Breast Cancer (BC) is the most common cancer among women and is the second leading cause of cancer deaths in women. Breast cancer affects women worldwide. In 2021, the United States estimated over 280,000 new cases of the invasive disease and over 40,000 deaths (1, 2). This is primarily due to the high metastasis rates, recurrence, and chemoresistance. BC is a heterogeneous and complex disease exhibiting a great degree of intra- and inter-tumoral heterogeneity (3). BC can be categorized into four subtypes: Basal, Human Epidermal growth factor Receptor 2 (HER2) positive, Luminal A, and Luminal B. Each is determined by different genetic or epigenetic factors (4). By such a classification, significant differences have been defined by their risk factors, incidence, treatment sensitivity, and prognosis as the basis for diagnosing and treating this disease (5). Basal, commonly known as Triple Negative Breast Cancer (TNBC), is the most aggressive and malignant subtype since it lacks estrogen receptors (ER), progesterone receptors (PR), and does not express the human epidermal growth factor receptor 2 (HER2) (6). Current traditional treatment options are not as effective at combating this specific breast cancer due to its characteristics. These characteristics make it difficult to treat this subtype with hormone therapies and HER2 inhibitors. Standard treatment options typically involve hormonal therapies and HER2 inhibitors, valid treatment options with other BC subtypes. While therapeutic hormonal therapies for other subtypes are effective, therapeutic resistance can form from prolonged treatment (7). Accordingly, new therapeutic treatments that target specific genes responsible for the regulation and metastasis of tumors are needed to improve the prognosis of patients diagnosed with BC.

This study will be focusing on NR4A (nuclear receptor subfamily 4A), an orphan member of the steroid/thyroid/retinoid nuclear receptor superfamily. These nuclear receptors act as transcription factors to control downstream gene expression and participate in diverse biological functions (8, 9). NR4A receptors are modified by various cell-signaling pathways depending on subcellular localization, levels of expression, transcriptional modulation by coactivators and/or corepressors, posttranslational modification, and interaction with other transcription factors (9). Modulating the expression levels of NR4A1, NR4A2, and NR4A3 can be a potential therapeutic approach for breast cancer. As we outline herein, elucidating the novel prognostic and regulatory roles of the NR4A family and their function as essential mediators regulating breast cancer progression.

The homologous orphan receptors, NR4A1, NR4A2, and NR4A3, are a subfamily of the eukaryotic transcription factors (10, 11). This nuclear receptor subfamily encodes 48 human genes and has many biological functions. They have physiological and pathological roles in the human body specifically regulating homeostasis, proliferation, cell migration, apoptosis, metabolism, DNA repair, glucose utilization, and tumorigenesis (12–14). No endogenous ligand has been identified for these nuclear receptors, but they are considered constitutively active (11, 15). Although ligands are not identified, some compounds characterized as ligands that bind the receptor (8, 15). One compound, Cytosporone B (CnsB), an NR4A agonist, has been shown to have increased pro-inflammatory mediators when compared to a control that did not have CnsB (15, 16). NR4A1-3 is commonly considered a molecular switch, and its actions are regulated by a complex network of cellular signaling pathways (11). They are an immediate early gene of stressors and stimulated by peptide hormone, growth factors, cytokines, inflammation, and most importantly, cellular stress (8, 15). They contain a potent sensor that detects changes in the cellular microenvironment, and the NR4A receptors trigger mitochondria biogenesis and improve mitochondrial functions (12, 17). Previous studies have stated that the receptors played roles in promoting DNA repair after cellular stress and can even stimulate protective cells that would aid in the prevention of further damage to cells (14). The same response is also seen in neurons (18). When neurons are exposed to oxidative stress, NR4A is induced through cAMP response element-binding proteins (CREB), upregulated neuroprotective genes, and increased neuronal survival (18). These nuclear receptors also have an important role in immune function and are linked to chronic inflammation, altered immune cell response, and cancer development (8, 11, 12, 19, 20). NR4A3 was identified as a direct transcriptional target of p53 (13). Once p53 is bound to NR4A3, it induces transcription and leads to an increase in expression. This overexpression has been determined to attenuate the proliferation of cancer cells and promote apoptosis through the increasing pro-apoptotic genes (13, 21). The role of the nuclear receptors has been mainly studied in two different cancers: blood-derived cancers and solid tumor cancers (15). The studies on blood-derived cancers have suggested that NR4A has tumor suppressor activity (15). In acute myeloid leukemia (AML), NR4A is silenced by blocking the transcriptional elongation rather than epigenetic promoter silencing (15, 22, 23). Treatment with dihydroergotamine was shown to reactivate NR4A expression and enable elongation of the NR4A promoter, previously hindered by RNA polymerase II (22, 23), and this led to increased survival. Similar trends were shown for Lymphomas, where NR4A3 expression was low and had poor survival (14, 15, 21). Therefore, with an ectopic expression and pharmacological activation, NR4A3 expression increased and was measured to have a higher proportion of apoptotic cells than before the treatment (21). In contrast, solid tumors showed higher levels of NR4A1 and NR4A2 and were exhibited to be prooncogenic (15). An experiment was performed and showed that chimeric antigen receptor T- cells (CAR T-cells) that lacked NR4A promoted tumor regression and prolonged survival of cancer patients when compared to CAR T-cells that expressed NR4A (24). It was determined that NR4A played a role in the cell-intrinsic program of t-cell hyperresponsiveness. Studies that have been performed concluded that an NR4A inducer would be helpful in blood-derived tumors since it would increase the pro-apoptotic genes. At the same time, N4A1/NR4A2 antagonists would benefit solid tumors (15).

Metabolic reprogramming is a typical hallmark of cancer cells (25). Despite adequate oxygen availability, tumor cells tend to generate aerobic glycolysis energy rather than consume the energy produced via mitochondrial oxidative phosphorylation (OXPHOS). This phenomenon is known as The Warburg Effect, which often results in increased glucose uptake, an accumulation of ATP, and lactate production in tumor cells in breast cancer, both elevated and reduced OXPHOS activity are observed. In the case of elevated OXPHOS, invasive metastatic breast cancer cells specifically favor mitochondrial respiration to increase ATP levels through a mechanism that involves overexpression of PGC-1α and increases mitochondrial biogenesis (26). It has been shown that OXPHOS activity increases concomitantly with the metastatic potential in primary breast cancer (27, 28). Consistent with this, Alpha CUB-domain containing protein 1 (CDCP1), a transmembrane glycoprotein, drives breast cancer cell metastasis by activating OXPHOS and fatty acid oxidation (26, 29). Therefore, mitochondrial metabolism represents an attractive target for anti-metastatic approaches.

The NR4A subfamily regulates cellular proliferation in a tissue-dependent manner in breast cancer, which means their roles are different in each subtype (19, 30). As described above, in some cancers, the NR4A subfamily can promote proliferation, but in others, they inhibit it. However, results about the roles of the receptors are not consistent. This could result from the nuclear receptors binding lipophilic molecules, which allows it to sense environmental, systemic, and local factors (31). Nuclear receptors derive their ligands from dietary derived factors and metabolism, allowing them to regulate glycolysis and oxidative phosphorylation (9). Previous studies showed an increase in glucagon, which led to an increase in NR4A1, NR4A2, and NR4A3 in a cAMP-dependent process (32). The overexpression of the nuclear receptors induces the expression of other target genes, including Glut2, leading to an increase in gluconeogenesis. This occurs since the expression of the receptors transcriptionally upregulates the rate-limiting enzymes in gluconeogenesis (33). This causes a shift that favors gluconeogenesis, leading to a suppression of glycolysis and, in conjugation, low ATP and cell arrest. In HCC, it has been shown that NR4A1 interacts with phosphoenolpyruvate carboxykinase (PEPCK1), the rate-limiting enzyme in gluconeogenesis, to increase gluconeogenesis and suppress glycolysis, resulting in ATP depletion and cell growth arrest (33). In addition, reduced expression of NR4A1 activated glycolytic pathway in acute promyelocytic leukemia cells (34).

As for the roles of NR4A receptors in the mitochondria, there have been direct links that prove the role of NR4A in mitochondrial production and fuel oxidation. The nuclear receptors are essential for the biogenesis and enzymatic components of the TCA cycle and the ETC (35). Through a downregulation of isocitrate dehydrogenase 1, NR4A1 had been shown to induce oxidative and endoplasmic reticulum stress (36). NR4A1 binds to the mitochondrial bcl-2. In doing so, it weakens the mitochondrial membrane potential. This causes a pro-apoptotic complex (36). These are some mechanisms in which the nuclear receptors can be viewed as having tumor suppression qualities (12). However, there is still a lack of comprehensive bioinformatic study on this topic. Altogether, NR4A receptors are important factors in the processes of carcinogenesis, apoptosis, DNA repair, proliferation, migration, inflammation, metabolism, and angiogenesis (9). Therefore, modulating the expression levels, activity, and nuclear export of NR4A receptors, can develop new cancer treatments. As we outline herein, the novel prognostic role of the NR4A1–NR4A3 receptors implicate these receptors as important mediators controlling breast cancer metabolic reprograming and its progression.

A significantly higher level of NR4A1 expression was found in tumor stage four relative to tumor stage one and three. However, there was no difference in the levels of the other receptors (p-value 0.0099 and 0.0218, respectively), ( Figure 4A ). NR4A1 and NR4A2 had significantly lower expression in grade 3 tumors when compared to grades 1 and 2, but there was no difference when evaluating the expression of NR4A3 (p-value 0.0011, 0.0338, <0.0001, and <0.0001, respectively) ( Figure 4B ). All three nuclear receptors’ expression levels did not influence tumor size ( Figure 4C ). NR4A1 showed a significantly higher gene expression in patients that were diagnosed lymph node-positive relative to those lymph node-negative. At the same time, there was no significant difference in the levels for NR4A2 and NR4A3 (p-value 0.0308) ( Figure 4D ). NR4A1 showed a significantly lower gene expression in those 65-85 when compared to those 20-45 and 55-65 (p-value 0.0167 and 0.0046, respectively). However, the opposite trend was shown for NR4A2, with those 65-85 having a significantly higher gene expression than all other groups (p-value 0.0003, 0.0001, 0.0453, respectively). Finally, there was no significant difference in NR4A3 expression between age groups ( Figure 4E ).

According to the 2021 American Cancer Society statistics, BC has the highest number of estimated new cases and the second-highest number of deaths (43). There are multiple available treatments for BC that have proven effective. However, there are often cases of relapse or drug resistance following the completion of treatment. Therefore, it is important to emphasize that BC diagnosis time is critical, as early detection will allow for quicker treatment intervention. The NR4A family of orphan receptors, NR4A1–NR4A3, belongs to the superfamily of nuclear receptors, which regulate genes involved in proliferation, cell migration, and apoptosis. Here, we report on the clinical and prognostic value of NR4As and their relevance in prognosis and therapeutics for BC patients. Using the large genomic studies, TCGA and METABRIC, we were able to analyze the expression of NR4A1–NR4A3 in all BC subtypes, including Basal, Her2, Luminal A, and Luminal B. Basal BC is considered to be aggressive and metastatic, and effective therapeutic treatments are minimal (44). NR4A1 and NR4A2 have been studied in breast cancer (30, 45, 46). Consistent with our analysis, it has been shown that NR4A1 reduced during the development of mouse basal-like mammary tumors and significantly downregulated in human TNBC samples. More specifically, TNBC cell lines with little endogenous NR4A1 inhibited cell proliferation, viability, migration, and invasion by down-regulating the JNK1–AP-1–cyclin D1 pathway (45). This is probably why this subtype has a very poor prognosis and is often associated with cancer relapse following completion of treatment. Also, the nuclear receptor NR4A2 participates in multiple metabolic regulations and plays paradoxical roles in tumorigeneses (11). For example, NR4A2 interacted and recruited corepressors, the SWI/SNF complex, to the promoters of CD36 and FABP4 to suppress their transcriptions. Because of this, the fatty acid uptake was hampered, leading to the inhibition of breast cancer cell proliferation (46). In another study, Llopis, S., et al. showed the dichotomous roles of the NR4A2 in breast cancer (30). They showed a higher NR4A2 expression level in the normal breast epithelium compared to breast carcinoma cells, which strongly correlated with an increase in relapse-free survival in a cohort of breast cancer patients. However, NR4A2-silenced breast xenografts models showed significantly decreased growth compared to the control model, showing a biphasic role for NR4A2 in breast cancer progression (30). NR4A3 has been previously shown to be a direct target for p53, inducing its transcription. Mechanistically, overexpression of NR4A3 attenuated the proliferation of breast cancer cells and promoted apoptosis by augmenting the expression of pro-apoptotic genes PUMA and Bax (13).

To date, there is no patient-centered clinical dataset validating the role of NR4A gene expression in breast cancer. Thus, we sought to investigate the role and functionality of NR4A in BC patients using publicly available databases containing records from large BC studies. Multiple research groups have extensively studied the role and therapeutic benefits of NR4As as a potent tumor suppressor or oncogene protein in breast cancer and, more specifically, how it depends on the tumor subtypes. In this study, we characterized gene expression of NR4A1–NR4A3 in all the breast cancer subtypes (Basal, Her2, Luminal A, and Luminal B) by using large genomic studies (TCGA and METABRIC). Previous studies found NR4A family gene expression critical in basal breast cancer progression and metastasis (30, 35, 36, 45). Our study found that NR4A1, 2, and 3 gene expressions are luminal breast cancer-specific. Low gene expression of NR4A1 is observed in basal, HER2, and luminal A subtypes compared to the Normal-like subtype. NR4A2 mRNA overexpressed in both luminal A and luminal B breast cancer relative to other subtypes. NR4A3 mRNA expression was lower in all cancer subtypes compared to Normal-like subtypes with no meaningful differential expression between cancerous subtypes. In addition, NR4A1 and NR4A2 mRNA expressions are lower in basal breast cancer when compared to other subtypes of breast cancer. Also, when samples from all BC intrinsic subtypes were analyzed as a group, NR4A1-NR4A3 expression is significantly downregulated in BC patients compared to normal control subjects. Previously, using publicly available clinical data and patient survival analysis has been shown that having high levels of NR4A3 expression positively correlates with increased survival rates for patients with breast cancer (13). Consistent with this, our results showed a positive link between NR4A1-NR4A3 expression and increased overall survival and relapse-free survival in breast cancer patients.

Interestingly, we found that NR4A1 expression is progressively downregulated with an increase in age while NR4A2 is upregulated. Previous studies have shown in HCC it has been demonstrated that NR4A1 suppresses glycolysis in different cancers (33, 34). The result of our correlation analysis validated that NR4A1-NR4A3 expression is reversely correlated with many glycolytic and oxidative phosphorylation targets, suggesting the probable role of these receptors in regulating these pathways in breast cancer. Finally, our methylation results showed no significant positive correlation between NR4A1-NR4A3 expression and their DNA methylation, suggesting that these genes’ epigenetic regulation might not be the case in breast cancer. In summary this review indicates prognostic role of the NR4A1–NR4A3 receptors and implicates these receptors as important mediators controlling breast cancer metabolic reprograming and its progression.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

HY and JF wrote the first draft and SA finalized the manuscript. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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