Patterns of mutations in nine cancer-related genes and PAF development among smoking male patients diagnosed with bladder cancer

Abbreviations

1 Introduction

Bladder cancer (BC) is one of the most prevalent global health concerns. It is the sixth most widespread type of cancer, representing high mortality rates; for example, 16,870 deaths from BC have been registered just this year [1, 2]. BC is characterized by several critical symptoms, e.g., blood in the urine, regular urination triggers, and urination-related pain [2]. BC is also associated with the following risk factors: inheritance and family history, previous bladder complications, history of radiation therapy, and smoking habits [2].

In Saudi Arabia, high rates of smoking (48–70.7%) are indicative of huge health problems for the local population, which is especially relevant in young adults, particularly males [3]. Despite clear evidence that smoking can provoke various types of cancer and shorten life, a considerable number of male adults continue to smoke [4, 5]. To date, one of the leading causes of BC development is tobacco smoking [6]. Previous research revealed that smoking contributes to elevated platelet-activating factor (PAF) development and PAF receptor (PAF-R) manifestation, thus increasing chances of tumors and metastasis formation among smokers [1, 7]. PAF has been demonstrated to play a key part in the inflammatory process, thereby affecting endothelium with inflammation. Other studies have shown PAF’s principal role in provoking tumors and metastasis [8–13].

As urothelial cells are biologically vulnerable to cigarette smoke, smoking initiates the release of PAF, suppresses hydrolysis functions, and intensifies PAF-R manifestation [14]. Experiments with mice and long-term cigarette smoking showed signs of damage to urothelial function and high concentration of PAF within the bladder [1, 14].

This study focused on the impact of cigarette smoke regarding nine cancer-related genes, utilizing next-generation sequencing (NGS) technology. The data was confirmed via the Sanger sequencing method, which was applied to both smokers and non-smokers diagnosed with BC. Moreover, smoking and PAF accumulation with PAF-R manifestation in BC areas was studied with immunohistochemistry and histopathological tests, based on H&E staining.

2 Materials and methods

3 Results

3.1 Analysis of biological mutations

DNA of 116 male participants (54 smokers and 62 non-smokers) diagnosed with BC has been sequenced to embrace each coding exon of 9 cancer-associated genes utilizing NGS technology. They were later evaluated for biological (somatic) mutations contrasting data to the control group. NGS technology revealed that 13 biological mutations were identified in 7 of 9 cancer-associated genes of the DNA samples regarding both smoking and non-smoking groups of male participants with BC. All the identified biological mutations (both gains/losses) were placed at repeated sites with alternative patterns/frequencies of the deviations defined, comparing between Group 2 and Group 3 (Fig. 1). Group 3 of smokers with BC demonstrated a considerable rise in biological mutations among the selected genes contrasted to Group 2 of non-smokers with BC. Moreover, male participants from Group 3 held between 3-4 exclusive mutations compared to 1-3 exclusive mutations from Group 2 male participants.

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Fig. 1

Identified somatic/biological mutations noticed in all coding exons of 9 cancer-associated genes through NGS technology; derived from non-smoking (green lines) and smoking (black lines) male participants diagnosed with BC.

Observable mutations have been detected in ARID1A gene (9.71%), RB1 gene (10.34%), CDKN2A gene (13.01%), NRAS (13.22%), KRAS (16.73%), FGFR3 gene (16.00%), TP53 gene (18.01%), PTEN gene (23.02%) and PIK3CA gene (24.00%) in Group 2 male participants.

Moreover, the prevalent mutations have been identified among Group 3 male participants, such as ARID1A gene (12.71%), RB1 gene (14.31%), CDKN2A gene (18.11%), NRAS (17.19%), KRAS (21.62%), FGFR3 gene (21.03%), TP53 gene (26.12%), PTEN gene (29.10%) and PIK3CA gene (32.04%).

Based on findings from NGS procedure, most frequently mutated genes were 7 of 9 selected genes, specifically TP53, PIK3CA, FGFR3, CDKN2A, NRAS, KRAS and PTEN. Furthermore, genes identified have been confirmed on validity with a help of SSV (Fig. 2). Two biological mutations were identified as frameshift pathologies presented in exon 2 for genes KRAS & NRAS with a considerable amount of mutation patterns in Group 3 compared to Group 2.

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Fig. 2

Patterns of various mutations have been detected in TP53, CDKN2A, PIK3CA, FGFR3, HRAS, NRAS, KRAS and PTEN genes with a help of SSV; they were observed in non-smoking participants (green lines) and smoking participants (black lines) diagnosed with BC.

Changes of SSCA in exon 2 within KRAS gene have been recorded in 16/62 (16.12%) among non-smoking participants diagnosed with BC (Group 2); meanwhile, they have been identified in 12/54 (22.2%) among smoking participants with BC (Group 3) because of 12 ins A mutation. Moreover, small changes of SSCA in exon 2 of NRAS gene were also identified in 8/62 (12.9%) among non-smoking participants diagnosed with BC (Group 2). Respectively, they have been recorded in 9/54 (16.66%) among smoking participants with BC (Group 3) because of 61 ins T mutation.

SSV revealed that the most regularly developing mutations (>25%) were observable in 3 of 9 studied genes (namely TP53, PIK3CA and PTEN); moreover, a considerable rise in number of mutations was registered in Group 3 contrasted to Group 2 (Fig. 3). In comparison, all 3 identified genes were associated with frequently developing mutations ranged 18–24% in Group 2 (Fig. 1).

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Fig. 3

Electropherograms derived through the Sanger sequencing technique indicated of biological mutations in such genes as TP53, CDKN2A, PIK3CA, FGFR3, NRAS, KRAS and PTEN genes.

Moreover, regularly developing mutations in 2 of 9 selected genes (namely, ARID1A and RB1) were associated with the lowest values (>10%) in both testing groups which was confirmed by NGS (Fig. 1). Importantly, the nucleotide sequencing of such genes as TP53, PIK3CA, FGFR3, CDKN2A and PTEN demonstrated no presence of frameshift mutations and pathologies in above-mentioned genes. Similarly, there were no mutations identified above in the study’s control group.

3.1 PAF production

The extent of PAF is considered one of the vital signs of the inflammation. Figure 4 showed a striking increase of PAF production (**P<0.01) in Group 2 consisting of non-smokers with BC as compared to the control group. PAF production levels also elevated noticeably (***P<0.001) in Group 3 of smoking male participants with BC contrasting to Group 1 and Group 2.

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Fig. 4

PAF production in three groups of male participants with normal and abnormal levels of PAF. They were calculated referring to the blood serum taken from non-smoking participants (green lines) and smoking participants (black lines) diagnosed with BC. Vlues were presented as means±SEM (n = 30) of three trials. ***P<0.001, **P<0.01, *P<0.05 comparing to the values of the control Group 1.

3.3 Histopathology and immunohistochemistry

Outcomes of histopathological tests have been evaluated by a professional histopathologist. They were later graded by three separate tumor scores (Table 2). Each section of carcinoma affecting human bladder revealed barely identifiable characteristics of BC tissues from Group 2 and Group 3. In addition, a distinct rise in mitotic numbers, sizes, abnormal extent of proliferation rates in covering typical tissues with a minor number of nuclei and nuclear pleomorphism tendencies has eventually increased the composition of RBCs with making veins overfilled, causing necrosis, initiating intrusion of intracellular and extracellular elements, and leading to damage of cellular polarity, yet without considerable histopathological differences between Group 2 and Group 3 (Fig. 5, A and B).

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Table 2

Categories of tumor for patients with BC in Group 2 and Group 3

Covariate	Group 2: Non-smoker patients were diagnosed as having bladder cancer	Group 3: Smoker patients were diagnosed as having bladder cancer
No. pathologic grade
Low (1 + 2 grades)	51/62 (82.25%)	18/ 54 (33.33%)
High (3 grade)	11/ 62 (17.74%)	36/ 54 (66.66%)
No. lymph node status
Positive	3/ 62 (4.47%)	31/ 54 (57.40%)

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Fig. 5

Microscopic histopathology provides images of cross sections of BC obtained from both Group 2 and Group 3 and analyzed under light microscopy, magnitude 100μm. H&E stain was added to A and B samples demonstrating imbalance in bladder cells, showing atypical sizes and forms for normal cells. Section A was obtained from Group 2; section B was obtained from Group 3, both demonstrating occasional textures of bladder tissues showing abnormal mitotic signs, decreased nucleoli, necrosis effects, presence of intracellular/extracellular elements and elevated level of RBCs with several overfilled veins. Sections C and D demonstrated immunity to stains by releasing PAF-R antibody. Section C was obtained from Group 2 demonstrating weak yet positive staining of PAF-R protein in non-smoking male participants with BC; meanwhile, Section D was obtained from Group 3 demonstrating strong and positive staining of PAF-R protein in male smokers with BC.

Later, immunohistochemistry evaluations indicated about nuclear staining in approximately 20% responses for weak positivity in relation to PAF-R antibody, which was observable in Group 2 (Fig. 5, C). Additionally, manifestation of PAF-R was identified as positive above 50% in all the rest bladder tissues affected by tumors (Fig. 5, D). It should also be mentioned that the practice of antigen sampling has been measured and performed in compliance with the latest registered quantitative H-scores for each case. It was completed by multiplying the values in relation to the following established categories: above 50% (robust), 20–40% (medium), 1–20% (weak), and 0% (none).

Current research relied on clinicopathological findings from different groups of participants (Table 1). This confirmed that smoking cigarettes raised health risks associated with BC development, which was supported by findings from another study that identified pyroptosis of urothelial cells via the ROS/NLRP3/caspase-1 signaling channel, provoked by cigarette smoke [23–25]. Moreover, the relation between nephropathy, renal failure, and BC has been confirmed. In addition, other research suggested that BC could be provoked among smokers and non-smokers depending on their medical history, e.g., hypertension, peripheral arterial disease, and diabetes [26–29]. Smoking also contributes to the progression of BC in late stages of life. As such, age remains a critical risk factor for eliciting umbilical cord blood (UCB) and accelerating the organism’s degradation with an official diagnosis [30]. Nonetheless, our data from Table 1 revealed that the diagnosis of BC is not differentiated by age among patients, although it was shown that various carcinogens (tobacco and smoking chemicals) contribute to its development, with a patient’s genetic risks and inheritance, independent of age [30]. In context of a patient’s smoking history, it was found that non-smoking patients have less active and severe forms of BC compared to their smoking counterparts, as shown in Table 2. Recent research showed that smoking may directly provoke development of invasive cancer cells with a much higher likelihood [30, 36]. Previous studies reported that the population-attributable risk with BC with tobacco smoking is 4 times higher in men than women, and current cigarette smoke triples BC risk compared to never smoking [30, 37].

The process of how cigarette smoke provokes metastasis, specifically in the lungs, has not yet been studied [32]. Smoking, despite being a factor of high morbidity and mortality, is still a popular habit at 22.1%, with Saudis using cigarettes each day. Statistically, 43.7% of smokers are males, with the female population of smokers representing 16.5% [33]. An average cigarette contains approximately 7,000 chemicals, and more than 70 are scientifically categorised as carcinogenic. This means that these chemicals can damage the cellular structure of DNA and initiate the process of tumorigenesis [34]. This not only damages DNA, but in mice studies it decreased recovery capacity in the lungs, heart, and bladder; in addition, similar degradation effects were noted in human studies in context of lung and bladder cells [35, 36].

Moreover, cancer-associated genes (comprising oncogenes and tumor suppressor genes) are mutated, causing cells to grow rapidly, thus leading to cancer. Among many cancer-associated genes, nine BC-associated genes were selected as the most common in this study [24, 38], relying on NGS technology, which identified thirteen observable mutations in 7 of 9 cancer-associated genes of DNA samples among non-smoking and smoking participants with BC. These mutations were confirmed by structural signature variation (SSV), detecting a similar number of mutation patterns in the studied genes. Other research determined growing mobility of tumor cells in the bladder, which increased urothelial cell damage with cigarette smoke’s chemicals [1, 36]. These findings uncover biological mechanisms by which smoke may generally induce BC progression, although details of that process are still unclear. Nonetheless, given that metastasis (not always the tumor itself) leads to 90% of deaths in cases with a BC diagnosis, with scientific efforts directed to study mechanisms of how cigarette smoke can prompt metastasis that in turn leads to cancer complications [32].

Other research demonstrated that PAF triggers pro-inflammatory cells, including macrophages and neutrophils through selected inflammatory channels; this was claimed to contribute to diverse biological processes, such as epithelial apoptosis, growth of vascular permeability, and smooth muscle contractions [33, 40]. In connection with reproductive function, anti-apoptosis, and DNA repair, it was suggested as a potential cause of cancer progression [36]. The research found that PAF suppression decreased adhesion to endothelial cells, reducing metastasis in melanoma and colon cancer cells [39, 40].

The current study presented outcomes of the ELISA assay: they identified that PAF was increasingly generated in the blood serum of participants from Groups 2 and 3, unlike participants from the control group. Specifically, the highest values of PAF (***P<0.001) were found in Group 3, or smokers with BC. Moreover, immunohistochemistry tests revealed a decline of PAF-R protein manifestation in BC samples among non-smokers (Group 2) in contrast to BC areas among smokers (Group 3). As histopathological tissues were stained with H&E, it was demonstrated that there were no considerable differences between Groups 2 and 3. With a close tie to cigarette smoking, inflammatory channels of spread are essential preconditions of BC development, with elevated PAF release potentially contributing to cancer cell development, spread, and tumor tissue production, confirmed and described by several past studies [11, 38–40].

In the end, it may be argued that smoking cigarettes activates PAF development and results in the urothelial inflammation process, which is further related to serious risks of BC and chances of mutation in nine cancer-related genes (shown in the data of this study, in which PAF levels were almost more than three times that of Group 3, compared to PAF levels in Group 2). These risk factors can be initiated in various signaling channels and pathways to eventually create the invasion of cancer cells in the bladder’s environment. In fact, the evaluation of nine cancer-related genes is a good prediction tool that defines risks of BC development in smokers, with a focus on PAF development and receptor (PAF-R) manifestation due to urothelial inflammation. Future studies must focus on other cancer-related genes to discover new somatic and biological mutations in smokers with a BC diagnosis to be able to compare them with non-smoking patients who have BC.