Method (ΔΔCt method). Each sample was analyzed in triplicate by qRT-PCR. The designed primer sequences were as follows:

Statistical analysis
All statistical analyses were performed through SPSS for Windows (version 25.0, Chicago, IL). Differences between the cases and controls in the distributions of demographic characteristics were accessed by Chi-square test (qualitative data) and an unpaired two-tailed Student's t-test (quantitative data). Also, using hierarchical analysis and unconditional Logistic regression model to generate OR with 95% CI for HCC risk as relative risk estimation [24]. Because it is impossible for some HCC cases to match individual, it is necessary to carry out unconditional logistic regression model instead of the conditional, with adjusting the matching factors. Likelihood ratio test is the difference between maximum logarithmic and likelihood statistics, and it is used to evaluate the signi cance of additional covariates in the model [24]. All p-values quoted were two-sided and signi cant differences were considered at a p < 0.05.

Tobacco smoking and its interaction in the causation of HCC
The distribution of factors including demographic characteristics of chronic HBV infection, HCV infection and serological evidence between HCC cases and hospital controls, as well as smoking status and alcohol consumption, was showed in Supplementary Table 1. Cases and controls were similarly distributed by age, gender and body mass index. There were, as expected, signi cant differences (p < 0.05) in gender status and prevalence of HBV and HCV infection markers, because these are the main reasons for HCC. Also, it was interestingly found that smokers were signi cantly more likely to the causation of HCC than non-smokers (χ 2 = 67.131, p = 0.000) and smoking was more common among cases than among controls.
We examined the cigarette smoking-HCC association. Table 1 showed the distribution of HCC cases and hospital controls by smoking habits. The distributions were gender-speci c, owing to the expected differences about these activities in terms of prevalence by gender. Smokers were signi cantly associated with HCC. The corresponding estimates were 3.500 (95% CI = 2.518-4.866) and 2.962 (95% CI = 2.014-4.355) for current smokers and former smokers. In terms of dose, there appeared to be a dose-response relationship between HCC and quantity smoked with adjusted OR estimates of 3.267 and 4.375 for smoking < 2 packs per day and ≥ 2 packs per day for current smoker, respectively. Likewise, signi cant OR variation was found when considering only a group of subjects among former smoker, the ORs for < 2 packs per day and ≥ 2 packs per day being 2.450 (95% CI = 1.572-3.817) and 4.667 (95% CI = 2.451-8.885), respectively. Altogether, it demonstrated that smoking was associated with an increased risk of HCC according to the adjustment of age and gender, but mutual confounding precludes alcohol intake and valid etiological inferences.

FASAY for detection of p53 mutations
p53 gene mutation is one of the most common genetic changes in human cancer. The advantage of these mutations (95%) is missense mutations, which are mainly located in the DNA binding domain (amino acids 94-292), with hot spots at codons R175, G245, R248, R249, R273 and R282 [25,26]. FASAY was proved to reach the highest sensitivity and speci city of p53 mutation detection [27,28], due to the fact that a larger region of p53 gene in exon 4-10 was tested. Then mutations can be detected in a large number of normal tissues, because there are hundreds of clones in each sample, and simple red and white readouts mean mutations are not easy to ignore [29]. In addition, FASAY can exclude p53 polymorphisms that do not affect their transcriptional activity [30]. To detect the status of p53 in HCC tumors, the tumor mucosa of 300 patients was FASAY examined (Supplementary Table 2). In total, 228 tumors (76.00%) were positive (mutant) by FASAY. Besides, analysis of plasmid sequencing from red yeast colony showed that 210 of 228 plasmids were point missense mutations, and 6 of 228 was frameshift deletion mutation, as well as 9 of 228 were in-frameshift deletion mutations, and 3 of 228 were splicing mutations (Fig. 1A). Moreover, hotspot mutation p53-RS that occurred 126 times in this study, accounting for 55.26% in total mutations. We further evaluated the relation between tobacco smoking and p53-RS mutation. A striking increase in the risk of p53-RS was observed for smokers (Fig. 1B). Furthermore, these differences about p53-RS between smokers and nonsmokers were statistically signi cant by Chi-square test (56.67% versus 20.00%; χ 2 = 38.246, p = 0.000). These results suggested that p53-RS mutation might be associated with the response to cigarette smoking. We also sought to determine whether the presence of p53-RS has a measurable effect on the expression of p53 major transcriptional targets (p21 and Bax), as well as p53-RS-dependent target genes including c-Myc [31] and STAT1 [32] by using qRT-PCR on mRNA from fresh frozen components. For p21 and Bax, we found a decreased pro le in p53-RS group compared with the WT p53 group (Figs. 1C and  1D). However, in contrast to WT p53 group, we observed an increased expression about c-Myc and STAT1 in the tumors with p53-RS, showing a loss of transcriptional p53 function and p53-RS gain-of-function (Figs. 1E and 1F).

Discussion
Whether smoking is a risk factor for HCC is currently controversial. Although an increased risk of HCC is observed in smokers, there are no signi cant difference [33]. Besides, researches also report that smoking is a potential risk factor for HCC [18].
However, it is limited about exploration of the combined effects of these exposures such as HBV and HCV infection, and alcohol consumption. Synergism among these common factors would have substantial clinical relevance to smoking because it would place some individuals at extremely high risk. Thus, our goal is to carry out a clear examination to independently in uence of the smoking on liver cancer in this study, without any alternative in uence on HBV and HCV infection, as well as alcohol consumption.
Indeed, our results also indicated that HBV and HCV infection, and alcohol consumption increase the risk for liver cancer. This observed association was in agreement with those of most recent studies on this topic [34]. it was also shown that smoking is an independent risk factor for HCC in the present study (  Table 2). Also, we found evidence that smoking is an important cause of HCC in people with non-chronic HBV or HCV infections (< 2 packs per day, OR = 1.727; ≥2 packs per day, OR = 3.855, Table 3). However, HCC develops in only a few exposed populations and the complex interactions between many genetic and environmental factors may be the major cause of HCC.
p53 is a well-known tumor suppressor gene whose mutation plays an important role in the pathogenesis of HCC. Moreover, it is suggested that p53-RS mutant protein products may have speci c biological effects that promote hepatocyte transformation and/or liver cancer progression, resulting to the selection of cells expressing this mutant during HCC [35]. FASAY can use yeast cell yIG397 to evaluate the functional status of p53 protein expressed in tumor cells, thereby detecting important functional mutations of p53 as a transcription factor. Also, the assessment of p53 transcriptional activity by FASAY in cancer cells has recently emerged as a useful valuable alternative to p53 nuclear overexpression [36]. Furthermore, it is validated the assay as a new tool in mutagenesis research can be used to study the mutagenicity on the origin of the codon 249 hot spot of p53 mutation [37]. Remarkably, our studies showed the various types of p53 mutations in HCC by FASAY in the current work, and that most of them were p53-RS, accounting for 55.26% in total mutations (Supplementary Table 2). Moreover, it demonstrated the strikingly relation between tobacco smoking and p53-RS (Fig. 1B, p < 0.001). Also, at the functional level, the effect what we observed was the decrease of expression of p21 and Bax at the mRNA level in many mutated tumors as a direct consequence of the p53-RS, whereas the mRNA levels of the p53-RS targets (c-Myc and STAT1) seemed to be increased (Figs. 1C-1F). Altogether, it strongly demonstrated tobacco smoke as a causal factor of HCC development, which was attributed to p53-RS gain of function.
However, several limitations should be considered. First, our research enrolled the population by convenience sampling, which cannot represent the whole population in China, even in the world. Second, variables such as alcohol status is not available in the data, which is considered as potential risk factors of HCC.

Conclusions
Our results suggested that tobacco smoking is associated with HCC, independent from other major HCC risk factors, and this dose-response association was positive signi cantly. Besides, the present investigation indicated, that the p53-RS might confer genetic susceptibility that in uences HCC development especially in smoker's patients. Based on these ndings, it is conceivable that smokers have a high potential for HCC, who should receive clinical monitoring to p53-RS, and even treatment.

Supplementary Files
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