• This study reflects the pivotal role and prognostic consequence of RAR-α in BC pathogenesis.

The most frequent cancer diagnosed globally is now breast cancer (BC), surpassing lung carcinoma. In 2020, 2.3 million new instances of breast cancer were expected, accounting for roughly 11.7 percent of all new cancer cases, and 684,996 deaths were reported from it (Sung et al., 2021). Epidemiological research indicates that by 2030, there will be around two million people with breast cancer globally. In India, the incidence increased sharply, by about fifty percent, between 1965 and 1985 (Mehrotra and Yadav, 2022). There were an estimated 118000 incident cases in India in 2016, with 98.1 percent of the cases being women including the 526000 prevalent cases. From 1990 to 2016, there was a nationwide rise in the age-standardized incidence rate of BC in women over the preceding 26 years by 39.1 (Kaur et al., 2019). Global Cancer Observatory (GLOBOCAN) predictions for 2020 indicate that 19.3 million cancer cases occurred incidentally globally. India ranked 3^rd after China and the United States of America. By 2040, 2.08 million cancer cases are estimated to occur in India, indicating a 57.5 percent increase from 2020, according to GLOBOCAN’s prediction. Globocan data 2020 showed that breast carcinoma caused 10.6 percent (90408) of all deaths and 13.5 percent (178361) of all malignancy cases in India, respectively, with a 2.81 cumulative risk (Sofi et al., 2023). Although breast cancer is still the prominent reason for cancer death in females, recent data indicate a decrease in death rates (Printz, 2017). Increased knowledge, screening methods, and developments in targeted treatments are responsible for this. In contrast to morphological prognostic indicators, gene expression research has demonstrated that the tumor’s inherent molecular properties predominantly influence tumor response to therapy. As high-throughput platforms for gene expression research have emerged, molecular techniques like microarrays are utilized to investigate these inherent molecular characteristics (Weigelt et al., 2010). Therefore, a key element of treatment decision-making is the classification of BC into appropriate molecular subgroups. Perou and others categorised breast carcinoma into four major clinical subgroups based on receptor status ER, PR, HER2, gene expression patterns, and proliferative status as determined by Ki67 (Perou et al., 2000). These clinical subtypes include basal-like or basaloid or TNBC, HER2-enriched, and luminal (A and B). BCs with non-luminal A have a higher chance of recurring early. There is a prolonged dormancy period for potential recurrence in luminal-A tumors. TNBCs exhibit greater histological grade and advanced illness, making them aggressive (Jonnada et al., 2021).

Numerous risk factors, including genetic and inherited predisposition, are linked to the development of breast cancer. BCs are quite diverse. Treatment approaches for BC differ based on molecular characteristics, such as activation of the HER2, ER and PR hormonal receptors, gene mutations such as those in the BRCA1/2 and PIK3CA, and markers in the microenvironment of the immune system such as PDL1 and TIL. Early-stage BC is thought to be treatable and is treated mostly with local-regional treatments (surgery and radiation), with systemic therapy administered before or after surgery as needed (ACOG, 2019).

For the majority of early-stage HER2-positive and TNBC patients, preoperative or neoadjuvant therapy—such as immune checkpoint inhibitors or targeted therapy—is the accepted course of treatment. This is followed by risk-adapted post-surgical therapies. Endocrine treatment for five to 10 years is necessary for early breast cancer that is ER-positive. Presently, advanced BC with distant metastases is thought to be incurable. Systemic treatments in this case could involve immunotherapy, which is currently being used for TNBC, anti-HER2 targeted treatment for HER2 positive disease, poly (ADP-ribose) polymerase inhibitors for carriers of BRCA1/2 mutations, and endocrine therapy with targeted drugs like CDK4/6 inhibitors and PI3K inhibitors for hormone receptor + ve disease. In the future, precision medicine technologies may guide the escalation or de-escalation of personalized treatment (Hong and Xu, 2022). The development of therapeutic strategies for the treatment of BC and prognosis prediction depend heavily on the nuclear receptor superfamily of transcription factors, which include the ER and PR. There is mounting evidence that nuclear receptors other than ER and PR play a role in the initiation and progression of BC, according to recent research (Hua et al., 2009; Ni et al., 2011). Patients are frequently treated in clinics with medications that target nuclear receptors (Muscat et al., 2013). The anti-proliferative and anti-apoptotic effects of the glucocorticoid receptor in BC and its upregulation are linked to characteristics of extended survival (Abduljabbar et al., 2015).

Retinoids are both natural and synthetic compounds related to retinoic acids (RA), function through interacting with two different classes of nuclear receptors from the nuclear receptor superfamily: retinoic acid receptors (RAR-α, β, and γ) and retinoid X receptors (RXR-α, β, and γ) (Germain et al., 2006a; Pérez et al., 2012). These transcription factors are activated by ligands and belong to the superfamily of steroid hormone receptors. Retinoids are hypothesized to regulate cell development through both direct and indirect impacts on gene expression. Retinoic acid receptor (RAR-α, β, and γ), and retinoid X receptor (RXR-α, β, and γ) mediate these actions (Simeone and Tari, 2004). Retinoid receptors are essential for healthy development and are expressed by both healthy and malignant breast epithelial cells. Although the exact mechanism by which retinoid inhibits breast cell proliferation is yet unknown, it most likely involves several different signal transduction pathways. Several retinoids have the ability to bind to nuclear receptors and activate the AP-1 transcription pathway, which is triggered by growth factor signalling. Retinoids have a well-established function in the control of cell proliferation and differentiation, which is why numerous clinical trials are testing these drugs to prevent cancer (Decensi et al., 2003). Retinoids have been shown to prevent the development of cancer, slow the growth of tumors, and promote the differentiation and invasion of tumor cells in different tissues (Jan et al., 2023). Due to their capacity to prevent cell proliferation and induce morphological or phenotypic differentiation, retinoids, particularly RA, have been recommended as an adjuvant therapy for BC (Zanardi et al., 2006). A pleiotropic signaling protein called RA controls vital genetic programmes that regulate cell proliferation, differentiation, cell death, and/or survival (Clagett-Dame and Knutson, 2011; Samarut and Rochette-Egly, 2012). It also controls homeostasis, development, and other processes. Out of the three distinct RAR genes (RARα, RARβ, and RARγ) that have been identified, RARα interacts with target genes to contribute to tumor growth, metastasis, drug resistance, and other processes (Di Masi et al., 2015). According to the canonical model of RAR-mediated gene regulation, Retinoids (RXR) and RARα form heterodimers when ligand is absent. These heterodimers can engage constitutively specific response elements (RAREs) found in target gene promoters. RARα coupled to DNA is linked to the corepressors nuclear receptor corepressor 1 (NCoR1) and NCoR2, which serve as adaptors attracting additional subcomplexes with histone deacetylase (HDAC) activity, hence causing transcriptional repression (Perissi et al., 2010; Rochette-Egly, 2015). Following ligand binding, RARα experiences conformational changes that lead to its dissociation from corepressors and the recruitment of coactivators, including histone acetyltransferase (HAT) and nuclear receptor coactivator (NCoA)1/2/3. The recruitment of transcriptional machinery and the activation of target genes are made possible by this mechanism (Glass and Rosenfeld, 2000).They control the expression of certain target gene subsets related to apoptosis, proliferation, and cellular differentiation (Bour et al., 2006; Eifert et al., 2006; Delacroix et al., 2010; Gudas and Wagner, 2011). Accordingly, RARs are essential for many biological processes, such as development, immunity, reproduction, organogenesis, and homeostasis. This is demonstrated by pharmacological, vitamin A deficiency, and genetic research carried out in mice (Niederreither and Dollé, 2008; Su and Gudas, 2008; Dollé, 2009; Mark et al., 2009). Apart from their physiological functions, RARs are also linked to pathologies and tumors as a result of mutations, protein fusions, changes in expression levels, or abnormal post-translational changes that lead to aberrant functions and homeostasis breakdown. Given their capacity to control development and differentiation throughout life, it is obvious that aberrant expression of RAR and/or function may play a role in many malignancies. Tumor growth has thus been linked to RAR overexpression, deletion, mutations, or abnormal post-transcriptional alterations (Duong and Rochette-Egly, 2011). The retinoic acid receptor RAR-α is over-expressed in several malignancies. Increased invasion and migration in multiple cancer forms, such as HNSC carcinoma, pediatric low-grade gliomas, lung adenocarcinoma, and breast cancer, have been associated with its overexpression. Numerous authorised therapeutic regimens targeting RAR-α have been developed, improving patient survival rates (Jan et al., 2023). Identification of patient subpopulations who may be responsive to therapy and hence would benefit from it is necessary for the successful therapeutic usage of retinoids in the treatment of BC. High RAR-α levels in the tumor are associated with sensitivity to retinoid therapy, according to preclinical and clinical findings (Centritto et al., 2015). Co-amplification of the RAR-α gene, which results in enhanced production of the RAR-α protein is shown in a significant fraction of HER2 positive BCs. RA can effectively inhibit cell proliferation in these cases. In the context of ER negative cancers, which do not respond to hormonal treatments, this is especially important. Anti-HER2 positive medications are more effective in ER-negative/HER2positive/RAR-α+ tumors when RA is given at the same time (Paroni et al., 2012).

Trifarotene, a 4^th generation retinoid, on a topical basis, is used to treat acne vulgaris clinically. Compared to other retinoids trifarotene has a low toxicity and good stability, according to a recent study that examined its metabolic and pharmacological characteristics (Naik, 2022). Trifarotene has anti-inflammatory characteristics, is highly selective for RARγ, and regulates processes involved in the etiology of skin cancer (such as differentiation, proliferation, etc.). Additionally, this medication is well tolerated and relatively easily metabolized in the liver (Aubert et al., 2018; Ramchatesingh et al., 2022). Accordingly, trifarotene in the context of drug repurposing can be used in treating breast carcinoma including other cancers.

In the current work, for in silico investigation we explored the RAR-α expression and prognostic significance in breast carcinoma using bioinformatics tools. The expression pattern, genetic alteration, prognostic relevance, and immunological association of RAR-α in different BC tissues were assessed using the TIMER 2.0, UCSC XENA, Bc GeneExminer, ENRICHR, TISCH, UALCAN, KM-Plotter and Oncomine tools. In breast cancer tissues, RAR-α levels were elevated, and overexpression of RAR-α was linked with worse overall survival (OS) and relapse-free survival (RFS). Additionally, in-silico docking of RAR-α with trifarotene was conducted and showed a high binding score. Furthermore, trifarotene's effect was subsequently investigated in-vitro in breast cancer cells. This study shows that RAR-α is essential for breast tumorigenicity since it is linked with worse OS and RFS in breast carcinoma individuals with high RAR-α expression. Therefore, to reduce the chemoresistance development and improve the response of BC individuals to treatment can be achieved by targeting RAR-α in conjunction with other cancer pathways.

Cancer is the 2^nd main reason for mortality and a significant public health issue (Sofi et al., 2022). Breast cancer (BC), particularly has a significant occurrence and mortality rate, making it the most widespread form of malignancy among females globally. It is a type of cancer connected to a substantial number of deaths. The primary reason behind this elevated mortality is the emergence of drug resistance, which poses a major obstacle in the treatment of BC (Correia et al., 2018). The OS and RFS of BC patients have improved due to advancements in early detection and therapy, but the rise in metastatic and resistant malignancies need immediate attention. Thus, there is an urgent need to investigate new therapeutic targets and drug repurposing for established breast cancer targets (dos Santos Correia, 2019). RARs form heterodimers with RXRs, which are ligand-dependent transcriptional regulators (Germain et al., 2006b; Germain et al., 2006c). RARs regulates numerous biological functions, including development, reproduction, immunity, organogenesis and homeostasis. Apart from their physiological functions, RARs are also linked to pathologies and tumors as a result of mutations, protein fusions, changes in expression levels, or abnormal post-translational changes that lead to altered functions and homeostasis disruption. It is obvious that aberrant RAR expression and/or function may play a role in many malignancies (Niederreither and Dollé, 2008; Su and Gudas, 2008; Dollé, 2009; Mark et al., 2009). Tumor growth has thus been linked to RAR overexpression, deletion, mutations, or abnormal post-transcriptional alterations. The RAR-α is over-expressed in several malignancies (Duong and Rochette-Egly, 2011). Its over-expression has been connected to enhance the migration and invasion in several types of carcinomas including head and neck squamous cell carcinoma, pediatric low-grade gliomas, lung adenocarcinoma, and breast carcinoma. In the present study, several in silico procedures were used to investigate the RAR-α expression pattern in breast carcinoma. We have further analyzed the effect of the protein docking method to screen a fourth-generation retinoid based therapeutic molecules to target RAR-α.

TIMER 2.0 study results showed that RAR-α is highly expressed in many malignancies including BC, HNSC, KIRC, LUSC, LUAD, LGG, SARC, THCA, and UCEC (Figure 1). A heat map of important RARs and RXRs in breast cancer was created from UCSC Xena. The heat map results also demonstrated that, in comparison to other RARs and RXRs, RAR-α is significantly overexpressed in breast cancer (Figure 2). RAR-α expression analysis in connection to type of sample, age group, subtypes of breast cancer, and ethnicity was further explored using the UALCAN database. The study revealed that RAR-α is greatly expressed in primary tumors than normal patients. The patients with luminal breast cancer displayed increased expression of RAR-α when compared to HER2 or TNBC subtypes. According to the cancer stage, stage 3 showed the highest expression of RAR-α, followed by stage 2, stage 1, and stage 4. Additionally, RAR-α expression was augmented in women between the age of sixty-one to Eighty years old (Figure 3). Moreover, it was found that BC individuals with overexpression of RAR-α had aberrant (WNT, mTOR, and the p53-Rb) signaling pathways (Figure 4).

The bc-GenEXMiner portal was also used to explore the connection between RAR-α and clinicopathological characteristics in breast cancer patients. The findings demonstrated that RAR-α level is typically highly expressed in breast cancer individuals with ER+, PR + receptor expression, nodal negative status, SBR2, and NPI2 grades (Figure 5). The gene-gene interaction of RAR-α was examined using the GeneMANIA portal and the results depicted that RAR-α showed strong interactions with RXR-α, followed by TADA3, MED1, HOXD4, TMSBI10, and several other genes (Figure 6). The PPI of RAR-α was examined by the string database and the results showed a number of genes interacting with RAR-α and network’s top ten hub genes comprised of RARA, RXRA, NCOA1, NCOR1, NCOR2, PPARG, HDAC3, CREBBP, EP300, and SIN3A (Figure 7). Furthermore, the TISCH database revealed that RAR-α is greatly elevated in primary cancers than metastatic cancers. Furthermore, the primary tumor cell population’s expression patterns for RAR-α expression varied greatly and CD4T and CD8T cells expressed higher RAR-α expression in breast cancer patients who had primary tumors (Figure 8).

Using the Enrichr database RAR-α underwent additional analysis for KEGG and gene ontology. Among the biological processes, we analyzed that RAR-α was found to be associated with the positive regulation of T-helper two cell differentiation, IL-13 production, IL-5 production, T-helper cell differentiation, and type 2 immune response. In addition, RAR-α also regulates granulocyte differentiation and has an association with the retinoic acid receptor signaling pathway and cellular response to estrogen stimulus (Figure 9A). RAR-α was mainly abundant in the actin cytoskeleton followed by the cytoskeleton, nucleolus, and nuclear lumen among the cellular compartment (Figure 9B). Among molecular functions, RAR-α was involved in protein kinase B binding, retinoic acid binding, actinin binding, alpha-actinin binding, monocarboxylic acid binding, retinoid binding, transcription coactivator binding, chromatin DNA binding and transcription coregulator binding (Figure 9C). The KEGG pathway study revealed that RAR-α is related with acute myeloid leukemia, Th17 cell differentiation, estrogen signaling pathway, transcriptional mis regulation in cancer including pathways in cancer (Figure 9D).

To find out how RAR-α binds to trifarotene, molecular docking experiments were the next step. Molecular docking studies showed that trifarotene significantly bound to the protein RAR-α, with a binding energy of −8.35 kcal/mol (Figure 10). The stable RMSD plot indicated that the greater ligand affinity of RAR-α + trifarotene contributes to the complex’s stability. Residue changes were seen in the RMSF plot, with significant fluctuations occurring in the residual positions 5–20, 48, 115–125, and 130–148. The Rg decreased from 16.9 to 16.54 Å when the RAR-α Cα-backbone bound to the trifarotene ligand, indicating a highly compact orientation of the protein in the ligand-binding state. Remarkably, while bound with ligand, the SASA value dropped when compared to the unbound state (Figure 11).

Additionally, an in-vitro assays was done to confirm our in silico study results. Trifarotene showed strong anti-tumor action on MDA-MB-231 and 4T1 BC cell lines, with an IC50 of 9.558 µM and 11.63 µM, respectively, according to the findings of the cell viability MTT test. The low IC50 value indicated that trifarotene significantly inhibits the proliferation of cancer cell (Figure 12).

According to our research, RAR-α is overexpressed in BC and has carcinogenic features. Treating BC patients with trifarotene that targets RAR-α is an effective treatment approach.