1. Introduction
Cytochrome P450 enzymes (CYPs) catalyze various reactions and are of significant importance in the area of drug and carcinogen metabolism. The latter also include endogenous compounds such as estrogens, considered a major risk factor in breast cancer. It is estimated that ~75% of drug metabolism reactions involve CYPs, and this class of enzymes bioactivates ~66% of carcinogens. While in most of these reactions CYPs with well-characterized function and substrate specificity are involved, it is now clear 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 [Citation1], and it is estimated that of the 57 human CYPs and 58 pseudogenes discovered to date, 1/4 still remain ‘orphans’ in the sense that their function, expression sites, and regulation are still largely not elucidated [Citation2]. Interestingly, the expression of some of these orphan CYPs is tissue specific and increases in tumorigenesis. For example, CYP4Z1 was found to be frequently upregulated in primary mammary carcinoma and ovarian cancer and was associated with tumor grade/progression [Citation3]. Breast cancer is the leading cause of cancer-related deaths among women worldwide. Therefore, the orphan CYPs expressed in breast epithelial cells are of particular interest in targeted therapy.
This editorial focuses on describing and discussing current knowledge of orphan CYPs in this tissue and their possible application in diagnostics and drug targeting.
2. Expression of orphan CYPs in breast epithelium and cancer
Most data on the expression of orphan CYPs genes or enzyme present in the breast epithelium come from in vitro studies using breast cancer cell lines derived from cancer differing in tumor origin and receptor status. Only part of these results was confirmed in clinical samples.
In the most extensively investigated estrogen-dependent (ER+) MCF7 breast cancer cell line, the expression of three orphan CYPs, namely CYP2S1, 2W1, and 4Z1, was confirmed by several authors [e.g., Citation4,Citation5,Citation6,Citation7,Citation8]. The analysis of CYPs expression profile in ER+ T47D cell line, also derived from ductal carcinoma, revealed the presence of CYP4Z1 [Citation9,Citation10]. Stable expression of CYP4Z1 was achieved in BT-474 human HER2 positive breast cancer cells [Citation9]. This CYP enzyme has inspired particular interest because of its hypothetical role in breast cancer by forming the signaling molecule 20-hydroxyeicosatetraenoic acid (20-HETE). The expression of this orphan isoform as well as of CYP2S1and 2W1 was also described in triple-negative i.e., negative for ERs, progesterone receptors (PR), and HER2 cells, lines MDA-MB-231 and MDA-MB-468 [Citation4,Citation5,Citation7]. Moreover, in these cells, higher levels of expression of orphan CYPs compared to estrogen and progesterone positive breast cancer cells were observed.
The mRNA of several orphan CYPs (e.g., CYP2S1, 2U1, 4V2, 4X1, 4Z1) was also detected in non-tumorigenic breast epithelial MCF10A cell line. However, CYP4Z1 protein was not found on the cells’ surface [Citation8,Citation11].
In order to establish the possible role of orphan CYPs in breast cancer development and/or chemotherapy resistance, the group of Tao Xi [e.g. 7] 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 ER α activity, apoptosis, stemness, and resistance to tamoxifen in breast cancer cells. Moreover, stable overexpression of CYP4Z1 in breast cancer cells has been reported to promote angiogenesis and tumor growth in mice [Citation9].
Clinical studies describing the orphan CYPs expression profile and its relationship with clinicopathological variables, although limited, showed an interesting trend. Murray et al. [Citation3] 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. CYP4V2, CYP4X1, and CYP4Z1 showed a correlation with the tumor grade. Correlation with survival was identified for CYP2S1, CYP3A4, CYP4V2, and CYP26A1, although none of these P450s was an independent prognosis marker.
In some smaller pools of patient samples, the increased transcript level of CYP2S1, CYP2W1, and CYP4F11 both in breast cancer, adjacent and normal breast cells were found, although the protein levels were inappropriately low to confirm the results [Citation12].
As it was mentioned above, much attention is focused on the role of CYP4Z1 and an associated pseudogene CYP4Z2P because of a breast-epithelium specific expression. In this regard, Cizkova et al. [Citation13] noticed in the series of 249 ER+ breast cancer patients correlation between the mutation status of oncogene PIK3CA and overexpression of these CYPs. Moreover, another preliminary investigation on the sera of breast cancer patients noticed the increased anti-CYP4Z1 antibodies level compared to healthy controls [Citation8].
Finally, the expression of CYP2A7, an orphan CYPs of so far smaller interest in the context of breast cancer, showed association with poorer survival of triple-negative breast cancer patients [Citation14].
3. Orphan therapeutic targets?
So far, the most studies, as it was described above, concentrated on the prognostic value of orphan CYPs in breast cancer cells. However, attempts are made to understand the mechanism of catalytic activity of the most promising CYP4Z1 to design the specific inhibitor. In this regard, it was shown in a combined in vitro and in silico approach that residue Arg487 plays an important role in the substrate recognition and binding of CYP4Z1 [Citation15]. Stable overexpression of CYP4Z1 in breast cancer cells was linked with a concomitant increase in cellular 20-HETE [Citation9]. However, analysis of arachidonic acid (AA) metabolism by recombinant CYP4Z1 expressed in Saccharomyces cerevisiae did not show detectable oxidized metabolites at the v-terminus, but instead the epoxidation of AA at the 14,15-double bond with high regioselectivity and stereoselectivity. These data suggested that the mechanism of CYP4Z1 activity did not involve direct 20-HETE generation but rather the formation of 14,15-epoxyeicosatrienoic acid (14,15-EET). Since this AA metabolite is known to influence cellular proliferation, migration, and angiogenesis, the same group developed a selective mechanism-based inhibitor, 8-[(1 H-benzotriazol-1-yl)amino]octanoic acid, which in low micromolar concentrations inhibited 14,15-EET production in T47D breast cancer cells transfected with CYP4Z1 [Citation10]. These recent studies may pave the path to rational mechanism-based inhibitors – prodrugs design.
The modulation of CYP2S1 and CYP2W1 was described only in one study by Szaefer et al. [Citation5]. The increased CYP2S1 and 2W1 expression in ER+ MCF7 in the presence of some methoxystilbenes, and oppositely, a decreased favorable levels of these CYPs in ER- MDA-MB-231 cells as a result of treatment with methoxystilbenes and resveratrol were observed.
4. Expert opinion
Most of the orphan CYPs seem to be expressed in different cancer tissues, but only a few could be named the breast epithelium specific. These are CYP4Z1 and perhaps CYP4X1 and CYP4F11.
Based on the currently available data, CYP4Z1 may be considered the most promising target for preventing and treating breast cancer. Firstly, because inhibition of its breast specific expression may reduce the growth, progression, angiogenesis, and invasiveness of breast cancer. Secondly, due to its unique expression in all subtypes of breast cancer, CYP4Z1 could serve as a base for the design of prodrugs activated explicitly to the active drug in the neoplastic tissue of the breast gland, reducing the potential systematic side effects of chemotherapy. However, the main challenge for CYP4Z1 based prodrug strategy is to identify candidate prodrugs that can be activated by this enzyme. Recently elaborated CYP4Z1 homology models should bring the solution of this problem closer.
Thirdly, the generation of CYP4Z1 antibodies on breast cancer cells’ surface may contribute to the invention of effective immunotherapy of this cancer. Moreover, the potential role in reversing TAM-resistance in CYP4Z1 positive cells could give an important tool for enhancing the effectiveness of existing adjuvant therapies.
The presence of CYP2W1 and CYP2S1, although it is not so specific for breast epithelium as CYP4Z1 is, shows much higher expression in tumor 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 triple-negative cases of breast cancer, for which treatment options are limited and survival prognosis poorer.
Unfortunately, the knowledge about orphan CYPs and their role in breast cancer is restricted to cell cultures and clinical research with a relatively small pool of patients. Identifying endogenous ligands and hence the deorphanization of these isoforms seems to be crucial in understanding their role in the 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 to inhibit tumorigenesis initiation and 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 hope for effective prevention and treatment of breast cancer.
Declaration of interest
The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.
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