The association of genetic factors with serum calretinin levels in asbestos-related diseases

Prolonged asbestos exposure can lead to occurrence of different asbestos-related diseases, including pleural plaques and asbestosis, as well as several cancers. Use and production of asbestos was largely banned after it was classified as a carcinogen, but it is still legally used in mostly developing countries and it can still be found in the environment.^1,2 Asbestos-related diseases often occur long after initial asbestos exposure and their incidence continues to rise.¹

The most problematic asbestos-related disease is malignant mesothelioma (MM), a rare but very aggressive cancer. However, only a minority of asbestos-exposed people develops MM. Other factors, such as genetic variability may contribute to carcinogenesis and development of MM.³ Among asbestos-exposed workers, several familial cases of MM were described, emphasizing that genetic factors could contribute to MM development.⁴ In recent years, germline BRCA1-associated protein 1 (BAP1) mutations were shown to predispose to the development of MM and other cancers. Additionally, studies suggest that numerous chromosomal deletions can accumulate in most MM cases, usually associated with the loss or inactivation of tumor suppressor genes.^5,6 Despite advances in treatment, prognosis and survival of MM patients remain poor.^7,8 Therefore, MM diagnosis and treatment have become increasingly focused on molecular mechanisms.⁹

To confirm MM diagnosis, several tumor markers are routinely analysed using immunohistochemical staining.¹⁰ One of the established immunohistochemical markers is calretinin¹⁰, a calcium binding protein and calcium sensor crucial for neuron function that is also expressed on mesothelial cells.¹¹ It has been shown to affect mesothelial cell proliferation and migration and epithelialto-mesenchymal transition. It was also associated with focal adhesion kinase signaling pathway and signaling pathways associated with response to asbestos.¹² Calretinin is encoded by the CALB2 gene.¹³

As MM diagnosis is usually made when the disease is already advanced, blood-based biomarkers such as mesothelin and fibulin-3 that would enable an earlier diagnosis and better prognosis of MM are extensively studied.^14,15 Recently, calretinin was also proposed as a soluble biomarker in MM, as increased plasma or serum levels were observed in MM patients compared to subjects with other asbestos-related diseases or healthy controls.^8,16,17,18 However, interindividual variability limits the sensitivity and specificity of calretinin as a diagnostic biomarker and several clinical characteristics were previously associated with soluble calretinin levels.¹⁹ Low tumor calretinin expression was associated with lower protein concentration in the bloodstream, but there was no clear correlation with tumor size.²⁰ Higher calretinin concentrations were observed in patients with epithelioid or biphasic MM compared to patients with sarcomatoid MM.^8,20,21 Calretinin levels were also higher in women compared to men and in subjects with renal dysfunction.²²

Molecular mechanisms regulating calretinin expression in various tissues or in cancer could also contribute to interindividual variability of serum calretinin concentration, but the knowledge of these processes is limited.²³ Calretinin expression may be affected by several factors, including transcription factors or miRNAs. Among transcription factors, calretinin expression was found to be influenced by septin 7, E2F transcription factor 2 (E2F2) and nuclear respiratory factor 1 (NRF-1) in previous studies.^23,24 Additionally, miR-335-5p was proposed as a regulator of CALB2 expression²⁵ and miR-30e-5p was negatively correlated with the calretinin expression in pleural MM patient samples.²⁶ Gene expression can also be modified by genetic variability in the promoter 5′ untranslated region (UTR) of the gene affecting binding of transcription factors, or genetic variability in the 3′ UTR affecting miRNA binding. Polymorphisms in genes coding for miRNAs or transcription factors involved in calretinin regulation could also influence calretinin expression. In previous studies, genetic factors affecting expression and circulating levels of other important MM biomarkers such as mesothelin have already been identified.^27,28,29 On the other hand, very little is known about the role of single nucleotide polymorphisms (SNPs) in the CALB2 gene. An intronic polymorphism in CALB2 gene was previously proposed as a risk factor for colon cancer.30 To date, no studies have been performed to evaluate if genetic factors influence calretinin expression or if they could modify susceptibility to develop asbestos-related diseases.

Our aim was to determine whether genetic polymorphisms in the CALB2 gene and in the genes coding for miRNA and transcription factors regulating calretinin expression are associated with MM susceptibility or serum calretinin levels in patients with asbestos-related diseases.

In the present study, we evaluated the role of genetic variability in CALB2 and its regulatory miRNA and transcription factors genes with serum calretinin levels and MM susceptibility. Genetic variability of CALB2 was associated with calretinin concentration, but not with MM susceptibility. For SNPs in genes regulating calretinin expression, differences in genotype frequencies among MM and other subjects were also observed. Additionally, genetic factors influenced optimal serum calretinin cut off values differentiating MM patients from other asbestos-exposed subjects.

Using bioinformatic analysis, we identified seven common putatively functional SNPs that could affect calretinin expression: three SNPs in CALB2 gene, one SNP in transcription factor E2F2, one SNP in transcription factor NRF1, one SNP in transcription factor SEPTIN7 and one SNP in miRNA MIR335. In previous studies, demographic and clinical factors such as sex and renal function affecting plasma or serum calretinin concentration in asbestos-related diseases were already identified^21,22,41, but the role of genetic variability is largely unexplored.

Among CALB2 SNPs investigated in our study, CALB2 rs1862818 and CALB2 rs889704 may influence transcription factor binding, while CALB2 rs8063760 may influence miRNA binding. In our study, CALB2 rs889704 was associated with lower serum calretinin levels in all subjects and subjects without asbestos-related diseases, while there was no association in patients with MM. None of the selected CALB2 SNPs or haplotypes were significantly associated with MM susceptibility. To the best of our knowledge, the functional role of CALB2 SNPs and their association with asbestos-related diseases was not investigated yet. However, one intronic SNP in CALB2 was previously associated with calretinin expression in tumor cell lines and the development of colon cancer, but no association with lung cancer was observed.³⁰ Data on CALB2 genetic variability are therefore lacking and further studies are needed to evaluate its role in MM and serum calretinin levels.

Three important transcription factors were previously associated with regulation of calretinin.^23,24 E2F2 is a transcription factor that binds to CALB2 promoter and was associated with calretinin expression in mesothelioma cell lines.²³ In our study, E2F2 rs2075995 was associated with decreased MM risk. When comparing MM patients to only subjects without disease, the association remained significant even after taking into account the age of the subjects. E2F2 rs2075995 was also associated with higher serum calretinin level among subjects with asbestosis. E2F2 has an important role in the regulation of cell cycle, but also affects other important processes such as cell proliferation, apoptosis and inflammation.⁴² In cancer, it was mostly associated with promoting tumor progression in various malignancies, including lung cancer.⁴² E2F2 could also contribute to the cell cycle-dependent differences observed for calretinin expression.²³ E2F2 rs2075995 is a nonsynonymous SNP and may influence splicing. So far, E2F2 rs2075995 was only evaluated in patients with colorectal cancer, where no association with cancer risk was observed.^43,44 However, no studies evaluated the association of E2F2 rs2075995 with MM. Still, the E2F gene family was often associated with different types of cancer. Several other E2F2 polymorphisms were associated with oral and oropharyngeal squamous cell carcinoma risk and might also affect the course of the disease.⁴⁵ Combinations of different E2F2 gene SNPs were proposed as a risk factor for squamous cell carcinoma of the head and neck.⁴⁶ The E2F2 gene was also associated with ovarian cancer risk.⁴⁷ Additionally, E2F2 genetic variability was proposed as recurrence biomarker in squamous cell carcinoma of the oropharynx.⁴⁸ Among other E2F2 SNPs, rs3218211 was in very high LD with rs2075995 investigated in our study. E2F2 rs3218211 was associated with T stage in oral and oropharyngeal squamous cell carcinoma and decreased head and neck squamous cell carcinoma risk, consistent with our results.^45,46 Taken together, this suggests further studies regarding the role of E2F2 genetic variability in asbestos-related diseases and its association with calretinin are needed.

The second important calretinin-related transcription factor is NRF-1. It binds to CALB2 promoter and might be important for the transcriptional control of calretinin expression in MM.²³ In our study, NRF1 rs13241028 was associated with lower serum calretinin level in subjects without MM, but it was not associated with MM susceptibility. NRF-1 regulates expression of various genes involved in oxidative phosphorylation, mitochondrial biogenesis and other mitochondrial processes, including transcription of mitochondrial DNA.⁴⁹ Additionally, NRF-1 can modify different aspects of carcinogenesis, including proliferation, invasion, and apoptosis.⁵⁰ NRF1 rs13241028 may influence miRNA binding.⁵¹ So far, NRF1 genetic variability has been associated primarily with increased susceptibility to diabetes.^52,53 NRF1 has also been associated with epithelial ovarian cancer risk.⁵⁴ Further studies are needed to better evaluate the role of NRF-1 and its genetic variability in asbestos-related diseases.

Septin 7 has also been identified as a factor that binds to the CALB2 promoter region, resulting in decreased calretinin expression in mesothelioma cell lines.²⁴ Septin 7 is a GTP-binding protein that is involved in cytokinesis, cytoskeleton organization and other cellular processes.^24,55 It was also implicated in calcium homeostasis.⁵⁶ Several studies also reported that septin 7 plays an important role in cancer development, especially glioma.^55,56 In our study, SEPTIN7 rs3801339 was not significantly associated with MM susceptibility or with serum calretinin levels. The functional role of SEPTIN7 rs3801339 is not yet understood: it was previously classified as a non-synonymous variant, while it is now described as a genic downstream transcript variant. Interestingly, SEPTIN7 rs1143149 in moderate LD with rs3801339 was proposed as a risk factor for the development of non-small cell lung cancer and was associated with shorter survival in long-term smokers.⁵⁵ SEPTIN7 was often mutated in breast ductal carcinoma in situ cell lines and these mutations might participate in the progression of breast ductal carcinoma.⁵⁷ Recent studies therefore suggest that SEPTIN7 variability may play a role in some cancers, but it was not an important risk factor in asbestos-related diseases in our study.

MiRNAs affect gene expression on the post-transcriptional level and are often deregulated in cancer.⁵⁸ Among miRNAs predicted to modify calretinin expression, common polymorphisms were only described for miR-335. In our study, carriers of two polymorphic MIR335 rs3807348 alleles were more likely to develop MM compared to subjects with asbestosis, even after adjustment for age. MIR335 rs3807348 was also associated with serum calretinin level in subjects without MM. MiR-335 can modulate cell proliferation, apoptosis, migration and invasion through various signaling pathways. It mostly acts as a tumor suppressor and is downregulated in different cancer types.⁵⁸ MIR335 rs3807348 may influence transcription factor binding, but its role has not been experimentally confirmed. To date, no research has been done on the association of rs3807348 with MM. MIR335 rs3807348 was not associated with breast cancer risk in a previous study⁵⁹, but more studies would be needed in this field.

As several genetic factors were associated with calretinin, we also evaluated how these factors influence serum calretinin cut off values. We found that four SNPs, CALB2 rs889704, E2F2 rs2075995, MIR335 rs3807348, and NRF1 rs13241028 could be used to fine tune serum calretinin cut off values predicting MM. Calretinin as a biomarker could thus have higher sensitivity and specificity in individuals with known genetic variability. Similar results were observed for mesothelin, where predictive value was improved when taking into account polymorphisms located in 5′ UTR and 3′ UTR of the MSLN gene.27–29 In the future, combination of clinical and genetic factors could thus help guide calretinin cut-off values and decrease false negative or positive results.

This is the first study to show that genetic factors can affect serum calretinin levels and that accounting for these genetic factors may improve the predictive value of serum calretinin. We have also shown that genetic factors associated with calretinin may play a role in the development of mesothelioma. A limitation of our study is that we only had serum calretinin concentrations available for a subgroup of participants included in the study. On the other hand, we performed a comprehensive analysis of the factors that could affect calretinin expression using literature review and detailed bioinformatics analysis. Genetic variability was evaluated in a large cohort, which gives additional power to the study. However, other polymorphisms in the investigated genes could also affect calretinin concentration and other factors could affect calretinin regulation. In the future, further studies in this field and validation of these results in an independent population are needed.

The present study showed that genetic variability in CALB2 gene and genes coding for transcription factors and miRNAs that regulate calretinin expression could contribute to interindividual differences in serum calretinin levels in MM patients or asbestos-exposed subjects. These results could contribute to a better understanding of calretinin regulation and could potentially contribute to an earlier diagnosis of MM.