Genomic Investigation for Potential Therapeutic Use of Abiraterone in the Amazonian Indigenous Population

1. Introduction

The worldwide incidence of prostate cancer, as reported by the World Health Organization, reached 1.2 million individuals, securing the fourth position in terms of case numbers. The mortality rate stands at approximately 40%, with studies suggesting that poorer prognoses are more closely associated with the European population [21].

The clinical approach to addressing this type of cancer depends on the stage of the disease. In more advanced stages, according to the National Comprehensive Cancer Network (NCCN), one of the approaches involves implementing androgen deprivation therapy (ADT). In this context, one of the drugs used is abiraterone [17], which acts as a selective inhibitor of the enzyme CYP17.

Even after the implementation of ADT protocols, some patients develop a more aggressive form of the disease called castration-resistant prostate cancer (CRPC), in which there is high expression of steroid hormones that ultimately promote disease recurrence and shorter survival time.

After studies, it has been observed that individuals who progress to CRPC exhibit variants in the HSD3B1 gene. This gene is already described as a biomarker for the occurrence of CRPC and has clinical indications on the PharmGKBD portal [1,3] and regulatory bodies such as the FDA.

Focusing on the ADT protocol, the signaling pathway of the drug abiraterone was examined, revealing five related genes: SLCO2B1, SULT2A1, CYP3A4, HSD3B1, HSD3B2, and UGT1A4. As can be seen, the HSD3B1 gene, associated with CRPC occurrence, is present in this pathway.

The aim of this article is to assess variants found in genes of the abiraterone signaling pathway in the indigenous population of the Amazon, including the variant of the HSD3B1 gene related as a biomarker for CRPC, and compare the frequencies found with those already verified in other global populations. The analysis also seeks to validate the unique character of the indigenous population studied, reinforcing the need for a specific approach to precision medicine and hormone therapy to benefit this population.

2. Materials and Methods

2.1 Population Analyzed in the Study

The analyzed indigenous population comprises indigenous peoples from the Amazon, totaling 64 individuals who are part of 12 indigenous tribes from the northern region of Brazil: Asurini from the Xingu and Tocantins localities, Arara, Araweté, Awa-Guajá, Juruna, Kayapó, Xikrin, Karipuna, Munduruku, Phurere, Wajãpi, and Zo’é. These tribes were collectively grouped as the Indigenous (INDG) population for statistical analyses. Genetic data were obtained with the consent of all individuals and their leaders, who completed and signed a specific form for this purpose. The entire study and information gathering were approved by the national ethics committee (CONEP) and the ethics committee of the Center for Tropical Medicine at the Federal University of Para (CAE: 20654313.6.0000.5172).

The frequencies of indigenous peoples were compared with those of other continental populations: Europe (EUR), Africa (AFR), East Asia (EAS), South Asia (SAS), and the Americas (AMR). These datasets were sourced from the 1000 Genomes Database, version 3 (available at: http://www.1000genomes.org; accessed on Feb 2, 2024). The study included 503 individuals from Europe, 661 from Africa, 504 from East Asia, 489 from South Asia, and 347 from the Americas.

2.2. DNA Extraction and Exome Analysis

DNA extraction was conducted following the Phenol-Chloroform method [19] with modifications. The extracted product's quantity was measured using a Nanodrop-8000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA), and the prospective analysis of the extracted material's quality was performed through 2% agarose gel electrophoresis.

2.3. Gene Selection

Gene selection was performed by consulting the PharmGKB database (https://www.pharmgkb.org/pathway/PA166310681; accessed on March 20, 2024). The five selected genes (SLCO2B1, SULT2A1, CYP3A4, HSD3B1, HSD3B2, and UGT1A4) are related to the signaling pathway of the drug abiraterone, which is the focus of this study in prostate cancer treatment.

2.4. Statistical and Bioinformatic Analysis

The allele frequency of the studied population was obtained by gene counting and compared with other major populations already investigated (EUR, AMR, EAS, SAS, and AFR). Fisher's exact test was used to assess the statistical significance in frequency differentiation between populations. The population variability of polymorphisms was observed through Wright's fixation index (FST). A p-value ≤ 0.05 was considered significant. The entire investigation was conducted using RStudio v.3.5.1. Bioinformatic analyses followed the procedures described by Cohen-Paes et al., 2022 [22].

2.5. Inclusion Criteria

The inclusion criteria for SNPs were as follows: (i) a minimum of 10 coverage reads (fastx_tools v.0.13 http://hannonlab.cshl.edu/fastx_toolkit/, accessed in January 2024); (ii) variant impact: modifier, moderate, or high (SNPeff classification (https://pcingola.github.io/SnpEff/, accessed in March 2024); and (iii) allelic and genomic frequency in worldwide populations (http://www.1000genomes.org, accessed in March 2024).

3. Results

One of the treatment approaches for prostate cancer involves androgen deprivation therapy (ADT), aiming to reduce testosterone production [1]. Among the drugs used to implement this protocol are those containing the substance abiraterone [2]. However, in some cases, despite the application of this protocol, some patients develop castration-resistant prostate cancer (CRPC). Upon investigating possible mechanisms underlying this resistance, a mutation in the HSD3B1 gene was discovered, which has been identified as a potential biomarker for poor prognosis when ADT is implemented [4].

Taking the gene identified as the biomarker HSD3B1 and other genes involved in the drug signaling pathway, including SLCO2B1, SULT2A1, CYP3A4, HSD3B1, HSD3B2, and UGT1A4, as a starting point, we investigated the described variants, their distribution across the global population. Our objective was to identify which of these variants are present in the indigenous population of the Amazon, with the aim of mapping whether variants associated with the HSD3B1 gene and other genes in the pathway are also prevalent in this demographic.

Based on the genes outlined in the signaling pathway illustrated in Figure 01, 46 variants were identified, among which a mutation was found that had only been described in the indigenous population, initially indicated as having low impact. As shown in Figure 02, there is a predominance of variants with modifier impact, comprising 64.3% of the total variants, followed by 21.4% of variants with moderate impact and 14.3% of variants with low impact. Upon analyzing the genes separately, it can be observed that the SULT2A1 gene has the highest percentage of variants with moderate impact, accounting for 80% of all variants described for this gene, followed by the UGT1A4 gene (75.86%), CYP3A4 (66.67%), SLCO2B1 (60%), and HSD3B1 (22.22%).

Out of a total of 56 variants, 48 were selected with modifier and moderate impacts, and one variant not described in the databases used, initially classified as having low impact. In terms of variant allocation by gene, there are three for the CYP3A4 gene, five for the HSD3B1 gene, ten for the SLCO2B1 gene, five for the SULT2A1 gene, and twenty-six for the UGT1A4 gene. The new variant found in the indigenous population, associated with rfseq NM_017460.5, SNV type, in the CDS region, was identified in the CYP3A4 gene.

Table 1 presents the characteristics of these variants, including their reference number, chromosomal region, nucleotide exchange, predicted impact by the SNPeff software, and allelic frequency related to the indigenous group (IND) and the five continental populations present in the 1000 Genomes Program (AFR, AMR, EAS, EUR, and SAS).

Regarding variant types, thirty-six variants are predicted to have a modifier impact, and twelve have a moderate impact. In terms of location, thirty-three were found in intronic regions, thirteen in the CDS region, two in the 5'UTR region, and one in the 3'UTR region. It was observed that variants predicted to have a modifier impact are all located in the 3'UTR, 5'UTR, and intronic regions, while all variants with a moderate impact are located in the CDS region.

Focusing on the HSD3B1 gene, which is identified as a biomarker for increased CPRC recurrence, only one variant was found in the indigenous population.

Data for all identified variants will be available in a supplementary table.

Table 2 presents all variants identified in the indigenous population in a detailed manner, including: gene, refseq, dbsnp, VarType, Impact and detailed region.

Through multidimensional scaling analysis (MDS) (Figure 3), using the FST values (supplementary table) for the 46 variants related to the genes SLCO2B1, SULT2A1, CYP3A4, HSD3B1, HSD3B2, and UGT1A4, the distribution of the five population groups was evidenced (Figure 2). Based on the distribution of populations, the African population is isolated, as expected due to genetic diversity. The American and European populations show significant similarity, followed by populations from South Asia and East Asia with little divergence. An important point to note is the indigenous population, which is also isolated from all other populations, indicating a significant genetic distance from them. The isolation of the studied Amazonian indigenous population contributes to the existence of the variant described only in this population.

4. Discussion

According to the World Health Organization (WHO), in 2020 the global incidence of prostate cancer was 1.2 million individuals, ranking fourth in terms of the number of cases and resulting in 375,000 deaths. Despite its high incidence, the survival rate is 78%, achieved through effective therapeutic approaches including early diagnosis and protocols based on androgen deprivation therapy (ADT) and prostatectomy procedures.

In cases with a poor prognosis, such as castration-resistant prostate cancer (CRPC), individuals may develop resistance to treatment despite adherence to protocols. This resistance often leads to high levels of steroid hormones, such as testosterone, even after ADT and prostatectomy protocols are implemented. Increased steroid hormone expression is a key indicator of CRPC progression and is associated with a shorter survival time.

To understand the mechanisms underlying resistance, genetic studies have focused on genes involved in steroid hormone regulation linked to CRPC incidence. Tissue analysis of these tumors revealed variants in the HSB3D1 gene, which plays a crucial role in steroid hormone production regulation. Due to the significance of this gene and the recurrent mutations observed in individuals with CRPC, it has been identified as a biomarker for poor prognosis in ADT protocols.

In accordance with the guidelines for prostate cancer management provided by the National Comprehensive Cancer Network (NCCN), the primary medication used for treating CRPC is abiraterone acetate. Building upon this protocol, an examination of mutations in this gene and other genes implicated in its signaling pathway was conducted, along with an assessment of the prevalence of these variants in global populations. Additionally, the study delved into the evaluation of these variants within indigenous populations of the Amazon, renowned for their extreme genetic isolation, which often yields divergent treatment responses compared to other populations, including heightened drug toxicity and diminished efficacy.

The distinctive and secluded genetic profile of the indigenous population is highlighted through the multidimensional scaling analysis (MDS) graph (Figure 3), where this population emerges as distinctly separate from others in terms of the identified variants within the studied genes. Notably, the population most genetically akin to the indigenous population is the American population, owing to greater amalgamation with Latino populations.

This genetic distinctiveness underscores the imperative for a highly tailored approach in precision medicine. Analysis of the variants present in the target genes revealed occurrences across all genes studied. For the HSD3B1 gene, three occurrences were identified, two initially classified as having low impact (rs33937873 and rs6203), yet recent studies indicate they serve as risk factors for prostate cancer development and recurrence. Additionally, a variant with moderate impact (rs6205) was identified.

In the SLCO2B1 gene, seven variants were detected, all of which play a pivotal role in ADT protocols by facilitating the transport of abiraterone. Variants within this gene have been associated with cancer progression and poor response to ADT.

Among the five variants described in the SULT2A1 gene, three were found within the indigenous population, all characterized by a modifier impact. Variants within this gene have been linked to an increased risk of prostate cancer development.

The gene harboring the greatest number of described variants within the studied pathway was UGT1A4, with twenty-six variants described, ten of which were identified within the indigenous population. These variants have been associated with the recurrence of localized prostate cancer following radical prostatectomy.

The CYP3A4 gene exhibited the fewest occurrences, with three detected across all studied populations and two within the indigenous population. One occurrence pertained to a new variant lacking description within the examined databases, initially classified as having a low impact. This discovery underscores the genetic distinctiveness of the Amazon indigenous population, as evidenced by the multidimensional scaling analysis (MDS).

Overall, the indigenous population lacks the variant associated with HSD3B1-mediated CRPC, suggesting a potentially more favorable prognosis for prostate cancer treatment. However, variants indicating a risk of developing this type of cancer were uncovered, accentuating the importance of implementing strategies for early diagnosis to enhance treatment efficacy. The identification of a new variant within the CYP3A4 gene underscores the uniqueness of the Amazon indigenous population in genomic studies, rendering this study singular in its field.

These findings and discussions contribute to the development of novel clinical protocols for precision medicine in hormone therapy, aimed at facilitating more efficacious management of this population and mixed populations.5. Conclusions

This section is not mandatory but can be added to the manuscript if the discussion is unusually long or complex.