Are Orphan Cytochromes P450 Suitable Targets for Breast Cancer Prevention and Treatment?

1. Introduction

Cytochromes P450 (CYPs) catalyze a variety of reactions and are of significant importance in the areas of xenobiotics metabolism, including drugs and carcinogens. It is estimated that ~75% of drug metabolism reactions involve CYPs, and ~66% of carcinogens are bioactivated by this class of enzymes [1]. CYP enzymes play an important role in carcinogenesis initiation through the activation of several environmental and endogenous carcinogens, such as estrogen metabolites, as well as in the latter stages of cancer development. Therefore, CYPs are considered promising targets in cancer prophylaxis and chemotherapy. Moreover, they may activate or inactivate prodrugs [2,3]. While most of these reactions involved CYPs with well-characterized function and substrate specificity, it is clear now that some orphan CYPs may be equally important for drugs and carcinogens transformation. The term orphan CYPs was adopted from descriptions of the steroid nuclear superfamily [4] and it is estimated that of the 57 human cytochromes P450 (P450) and 58 pseudogenes discovered to date, 1/4 remain “orphans” in the sense that their function, expression sites, and regulation are still largely not elucidated [5]. Interestingly, the expression of some of these orphan CYPs is tissue-specific and increases in the process of tumorigenesis. For example, CYP4Z1 was found to be frequently upregulated in primary mammary carcinoma and ovarian cancer and was associated with tumor progression and metastasis [6,7]. The others, such as CYP2W1, CYP2S1, CYP2U1, CYP4X1, and CYP4V2, are less specific, but their overexpression in breast cancer has also been found. Attempts are made to deorphanize some of them, but so far only partly successful [8]. Breast cancer is the leading cause of cancer-related deaths among women worldwide. Although breast cancer mortality has been dropping since the 1990s [9], much still has to be done to reduce these numbers. Estrogens play a key role in the pathogenesis of breast cancer. Immunohistochemically, breast cancer has been classified into four subtypes: estrogen receptor-positive (ER+), progesterone receptor-positive (PR+), human epidermal growth factor receptor 2-positive (HER+), and triple-negative (TNBC) [10]. TNBC accounts for 10-20% of all cases and is resistant to conventional endocrine therapy [11]. Therefore, TNBC has a particularly unmet need for highly effective therapeutic agents.

Thus, the new druggable targets, such as the orphan CYPs expressed in breast epithelial cells, are of particular interest in targeted breast cancer prophylaxis and therapy [12]. This review focuses on the description and discussion of current knowledge of orphan CYPs in breast tissue and their possible application in drug targeting.

2. Expression of Orphan CYPs in Breast Epithelium and Cancer

Most data on the expression of orphan CYPs genes or enzyme activity in the breast epithelium come from in vitro studies using breast cancer cell lines derived from cancer differing in tumor origin and receptor status. In the most extensively investigated ER+ MCF7 breast cancer cell line, the expression of three orphan CYPs, namely CYP4Z1, CYP2S1, and CYP2W1, was confirmed by several authors [13,14,15,16,17,18,19,20]. The analysis of the CYPs expression profile in the ER+ T47D cell line, also derived from ductal carcinoma, revealed the presence of CYP4Z1 [7,16,21,22]. Stable expression of CYP4Z1 was achieved in BT-474 human HER+ breast cancer cells [21]. CYP4Z1 isoform has triggered particular interest because of its hypothetical role in breast cancer through the formation of the signaling molecule 20-hydroxyeicosatetraenoic acid (20-HETE). The expression of this orphan isoform, as well as of CYP2S1 and CYP2W1, was also described in triple-negative cell lines MDA-MB-231 and MDA-MB-468 [13,14,17,18]. Moreover, in comparison to ER+ and PR+ breast cancer cells, higher levels of expression of orphan CYPs were observed in these cell lines. Concurrently, in the non-tumorigenic breast epithelial MCF10A cell line, several orphan CYPs mRNA were detected, in particular CYP2S1, CYP2U1, CYP4V2, CYP4X1, or CYP4Z1. However, CYP4Z1 protein was not revealed on the MCF10A cells’ surface [23,24].

To establish the possible role of orphan CYPs in breast cancer development and/or chemotherapy resistance, the group of Tao Xi [17,25,26] analyzed the synergic expression of CYP4Z1 and pseudogene CYP4Z2P in the cells of regular MCF7, tamoxifen-resistant MCF7-TamR, and MDA-MB-231 cell lines. They concluded that overexpression of CYP4Z1 and/or 4Z2P may enhance the transcriptional ERα activity, apoptosis, stemness, and resistance to tamoxifen of breast cancer cells. These observations confirmed the study, which used a human transgenic model, showed that overexpression of CYP4Z1 in lactating female transgenic mice did not result in tumor formation or other mammary abnormalities, but upregulated estrogen receptor (ERα) expression was observed [27]. Additional in vitro studies of this group suggested that human CYP4Z1 might metabolize a small molecule into a transcriptional activator of ERα. Moreover, stable overexpression of CYP4Z1 in breast cancer cells has been reported to promote angiogenesis and tumor growth in mice [21].

Table 1 summarizes the in vitro studies on the orphan CYPs in human breast epithelial cell lines.

A more recent clinical study showed a high incidence of CYP4Z1 expression in TNBC patients with advanced grades, later stages, and larger tumors [31]. Clinical studies of the other orphan CYPs expression profile and its relationship with clinical pathological variables, although limited, showed an interesting trend. Murray et al. [6] performed immunostaining of a tissue microarray containing 170 breast cancers of no special type for a panel of 21 CYPs.

The highest percentage of strong immunopositivity in these samples was seen for CYP4X1, CYP2S1, and CYP2U1. At the same time, CYP4V2, CYP4X1, and CYP4Z1 showed correlation with the tumor grade. Association with survival was identified for CYP2S1, CYP3A4, CYP4V2, and CYP26A1, although none of these P450s were an independent prognosis biomarker [6]. In some smaller pools of patients, the increased transcript levels of CYP2S1, CYP2W1, and CYP4F11 both in breast cancer, adjacent, and normal breast cells were found. Unfortunately, protein levels were inappropriately low to confirm the results [32,33]. Table 2 presents the results of the orphan CYPs analyses in clinical samples.

As it was mentioned above, much attention is focused on the role of CYP4Z1 and an associated pseudogene, CYP4Z2P, because of their breast-epithelium-specific expression. In this regard, Cizkova et al. [43] noticed in the series of 249 ER+ breast cancer patients a correlation between the mutation status of oncogene PIK3CA and overexpression of these CYPs.

Finally, the expression of CYP2A7, an orphan CYPs of so far smaller interest in the context of breast cancer, showed an association with poorer survival of TNBC patients [35].

3. Orphans CYPs as the Therapeutic Targets - Mechanistic Studies

As was described in the previous section, several orphan CYPs were detected in breast epithelium. Here, the most studied isoforms’ characteristics and their potential for prevention or therapeutic purposes are provided.

3.1. CYP4Z1

Some experimental data, including the analysis of Human Protein Atlas for CYP4Z1 expression in normal and cancer tissues, confirm that CYP2Z1 is preferentially localized in the breast and its activity is dramatically increased in breast cancer [7]. Therefore, modulation of CYP2Z1 activity appears to be an interesting target, even a kind of “silver bullet” for breast cancer treatment [46]. The CYP4Z1 gene is located on chromosome 1p33, in a cluster containing the CYP4A11 and CYP4X1 genes. CYP4Z1 shares 54% and 52% sequence identity with CYP4X1 and CYP4A11, respectively [16]. Comparative genomics analysis demonstrated that tumor-specifically expressed sequences, such as CYP4Z1, are either evolutionarily new (primates or humans) or relatively young (mammals) [47]. Consequently, no orthologs have been found in mice or rats, so CYP4Z1 appears to be specific to humans and primates [48], which, to some extent, limits the investigation of animal models. On the other hand enables to creation of human transgenic models mentioned in the previous section. Designing suitable small molecules as potential drugs or chemopreventive agents requires knowledge of the metabolically based role of CYP4Z1 in breast cancer progression and identifying its substrates.

In this context, it is worth noticing that in contrast to the other members of the CYP4 family, CYP4Z1 characterizes the presence of heme not covalently linked [49].

The first functional study of CYP4Z1 was performed in 2009 by Bureik’s research group. Using human CYP4Z1 expressed in yeast, they found a unique pattern of metabolites with lauric acid generating mainly the 8-hydroxy (ω-4) product and myristic acid forming 12-hydroxy (ω-2) [50].

Subsequent studies focused on the possibility of generation by CYP4Z1 of arachidonic acid metabolites such as pro-angiogenic epoxyeicosatrienoic acids (EETs) and HETE acids considered critical modulators of cancer progression, acting in concert with endothelial growth factor and others growth factors ultimately promoting cellular proliferation, neovascularization, angiogenesis, and metastasis. Of particular interest is 20-HETE, which, as it was postulated, is responsible for proliferative, angiogenic, and tumor growth effects [21].

However, CYP4Z1 is considered an EET synthase. Therefore, it was not surprising that the major products of internal oxidation of arachidonic acid (AA) in breast ductal carcinoma T47D cells engineered to express CYP4Z1 were derivatives 14,15-EET and 14,15-dihydroxy eicosatrienoic acid. Only a trace of 20-HETE was detected, leading to conclusions that CYP4Z1 is distinct from other CYP4 enzymes and 20-HETE is not the major AA metabolite produced in a reaction catalyzed by CYP4Z1 [7,22].

Besides, fatty acids screening for the other substrates was performed in yeast enzyme bags (permeabilized cells from recombinant fission yeast cells) and indicated the ability of CYP4Z1 to catalyze 11-O -dealkylation cleavages and 2-hydroxylation reactions of pro-luciferin compounds [7,29,51]. Through docking experiments and site-directed mutagenesis, Asn381 and Arg487 were pointed out as key active sites of CYP4Z1 [7].

The subsequent search for inhibitors was based on the fatty acid hydroxylase activity of CYP4Z1. One of the tested compounds was N-hydroxy-N’-(-butyl-2-methylphenyl)-formamidine (HET0016), a highly potent inhibitor of 20-HETE synthase. Although the results of initial studies [21] were promising, further investigations indicated only weak inhibition of CYP4Z1 [52]. It was in contrast to results obtained with the other members of the CYP4 family and might be related to their covalent linkage with heme. The first selective mechanism-based CYP4Z1 inhibitor was discovered by Kowalski et al. in 2020, namely 8-[(1H-benzotriazol-1-y)amino]octanoic acid [22]. Analysis of major metabolites of this compound in rats’ plasma showed that products of β-oxidation, with a probable higher inhibitory effect than the parent compound [7]. One of the latest proposed CYP4Z1 inhibitors is a derivative of HET0016 with an esterified 4-carbon carboxylate tail [53]. This compound, tested in MCF7 and MDA-MB-231 cells, showed the ability to reduce metastatic potential, spheroid formation, and expression of stemness markers. Low constitutive expression of CYP4Z1 in these cell lines requires careful interpretation of these data, although even minimal activity of this enzyme might be sufficient to obtain the desired effect [7]. Previously, Rieger et al. [15] suggested utilizing the tissue and cancer-specific expression of CYP4Z1 to bioactivate prodrugs into active agents for breast carcinoma treatment. However, despite attempts, no fruitful results have been obtained so far that would allow the use of CYP4Z1 activity to activate antibreast cancer prodrugs.

The observation that MCF7 breast cancer cells, in contrast to non-tumorigenic MCF10A cells, display CYP4Z1 on their surface might indicate its usefulness in breast cancer immunotherapy [24].

3.2. CYP2S1

As was mentioned in section 2, orphan CYP2S1 was reported to be upregulated in breast cancer cells and clinical samples. Moreover, its association with patient survival was found [36]. However, CYP2S1, in contrast to CYP4Z1, is not unequivocally linked with this tissue. Therefore, the data on its usefulness as a drug in breast cancer was not extensively studied.

The human CYP2S1 gene is located on chromosome 19, at the 19q13.2 region, encodes a protein of 504 amino acids [54], and is mainly expressed in the endoplasmic reticulum. The highest expression of CYP2S1 was found in the epithelium of portal entry organs, but the lower expression was described in several organs, including breast epithelium. CYP2S1 catalyzes the oxidation of AA into 19-hydroxy-5Z,8Z, 11Z, and 14Z-eicosatetraenoic acid, among other polyunsaturated fatty acids (PUFA) with ω-1-PUFA [55]. CYP2S1 also possesses epoxygenase activity involved in the metabolism of prostaglandins, modulating the inflammatory process [56].

As CYP1 family members, AhR and ARNT regulate the induction of CYP2S1 [57]. Moreover, CYP2S1 peroxidase activity linked this CYP isoform with the metabolism of xenobiotics, such as environmental toxins/pollution and small-molecule drugs [58]. CYP2S1 can activate some anticancer prodrugs, e.g., ellipticine to 12-hydroxyellipticine and 13-hydroxyellipticine [8,59,60,61]. Therefore might be considered a more general therapeutic target. Analysis of the antitumor activity of GW-610 (2-(3,4-dimethoxyphenyl)-5-fluorobenzothazole) and 5F-203 (2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole) in breast cancer cell lines selectively depleted of CYP1A1, CYP2S1, and CYP2W1 showed that in contrast to CYP1A1 and CYP2W1, CYP2S1 mediates these drugs inactivation [13,62]. More detailed analysis of the metabolites formed from these drugs suggested that in its activation-deactivation hydroxylamine metabolite is involved, which can either be reduced back to the parent compound by CYP2S1 or progress to the formation of DNA adducts, mainly dGuo [62].

As was mentioned in the previous section, CYP2S1 expression was found in breast cancer cells differing in hormone receptors status. Interestingly, treatment of these cells with some methoxy stilbenes or resveratrol increased the expression of CYP2S1 in ER+ MCF7 cells, but not in ER- MDA-MB-231, where a decrease of the favorable level of this CYP was observed [14].

3.3. CYP2W1

Similarly to CYP2S1, the expression of CYP2W1 was described in breast cancer cells differing in hormone receptors status, and its upregulation was found in breast cancer samples [36]. Moreover, cells with higher levels of expression of these orphan CYPs in comparison to ER+ and PR+ breast cancer cells were observed in MDA-MB-231 and MDA-MB-468 cells.

The CYP2W1 gene is located on chromosome 7 p22.3 and encodes a protein comprising 490 amino acids [63]. The highest expression of CYP2W1 was noticed in the prostate and pancreas. The CYP2W1 expression is, to a great extent, regulated through epigenetic mechanisms mainly by DNA methylation. DNA hypermethylation is a pivotal epigenetic mechanism that silences many genes, including those regulating cell cycle, inflammation, stress response, DNA repair, and apoptosis [64]. Hypermethylation of certain genes, particularly tumor suppressor genes, is known to be associated with the inactivation of various pathways involved in tumorigenesis. CYP2W1 gene expression is supposed to be regulated by epigenetic modification, namely DNA methylation of its promoter CpG islands. In this regard, the early study of Gomez et al. [65] showed that the expression of CYP2W1 in colon cancer was associated with the methylation status of its promoter and suggested a causal link between the gene CpG island demethylation and CYP2W1 enhanced expression. This suggestion is further supported by the fact that CYP2W1 is expressed in the course of development of the gastrointestinal tract, silenced after birth in the intestine and colon by its promoter CpG island hypermethylation, but activated following demethylation. Therefore, it seems that demethylation is a prerequisite for CYP2W1 expression, probably also in breast cancer cells [65].

CYP2W1 is induced by AhR and ARNT pathway, as shown in the experiments in which treatment of MDA-MB-468 and MCF7 breast cancer cells with AhR ligands such as 5F-203 and GW-610 increased expression of CYP2W1 [13,66]. Moreover, in contrast to CYP2S1, CYP2W1 was involved in the activation of these drugs [6].

Untargeted substrate searches showed that CYP2W1 catalyzes both hydroxylation and epoxidation of several fatty acids and phosphatidylcholine [68]. Moreover, among the numerous other endogenous substrates were also steroids, including 17β-estradiol. However, its binding to CYP2W1 was significantly weaker than that of retinoids [69]. CYP2W1 is involved in drug metabolism, facilitating such reactions like N-demethylation and aromatic hydroxylation e.g., benzphetamine [70]. High expression of CYP2W1 in tumor tissue, particularly in breast cancer (overexpression ~230 fold) in comparison to normal mammary gland tissues [33], makes this CYP isoform an attractive anticancer drug target. CYP2W1 having the ability to activate prodrugs can increase the selectivity of chemotherapeutic agents to prevent tumor growth or metastasis as well as reduce the tumor volume before surgery [71]. Besides, CYP2W1 can be a potential target for immunotherapy, since it is located on the cell membrane surface. A specific antibody, derived from the peptide sequence of CYP2W1, was already developed and tested in MCF7 breast cancer cells [72].

3.4. CYP2U1

Immunohistochemical analysis, along with survival analysis based on clinical-pathological features, showed that CYP2U1 is engaged in the malignant progression of breast carcinoma.

Interestingly, CYP2U1 protein level was inversely linked with the state of ER, i.e., much higher in ER- in comparison with ER+ cancer tissue [37]. Therefore, it might be considered another druggable target for the treatment of advanced breast cancer. The human CYP2U1 gene is located on chromosome 4q25. Its protein product comprises 544 amino acids with the region containing 8 proline residues before the transmembrane helix and an insert of about 20 amino acids rich in arginine residues located after the transmembrane helix [73]. CYP2U1 is the only member of this subfamily, and it seems to be very old and highly conserved across species [74]. Similarly to the orphan CYPs described above, CYP2U1 catalyzes fatty acids hydroxylation. These include AA, docosahexaenoic acid, and eicosapentaenoic acids. CYP2U1-mediated metabolism of AA leads to the formation of 19- and 20-HETE. It was also shown that CYP2U1 efficiently catalyzes the hydroxylation of leukotriene B4 (LTB4) predominantly on its ω-position. The involvement of CYP2U1 in the metabolism of LTB4 could have significant physiologic consequences, as LTB4 is an important inflammatory mediator involved in the pathogenesis of many diseases, including cancer [75]. Specific inhibitors of CYP ω-hydroxylases (e.g., 17-octadecenoic acid) decrease CYP2U1-mediated activity [37].

3.5. CYP4X1

As was mentioned in the previous section, CYP4X1, along with the described above orphan CYPs, showed the highest expression in breast cancer samples and correlation with the tumor grade [6].

The CYP4X1 gene is located in the cytochrome P450 ABXZ gene cluster along with CYP4Z2, and according to recent studies, this enzyme catalyzes epoxidation of endogenous cannabinoid anandamide and arachidonic acid [39,76]. The lower gene expression of CYP4X1 was associated with shorter overall survival of Chinese gastric cancer patients treated with capecitabine and oxaliplatin [77].

A recent study by Hlavac et al. [2021] pointed out the association of a specific variant, rs17102977 in the CYP4X1 gene, with the response of breast cancer patients to the neoadjuvant cytotoxic chemotherapy. The substitution rs17102977 in CYP4X1 intron was associated with both the response of the patients to the neoadjuvant cytotoxic therapy and the disease-free survival of hormonally treated patients. It is also prognostic in patients unselected according to the therapy. The endocannabinoid system is involved in various physiological processes, including inflammation, immunomodulation, and suppression of different cancers, including breast cancer, thus, CYP4X1 may play a role in the response to anticancer chemotherapy via physiological processes. The role of rs17102977 in cancer is, however, unknown [41].

4. Conclusions and Future Direction

Most of the orphan CYPs described in this review seem to be expressed in different cancer tissues, and only one, i.e., CYP4Z1, could be named specifically for the breast epithelium. Therefore, this CYP isoform may be considered the most promising for the treatment of breast cancer. The currently available data indicate that inhibition of CYP4Z1 breast-specific expression may reduce the growth, progression, angiogenesis, and invasiveness of breast cancer. Moreover, the knowledge of the biological mechanism of CYP4Z1 activation could serve for the design of prodrugs activated explicitly to the active drug in the neoplastic tissue of the breast gland, reducing the potential systemic side effects of chemotherapy. Furthermore, a potency to reverse TAM-resistance in CYP4Z1-positive breast cancer cells may be an important tool to enhance the efficacy of existing adjuvant therapies. Finally, the generation of CYP4Z1 antibodies on the surface of breast cancer cells may contribute to the invention of effective immunotherapy for this cancer, employing anti-cancer vaccines. Since CYP4Z1 facilitates breast cancer development by induction of ERα expression, its inhibition may have a double effect, eliminating the major breast cancer risk factor.

CYP2W1, CYP2S1, CYP2U1, and CYP4X1, although not as specific for breast epithelium as CYP4Z1 is, show much higher expression in tumors than in normal tissues. Therefore, they may be considered more general cancer therapeutic targets. The most promising are the results of the studies confirming the importance of these CYPs in TNBC cases, for which treatment options are limited, and the survival prognosis is poorer.

Although the functions and substrates of these CYPs are still not known, it seems that all catalyze fatty acids hydroxylation. Their products, such as EETs or HETEs, are considered critical modulators of cancer progression involved in promoting cellular proliferation, neovascularization, angiogenesis, and metastasis. Therefore, inhibition of the expression and activity of these orphan CYPs might be more useful in cancer treatment than prophylaxis. On the other hand, CYP4Z1 induction of ERα expression may affect tumorigenesis initiation, thus, its inhibition may prevent tumor development.

Unfortunately, the knowledge about orphan CYPs and their role in breast cancer is limited to cell cultures and clinical research with a relatively small pool of patients. The identification of endogenous ligands of these isoforms is still topical and seems to be crucial in understanding their role in breast epithelium homeostasis. The future scope depicts a great need for a broader panel of cell lines as well as clinical trials with large numbers of patients with a reliable non-cancer base. Conceptual studies involving the simultaneous assessment of orphan CYPs transcript and protein levels and the presence of antibodies on the surface of both normal and cancerous breast epithelial cells would be significant.

In conclusion, orphan CYPs expressed both in tumor and non-tumor breast tissues may serve as potential targets for inhibition of tumorigenesis, particularly the delay of progression of breast cancer. Moreover, the orphan CYPs isoforms influencing the activation of potential prodrugs and sensitizing cells resistant to adjuvant therapy are hopes for effective prevention and treatment of breast cancer.