Adrenal Steroids and Resistance to Hormonal Blockade of Prostate and Breast Cancer

Abstract

Prostate cancer and breast cancer are sex-steroid–dependent diseases that are driven in major part by gonadal sex steroids. Testosterone (T) is converted to 5α-dihydrotestosterone, both of which stimulate the androgen receptor (AR) and prostate cancer progression. Estradiol is the major stimulus for estrogen receptor-α (ERα) and proliferation of ERα-expressing breast cancer. However, the human adrenal provides an alternative source for sex steroids. A number of different androgens are produced by the adrenals, the most abundant of which is dehydroepiandrosterone (DHEA) and DHEA sulfate. These precursor steroids are subject to metabolism by peripherally expressed enzymes that are responsible for the synthesis of potent androgens and estrogens. In the case of prostate cancer, the regulation of one of these enzymatic steps occurs at least in part by way of a germline-encoded missense in 3β-hydroxysteroid dehydrogenase-1 (3βHSD1), which regulates potent androgen biosynthesis and clinical outcomes in men with advanced prostate cancer treated with gonadal T deprivation. The sex steroids that drive prostate cancer and breast cancer require a common set of enzymes for their generation. However, the pathways diverge once 3-keto, Δ⁴-androgens are generated and these steroids are either turned into potent androgens by steroid-5α-reductase, or into estrogens by aromatase. Alternative steroid receptors have also emerged as disease- and treatment-resistance modifiers, including a role for AR in breast cancer and glucocorticoid receptor both in breast and prostate cancer. In this review, we integrate the commonalities of adrenal steroid physiology that regulate both prostate and breast cancer while recognizing the clear distinctions between these diseases.

Prostate and breast cancer are the most commonly diagnosed malignancy and second most common cause of cancer death in men and women, respectively (1). These malignancies also share mechanistic pathways, given the role of sex steroids in driving progression of disease. Gonadal hormone synthesis and signaling are therefore primary targets for treatment of both prostate and breast cancer. However, after inhibition of testosterone (T) or estrogen synthesis and/or blockade of signaling, oncologic progression continues in many patients (2-4). One important mechanism of resistance to hormonal treatment is the production of precursor steroids by the adrenal gland and their subsequent conversion to sex steroids (5, 6). Substantial work has been undertaken to evaluate the effect of the adrenal gland on these hormonally driven malignancies and to understand the clinical implications for improving treatment in the large number of patients diagnosed with these cancers. Here, we briefly review recent work and ideas concerning the involvement of adrenal steroids in treatment resistance in prostate and breast cancers.

Androgen Physiology In Men

Under normal conditions, the majority of T is synthesized by the Leydig cells of the testes and is derived from cholesterol through a series of cascading reactions (Fig. 1). The synthesized T is then released into circulation and converted to 5α-dihydrotestosterone (DHT) within the prostate by steroid-5α-reductase enzymes (SRD5A), which are encoded by 2 genes: SRD5A1 is expressed in a variety of peripheral tissues, whereas SRD5A2 is primarily expressed in prostatic tissues (7). DHT is the primary driver of androgen receptor (AR) activity in the prostate, as evidenced by developmental abnormalities in male external genitalia and the absence of a prostate resulting from loss-of-function germline mutations in SRD5A2 (8). Additionally, SRD5A inhibitors are commonly used in treatment of benign prostatic hyperplasia to reduce the size of the prostate and associated symptoms (9).

Figure 1.

Steroidogenesis overview, beginning from cholesterol with metabolism to sex steroids. 3β-Hydroxysteroid dehydrogenase-1 (3βHSD1) serves as a critical enzyme in the steroid-metabolizing pathways in peripheral tissues. In steroidogenic endocrine organs, such as the testes, ovaries, and adrenals, the same metabolic step is catalyzed by 3βHSD2.

The adrenal gland acts as a secondary source for androgen production. Although the adrenal reticularis produces a small amount of T, adrenal androgen synthesis primarily occurs by way of dehydroepiandrosterone (DHEA) in its unconjugated and sulfated (DHEA-S) forms. Although DHEA is a weak AR agonist, this adrenal precursor can be metabolized to potent androgens to stimulate AR. DHEA can be metabolized to DHT directly by way of T, but the major pathway is likely through conversion from Δ⁴-androstenedione (AD) to 5α-androstanedione, occurring to a greater extent in the setting of reduced testicular androgen production (5, 10). In recent years, the adrenal also has been increasingly appreciated to generate 11-OH-androgens, which are also metabolized in peripheral tissues (11). However, those androgens will not be covered extensively here.

Estrogen Biosynthesis in Premenopausal and Postmenopausal Women

In premenopausal women, the primary source of estrogens is the granulosa cells of the dominant follicle in the ovaries (12). Three estrogens are produced physiologically: estrone (E1) and estradiol (E2) during the menstrual cycle and at extragonadal sites, and estriol (E3) by the placenta during gestation. The most potent estrogen that binds estrogen receptors (ERs) is E2, and it is also the most abundant estrogen found in circulation in premenopausal women (12). E2 is synthesized from cholesterol in a stepwise process involving both the ovarian theca and granulosa cells, similar to how T is synthesized in the Leydig cells of the testes. In fact, they share many of the same biochemical pathways. For example, CYP11A1 is responsible for the conversion of cholesterol to pregnenolone both in Leydig cells and theca cells. Both subsequent 17-hydroxylation and conversion from 21-carbon progestogens to 19-carbon androgens are mediated by CYP17A1. Importantly, both cell types express 3βHSD2, which is required to make AD (13, 14). In postmenopausal women, the most abundant precursor for estrogen biosynthesis is adrenal DHEA-S. Although DHEA-S may have direct effects on breast cancer (15), it can be metabolized to AD in peripheral tissues and then undergo subsequent conversion by aromatase, primarily in adipose tissues, to form E1 (16). E3 is most relevant during pregnancy when the placenta converts E1 to E3 through 16α-hydroxylation (12). Altogether, it is thought that estrogens in premenopausal women primarily are derived from the ovaries, whereas in postmenopausal women they are derived from extragonadal tissues (12). Finally, it is important to note that many studies show that estrogen production after menopause is largely intracrine or paracrine, thus placing a large importance on extragonadal estrogen production (12, 16, 17).

Sex Steroids and Prostate Cancer

Sex steroids play a considerable role in prostate cancer, with gonadal T being identified as a major factor in 1941, when castration was found to markedly improve prostatic acid phosphatase and alkaline phosphatase levels (18). Nearly 2 decades later in 1968, DHT was found to be the primary active androgen (19, 20). Consequently, androgen deprivation therapy (ADT) has been the mainstay for prostate cancer treatment for the past 80 years and was first therapeutically administered via bilateral orchiectomy or use of estrogens (18). Management, however, has become multifaceted with the addition of therapy for localized disease including surgical removal, external beam radiation therapy, brachytherapy, and a slate of various hormonal treatments targeting different components in the androgen production and signaling pathway for management of metastatic disease.

The utility of ADT is predicated on the importance of AR signaling for prostate cancer survival and proliferation (21). The androgen-regulated serine protease gene TMPRSS2 is upregulated in prostate cancer cells. Frequently, prostate cancer harbors a genetic translocation that places the AR-responsive element of TMPRSS2 next to an ETS family oncogene, which allows androgens to further drive oncogene expression (22). ADT confers clinical benefit by blocking testicular androgen production and downregulating AR-dependent transcription, which reverses the tumor prosurvival and proliferative effects of androgens. However, even in the setting of castration-resistant prostate cancer (CRPC), in which disease progression occurs despite low systemic levels of T, constitutive AR signaling by means of alternative activation pathways often remains the primary driver (23).

In the modern era, a number of medications including gonadotropin-releasing hormone (GnRH) agonists such as leuprolide are able to achieve castration levels of T through inhibition of testicular production of androgens by downregulating luteinizing hormone (24). The use of these GnRH agonists was noted to precipitate an initial “T flare” due to early T increase, which can be problematic, particularly with symptomatic disease, impending cord compression, or urinary obstruction. The potential consequences of a transient T increase are often managed using the combination of a GnRH agonist and direct AR antagonists or androgen synthesis inhibitors, such as CYP17A1 inhibition, or more recently, luteinizing hormone–releasing hormone antagonist monotherapy (25, 26).

Effects of Sex Steroids in Breast Cancer

The role that estrogens play in breast cancer is well established, with in vitro and in vivo evidence that estrogens directly promote tumor growth (27). When estrogens bind to the ligand-binding domain of estrogen receptor-α (ERα), the receptor forms a homodimer and translocates to the nucleus where it regulates gene expression and enhances tumor cell proliferation (28, 29). Thus, the rationale and strategies for targeting the estrogen receptor with selective estrogen receptor modulators (SERMs) was developed and became an important advance in the treatment of breast cancer (30, 31). As ERα is broadly expressed in many different tissue types, the advance of SERMs included their ability to act as antagonists or agonists depending on the target tissue; the complexity of this class of drugs is beyond the scope of this review (32).

Furthermore, breast cancer management has placed much attention on the role of aromatase, the final irreversible enzymatic step in estrogen synthesis, which primarily is thought to take place in adipose tissue in postmenopausal women (33, 34). Pursuing aromatase as a therapeutic target began with broad targeting of multiple cytochrome P450 enzymes; later in seminal work, Angela and Harry Brodie discovered inhibitors specific to aromatase (33, 35). Since then, newer generations of aromatase inhibitors with higher specificity and lower toxicity profiles have been developed (36). Clinically, these 2 classes of drugs have been heavily leveraged for the treatment of hormone receptor–positive breast cancer (37).

Roles of Nonestrogen Steroids in Breast Cancer

Typically, about 70% of breast cancers express ERα, and thus estrogens have been implicated in the pathogenesis of many breast cancers (38). However, the roles that the other steroids play, such as glucocorticoids and androgens, have gained increasing attention, given the expression of their respective nuclear receptors in breast cancer.

The role of glucocorticoids in breast cancer is quite complex. Glucocorticoid receptor (GR) expression tends to be associated with a better prognosis for women with ER-positive tumors compared to those with ER-negative tumors (39). Mechanistically, glucocorticoids are thought to interact with the estrogen-ER complex to dampen the proliferative effects of estrogens (39). However, in ER-negative tumors, glucocorticoids may mediate an increase in stimulation of kinases that are essential for tumor growth, including p38 mitogen-activated protein kinase (40). Furthermore, high GR expression in ER-negative tumors has been shown to decrease the effects of paclitaxel chemotherapy (39, 41). In fact, in the setting of triple-negative breast cancer (TNBC), work by West et al (42) shows that low GR expression is associated with prolonged relapse-free survival and that GR antagonism increases sensitivity to chemotherapy in vivo. Interestingly, a recent preclinical study demonstrated that GR activation can promote breast cancer metastasis and decrease survival via increased activation of the kinase ROR1 (43). Clearly, the role GR activation plays in different breast cancer subtypes is highly nuanced, and more work is needed to better understand the underlying mechanisms.

As mentioned earlier, 3-keto, Δ⁴-androgens are substrates for aromatase and are converted to estrogens, but their role in breast cancer has recently been shown to extend beyond this, with androgens themselves being highly active in breast cancer tissue. Approximately 90% of breast cancer tumors express AR although AR status is not currently used to classify tumors (44). AR activation seems to not only antagonize the nuclear receptor activity of ERα but also seems to reduce tumor growth in patient-derived models of endocrine-resistant hormone-positive breast cancer (45). These findings have been validated in the METABRIC and ROCK hormone-positive breast cancer cohorts, in which patients with increased expression of AR-target genes had a better prognosis (45). It is therefore hypothesized that some of the beneficial effects of aromatase inhibitors are mediated via decreased conversion of androgens to estrogens, with these androgens capable of activating AR in tumor cells. In TNBC, on the other hand, AR inhibition with enzalutamide decreases tumor cell proliferation in vitro (46). These data and work from many others have laid the groundwork for testing antiandrogen therapies in multiple clinical trials for the treatment of TNBC (47). Breast tumors are heterogeneous, but it is clear that steroid biology is an important component of this complexity.

Prostate Cancer Treatment Resistance

Adrenal Precursor Steroids Implicated in Resistance

Prostate cancer treatment relies on depletion of gonadal androgen synthesis, but disease progression occurs in the majority of patients despite circulating T being reduced to castration levels. Evidence shows that intraprostatic levels of T and DHT remain clinically significant even after initiation of ADT (48, 49). Physiologically, the adrenals provide a secondary source of androgen production in several forms, the most abundant of which are DHEA and DHEA-S (50). As mentioned previously, these steroid precursors are weak agonists of AR, but the downstream metabolism to T and DHT exerts a far greater effect on AR activity (51). During treatment with ADT, this secondary supply of circulating androgens is a major source of AR agonism and thus stimulation for prostate cancer progression.

Given the high intraprostatic levels of potent androgens, it was apparent that more intensified therapies were needed to inhibit androgen production from all sources. Historically, some patients received bilateral adrenalectomy in addition to bilateral orchiectomy (52). Patients undergoing bilateral adrenalectomy experienced pain relief and neurologic recovery, but survival rates remained poor. More recently, pharmacologic blockade of adrenal androgen biosynthesis has become a medical alternative to surgical adrenalectomy. Although now uncommonly used, ketoconazole is a nonsteroidal antifungal that also antagonizes CYP11A1 and CYP17A1. Abiraterone is a more specific and potent steroidal CYP17A1 inhibitor. Clinically, it has been shown that these medications further reduce potent androgen synthesis that is particularly important given persistent AR expression in CRPC tissues (53-56). Direct AR inhibition is another effective approach to oppose androgen activity for management of prostate cancer. A number of direct AR inhibitors have been developed, most recently drugs such as enzalutamide and apalutamide, which have shown overall survival (OS) benefit both for hormone-sensitive and castration-resistant disease (57-61).

Effects of Androgen Receptor Blockade on Glucocorticoid Receptor Activity and Treatment Resistance

A number of studies have made the observation that GR is upregulated when AR is presumably completely blocked with potent AR antagonists such as enzalutamide (62, 63). In this setting, GR may induce the expression of genes normally regulated by AR or elicit other mechanisms that enable the development of resistance and tumor progression through AR blockade. Some evidence suggests that GR is induced by a double-stranded RNA signaling response (64). However, GR upregulation is insufficient for resistance. Prostate cancer engages in steroid metabolic mechanisms that enable tumor tissues to sustain biologically active glucocorticoids that are required to stimulate GR, leading to enzalutamide resistance (65, 66). Nevertheless, thus far in clinical studies, GR antagonism using mifepristone has had disappointing results (67).

Antiestrogen Treatment Resistance Mechanisms in Breast Cancer

The mechanisms underlying antiestrogen therapy resistance are complicated. Antiestrogen resistance is typically seen in breast tumors that are ER positive and fail to respond (or recur) despite hormone treatment (68). One of the more straightforward possible explanations includes loss of ER expression (68). With the loss of ER, these tumors would not respond to antiestrogen strategies. Apart from ER loss, conformational changes in the receptor itself can explain some aspects of resistance (28). Further, in luminal type A tumors, a subtype of ER-positive tumors, it is thought that expression of the transcription factors FOXA1 and GATA-3 influences whether a tumor becomes resistant to antiestrogen therapies, with loss of expression of these transcription factors leading to increased resistance (68).

SERM drugs can have either stimulatory or suppressive properties on ER; however, this dual property can at times be another mechanism of resistance via estrogen-ER activation of AP-1 genes, which are thought to play a role in resistance to SERMs (38). Mechanisms of ligand-independent ER activation have also been described, including a potential signaling role of EGF receptor and Her2 in activation (69). Selective estrogen receptor degraders (SERDs), such as fulvestrant, are now showing promise in patients with ER-positive tumors resistant to other endocrine therapies. By causing degradation of the ER, SERDs are able to circumvent many mechanisms of resistance. In sum, endocrine therapy resistance in breast cancer is highly complex.

Gatekeeper for Sex-Steroid Synthesis of Adrenal Origin

Prostate Cancer

3βHSD1 is an enzyme located in peripheral tissues and functions to metabolize DHEA to AD, thereby providing a route to T and DHT synthesis from precursor steroids (5). This enzyme is encoded by the gene HSD3B1, for which there is a common germline missense-encoding variation (1245A->C) that impedes 3βHSD1 degradation by promoting resistance to ubiquitination. The effect of this is increased cellular levels of the 3βHSD1 enzyme and resultant increases in downstream T and DHT production, as shown in Fig. 2. This HSD3B1 allele variant (1245C) is termed the “adrenal-permissive” allele, whereas the 1245A allele is “adrenal restrictive” due to its relative limitation of potent androgen synthesis from adrenal precursors.

Figure 2.

HSD3B1 inheritance and metabolism of dehydroepiandrosterone (DHEA) to potent androgens and estrogens. The adrenal-permissive HSD3B1 (1245C) allele encodes for the more stable 3βHSD1 enzyme compared with the adrenal-restrictive HSD3B1 (1245A) allele and confers more rapid disease progression in men with prostate cancer treated with gonadal testosterone deprivation and who have only adrenal precursors as their source of androgens. 3β-Hydroxysteroid dehydrogenase-1 (3βHSD1) is also upstream of aromatase and could be predicted to have a role in postmenopausal estrogen-driven diseases. Created with biorender.com.

Inheritance of the adrenal-permissive allele is mechanistically implicated in and associated with worsened clinical outcomes across multiple prostate cancer cohorts managed with ADT. Progression-free (PFS), metastasis-free, and OS were initially assessed in a Cleveland Clinic cohort of 118 men who had undergone radical prostatectomy, subsequently developed biochemical recurrence, and were treated with ADT. For patients with 0, 1, or 2 adrenal-permissive alleles, median PFS was 6.6, 4.1, or 2.5 years, respectively, and all outcomes showed worsening with an increasing number of adrenal-permissive alleles. A confirmatory postprostatectomy Mayo Clinic cohort of 137 patients showed decreasing PFS with adrenal-permissive allele inheritance. A separate Mayo Clinic cohort of 188 patients with metastatic prostate cancer showed similar results with worsened PFS, metastasis-free, and OS associated with adrenal-permissive allele inheritance. Overall survival was 9.7, 6.8, and 4.6 years for 0, 1, and 2 adrenal-permissive alleles, respectively (70). A number of independent cohorts have further confirmed an association between inheritance of adrenal-permissive HSD3B1 and worse clinical outcomes in men treated with ADT, mainly in men with nonmetastatic or low-volume metastatic castration-sensitive prostate cancer (mCSPC) (71, 72).

Further studies have evaluated the association of HSD3B1 inheritance in practice-changing clinical trials evaluating treatment regimen efficacy, including ADT and docetaxel in mCSPC. The CHAARTED trial evaluated the effect of ADT with or without docetaxel in mCSPC with groups stratified according to low-volume and high-volume disease (73). The presence of 1 or more adrenal-permissive alleles was associated with a hazard ratio of 1.89 for development of CRPC in patients with low-volume disease. Notably, docetaxel had no effect on this association between HSD3B1 genotype and clinical outcomes (74). Thus, one of the next steps is to clarify the effect of additional hormonal therapies in patients stratified by inheritance of HSD3B1 alleles.

Mechanistically, it might be expected that tumors in men with adrenal-permissive allele inheritance are more dependent on extragonadal androgens. As such, extragonadal androgen ablation might result in greater clinical benefit. Ketoconazole is a nonsteroidal drug that inhibits CYP17A1 and CYP11A1 and, indeed, is associated with improved PFS in patients with the adrenal-permissive genotype (75). Such a relationship is not as clear for abiraterone, a steroidal CYP17A1 inhibitor that is converted to multiple downstream metabolites by 3βHSD1. Abiraterone is converted by 3βHSD1 to Δ⁴-abiraterone, which strongly inhibits steroidogenic enzymes as well as AR directly. However, a metabolite downstream of Δ⁴-abiraterone has partial AR agonist activity (76, 77). Consequently, the benefit of extragonadal androgen ablation may be limited by the increase of proandrogenic abiraterone metabolites that is due to the increased 3βHSD1 activity in patients with adrenal-permissive allele inheritance. Studies comparing response to abiraterone and HSD3B1 genotype have so far been limited by small cohort sizes and a low number of patients with the homozygous adrenal-permissive genotype (72, 78).

Taken together, these findings suggest that genotyping of HSD3B1 might provide predictive information in the context of ADT by identifying in the individual patient a tumor's relative dependence on extragonadal androgen activity. Although specific use in clinical practice will require further study, the current evidence suggests that presence of homozygous adrenal-permissive alleles may warrant more aggressive salvage therapy for those diagnosed with localized disease, given the worse clinical outcomes after the development of advanced disease. Conversely, more conservative management options may be reasonable to consider for patients who are homozygous adrenal restrictive, given that systemic treatment options may be more effective. In particular, some evidence suggests that abiraterone and enzalutamide may be less efficacious for patients who have homozygous adrenal-permissive inheritance. Treatment selection is becoming increasingly complex as the array of treatment regimens for metastatic hormone-sensitive disease continues to broaden, now including the option of triple therapies (79, 80).

Finally, direct targeting of 3βHSD1 remains an unexplored possibility for men who have adrenal-permissive inheritance. 3βHSD1-dependent androgen biosynthesis requires phosphorylation by the BMX tyrosine kinase (81). Based on these mechanistic data, a phase 2 clinical trial (NCT05361915) is in progress to test the combination of abiraterone plus BMX inhibition (using abivertinib) for men with CRPC who have inherited at least 1 copy of the adrenal-permissive allele.

Breast Cancer

3βHSD1 is necessary for synthesis of AD and testosterone from adrenal DHEA. AD and testosterone both are substrates for aromatase, which is required for the generation of estrogens (Fig. 2). One would expect that patients with the adrenal-permissive allele HSD3B1 would have greater peripheral production of estrogens, which would in turn promote tumor growth in these estrogen-dependent cancers. Recent studies in postmenopausal women with breast cancer suggest that the proportion of women with the homozygous adrenal-permissive genotype is lower among those with ER-negative disease but is elevated in women with ER-positive breast cancer (82). Women with the homozygous adrenal-permissive genotype may have worse outcomes after treatment for localized breast cancer (83). Although provocative, these studies require validation in other cohorts.

Any effect of 3βHSD1 in breast cancer could be mediated via effects on estrogen or androgen biosynthesis. Given what we know about androgens and breast cancer, this association is clearly thought-provoking. As discussed regarding 3βHSD1 in prostate cancer, it is understood that increased enzyme activity leads to increased androgen production. However, in hormone-responsive breast tumors, are these androgens acting as antiproliferative agents, or are these adrenal-derived steroids being directly aromatized and subsequently stimulating the estrogen receptor? Perhaps the tissue localization of aromatase and 3βHSD1 plays a role in determining this balance between androgen and estrogen production. Clearly, many more questions need to be answered before the role of 3βHSD1 biology can be fully understood in breast cancer.

Conclusions

Prostate cancer and breast cancer are hormonally driven cancers for which modern medicine has co-opted steroidogenesis and steroid signaling pathways for treatment and management. Resistance to these hormonal therapies occurs in a subset of patients with progressive prostate or breast cancer. The mechanisms of resistance are varied; however, adrenal precursor steroids are implicated in treatment failure for both malignancies. Further evaluation of the effect of these adrenal precursors as well as other steroids has shown the clinical effect of these pathways on patient outcomes. In particular, HSD3B1 adrenal-permissive allele inheritance has predictive value for PFS in prostate cancer and a possible role in ER-positive breast cancer. Further study is warranted, and preclinical and clinical studies are ongoing to further clarify these mechanisms to improve oncological outcomes.

Glossary

Abbreviations

3βHSD1

3β-hydroxysteroid dehydrogenase-1

AD

Δ⁴-androstenedione

ADT

androgen deprivation therapy

AR

androgen receptor

CRPC

castration-resistant prostate cancer

DHEA

dehydroepiandrosterone

DHEA-S

dehydroepiandrosterone sulfate

DHT

5α-dihydrotestosterone

E1

estrone

E2

estradiol

E3

estriol

ER

estrogen receptor

GnRH

gonadotropin-releasing hormone

GR

glucocorticoid receptor

mCSPC

metastatic castration-sensitive prostate cancer

OS

overall survival

PFS

progression-free survival

SERD

Selective estrogen receptor degrader

SERM

selective estrogen receptor modulator

SRD5A

steroid-5α-reductase enzymes

T

testosterone

TNBC

triple-negative breast cancer

Financial Support

This work was supported in part by the National Cancer Institute (grant Nos. R01CA172382, R01CA236780, R01CA261995, and R01CA249279 to N.S.), the Prostate Cancer Foundation (to N.S.), and the U.S. Army Medical Research and Development Command (Nos. W81XWH2010137 and W81XWH-22-1-0082 to N.S.).

Disclosures

Cleveland Clinic has patents pending on HSD3B1, and N.S. is a coinventor. N.S. has received research support from BMS and Astellas. M.P., G.R., and K.B. have nothing to disclose.

Data Availability

Data sharing is not applicable to this article as no data sets were generated or analyzed during the present study.