Breast cancer (BC) is one of the most commonly diagnosed global malignancies and is a leading cause of mortality among women. BC is a heterogeneous disease involving multiple environmental and genetic factors such as age, hormones, unhygienic diet, or toxic environmental exposure. The BRCA1 and BRCA2 tumor suppressor genes play a significant role in BC development (Li et al., 2017). Despite advances in treatments like chemotherapy, endocrine therapy, and human epidermal growth factor receptor-2 (HER2)-targeted therapy, the chance of relapse and BC metastasis remains a great challenge (Zhu and Yu, 2022). BC is a global health challenge as the most commonly diagnosed cancer, with an estimated incidence of 2.26 million cases worldwide according to GLOBOCAN 2020 global cancer statistics. The reported BC incidence rate is higher in Asia at 45.4% (Sung et al., 2021). There are different types of BC, depending on which cells in the breast become cancerous. Estrogen receptor positive (ERP) and triple-negative BC (TNBC) are the most aggressive types of BC. ERP BC is characterized by the presence of estrogen receptors (ERs) on tumor cells that help them grow and proliferate rapidly based on estrogen fueling. It is the largest subtype of BC as it involves the expression and activity of the estrogen receptor. It is estimated that approximately 80% of BCs are ERP (Lamb et al., 2019). TNBC is defined as a type of BC with a negative expression of ER, progesterone receptor (PR), and HER2. The mortality rate of TNBC is higher because of its high invasiveness and because approximately 46% of TNBC patients are more likely to have distant metastasis (Yin et al., 2020).

The ERP BC microenvironment (BCM) consists of immune cells, fibroblasts, adipocytes, mesenchymal stem cells, extracellular matrix, and tumor-associated macrophages (TAMs) (Munir et al., 2021). During breast tumorigenesis, tumor cells escape the immune surveillance by modifying surface antigens and altering their surrounding environment (Segovia-Mendoza and Morales-Montor, 2019). Chemokine, a family of signaling proteins, functions to induce leukocyte migration. Chemokine CC receptor type 5 (CCR5) is a cell surface receptor that has a high affinity for chemotropic cytokines called chemokines. A 32-bp deletion in this receptor (CCR5Δ32) results in a non-functional and deformed receptor which, in turn, results in the activation and invasion of immune cells at the site of tumorigenesis and ultimately leads to its progression (Fatima et al., 2019). In mammalian cells, alternative splicing (AS) is a key mechanism of gene expression regulation. AS occurs when intron and exon elements become rearranged by splicing at different splice-sites, resulting in multiple RNA transcripts. AS regulation is influenced by multiple factors such as cancer or other diseases (Vitting-Seerup and Sandelin, 2017). It occurs when there is differential usage of gene transcripts between different conditions (Baralle and Giudice, 2017). Thus, gene expression should be analyzed at the isoform level because isoform switching (IS) with predicted functional consequences is more common and important in dysfunctional cells (Kahraman et al., 2020).

RNA sequencing (RNA-seq) is a proven quantitative tool for the expression estimation of cells and facilitates the detection and identification of novel transcripts generated by AS. This study identified differential isoform usage (DIU) across conditions (ERP vs. TNBC) in immune system-related genes that may assist targeted therapies for ERP BC. Identifying novel biomarkers and isoform switching may pave the way for the early detection and successful treatment of ERP BC (Chen et al., 2022).

BC is the most prevalent type of cancer worldwide. It is thus essential to understand and explore ways to prevent its occurrence while identifying the genetic changes that are more susceptible to its incidence. The present study identified 9,771 DEGs, of which 86 genes were significantly upregulated and 117 were downregulated. The identified upregulated genes were FOXA1, RHOB, AR, CMBL, AGR2, ESR1, TFF3, SYBU, CBLC, and DNALI1; the downregulated genes were CENPW, EN1, A2ML1, TMSB15A, FOXC1, KRT16, SLC7A5, CDK6, MELTF, and CA9. Functional enrichment analysis of these DEGs revealed that the intracellular steroid hormone receptor signaling pathway, chemokine-mediated signaling pathway, kinetochore organization, pathways in cancer, BC, toll-like receptor signaling pathway, and cell cycle were the most dysregulated biological pathways and processes.

In the present study, FOXA1 was found to be upregulated and has been reported to inhibit STAT2, a transcription factor and its target IFN signaling pathway in BC; this may result in cancer progression due to suppressed immune response (He et al., 2021). Furthermore, upregulated RHOB results in ER-α (estrogen receptor alpha) overexpression that leads to increased estrogen uptake by BC cells which helps them grow and proliferate (Médale-Giamarchi et al., 2013). It has been reported that AR overexpression increases the transcription of genes involved in the cell cycle, resulting in increased proliferation of prostate cancer cells (Formaggio et al., 2021). This study found that CMBL (p-value: 0.00003) is suppressed in TNBC compared to non-TNBC types of BC, such as ERP. It encodes a cysteine hydrolase that cleaves cyclic esters which activate an angiotensin receptor blocker that helps lower blood pressure (Guo et al., 2017). Upregulated AGR2 is found in BC due to ER signaling and endoplasmic reticulum stress, and it results in increased cell proliferation, survival, and metastasis in BC (Ann et al., 2018). Moreover, ESR1 upregulation makes BC cells more prone to estrogen uptake which may lead to the increased growth and proliferation of cancer cells (Lei et al., 2019). According to the literature, TFF3 acts as an oncogene because it regulates other genes (FOXA1, HER2, and AR) involved in EMT, thus promoting invasiveness, survival, and increased proliferation in multiple carcinomas such as gastric cancer, mammary carcinoma, and prostate cancer (Yuan et al., 2017). It has been reported that SYBU, a microtubule-associated protein, is overexpressed in hepatocellular carcinoma (HCC), which results in disrupted cell cycle and increased proliferation (Zheng and Yu, 2021). Breast tumor formation is increased by CBLC overexpression, which suppresses TGF-β (transforming growth factor beta). This results in the deactivation of its target Smad3 pathway which is responsible for proliferation, differentiation, and apoptosis (Kang et al., 2012). This study found that DNALI1 (p-value: 0.0000148), a flagellar protein, is overexpressed in BC, which has not been reported previously for any other carcinoma.

According to the literature, CENPW was downregulated in BC and HCC. It is involved in kinetochore organization and centromere complex assembly. This downregulation results in subsequent function disruption, resulting in chromosomal instability due to mis-segregation of chromosomes (Liu and Liu, 2022). It has been reported that EN1 is downregulated in lung cancer due to altered DNA methylation which promotes cell proliferation and differentiation (Jiang et al., 2017). The downregulation of A2ML1, a protease inhibitor, results in MAPK pathway mutation, which leads to apoptotic resistance and uncontrolled cell division in BC (Li et al., 2016). FOXC1 suppression induces ER-α expression in BC cells, which helps in increased estrogen uptake, resulting in the growth and proliferation of tumor cells (Wang et al., 2017). It has been reported that KRT16 is overexpressed in basal-like TNBC, along with increased expression of EMT-associated proteins. In contrast, in luminal A and B subtypes of BC which include ER⁺ and PR⁺ tumors, KRT16 expression was suppressed, but E-cadherin (CDH1), an EMT protein was overexpressed, leading to metastasis due to increased cellular motility (Elazezy et al., 2021). SLC7A5 has been reported to be overexpressed in TNBC due to its glutamine transporting activity to tumor cells for energy production—TNBC is thus glutamine dependent and requires glutaminase for its catabolism. In addition, cells with increased proliferation use transaminases to catabolize glutamate, in contrast to glutamate dehydrogenase (GLUD), to reduce ammonia production. However, ER⁺ tumors are glutamine-independent and show increased GLUD expression (Wang et al., 2020). It has been reported that the downregulation of CDK6 also suppresses its interacting gene, RB1—a tumor suppressor gene. This results in dysregulated cell growth, apoptosis, and increased proliferation in tumor cells (Knudsen et al., 2020). MELTF downregulation also dysregulates its interacting genes such as ACO2, a gene-encoding Krebs’ cycle enzyme. The disruption of Krebs’ cycle enzymes leads to the production of oncometabolites, which stabilize hypoxia-inducible factor 1 and activate cell growth signaling by regulating DNA methylation—crucial factors in cancer progression (Sajnani et al., 2017). CA9 suppression leads to the disruption of interacting genes such as HIF3A and EPAS1 which are involved in regulating hypoxic conditions. Such conditions are favorable for the increased proliferation of tumor cells (Jun et al., 2017). TMSB15A has been reported to be upregulated in TNBC; it plays a crucial role in the organization of the cytoskeleton, which is responsible for cancer cell motility and is involved in cancer metastasis (Darb-Esfahani et al., 2012).

KEGG pathway enrichment analysis revealed protein digestion and absorption, pathways in cancer, chemical carcinogenesis, and ECM–receptor interaction as upregulated pathways, while downregulated pathways include chemokine signaling, toll-like receptor signaling, cell cycle, bladder cancer, IL-17 signaling, cellular senescence, cytokine–cytokine receptor interaction, and p53 signaling. We found that the protein digestion and absorption pathway was upregulated, which demonstrates the use of proteins as an alternative fuel by tumor cells to fulfill their metabolic needs. This occurs due to the limited supply and metabolism of glucose by cancer cells (Lieu et al., 2020). Moreover, it was found that pathways in cancer were upregulated that involve the disruption of the ErbB, p-53-mediated apoptotic, and GSK3 signaling pathways, which are involved in DNA repair, cell growth, migration, differentiation, and metabolism (Yip and Papa, 2021). The upregulation of chemical carcinogenesis activates certain hormonal pathways that make mammary glands more susceptible to carcinogenesis due to altered DNA repair genes (Rodgers et al., 2018). Furthermore, upregulated ECM–receptor interaction results in interaction with HMMR and SDC1 genes, the dysregulation of which promotes BC cell motility and differentiation (Yeh et al., 2018). A downregulated chemokine signaling pathway and cytokine–cytokine receptor interaction cannot recruit immune cells (leukocytes) to the tumor microenvironment, thus resulting in tumor progression (Gil Del Alcazar et al., 2020). The downregulation of toll-like receptor signaling pathways results in non-recognition and the escape of cancer cells from the immune system, leading to the invasiveness, migration, and angiogenic potential of cancer cells (Javaid and Choi, 2020). The disruption of the cell cycle at the G2/M phase results in cells that contain damaged DNA and genomic instability, a hallmark of cancer (Thu et al., 2018). It has been reported that increased ER-α in BC cells suppresses bladder cancer cell growth by downregulating INPP4B which, in turn, suppresses the AKT signaling pathway (Hsu et al., 2014). Research has found that the IL-17 signaling pathway becomes downregulated due to increased estrogen receptor expression, resulting in dysregulated PD-1/PD-L1 and CD8⁺ T cell expression—a suppressed immune response (Shuai et al., 2020). Furthermore, downregulated cellular senescence results in increased cell proliferation and tumor development (Milczarek, 2020). Moreover, a downregulated p53 signaling pathway cannot perform DNA damage repair and cell death, thereby facilitating the increased growth and metastasis of tumor cells (Marei et al., 2021).

According to the GTEx portal, cells express an average of 3.42 transcripts per gene (Tung et al., 2020). The expression dominance of major isoform transcripts compared to others from the same gene is crucial for normal cellular homeostasis (Hu et al., 2017). However, splicing regulation is often disrupted in cancer with a dominant expression of alternative transcripts in a tumor microenvironment (TME) which promotes switches that contribute to tumor progression and metastasis (Kahraman et al., 2020). The interaction pattern of the cancer-specific most dominant transcript (cMDT) differs from the generally expressed isoform of normal cells because of changes caused by alternative splicing; this could affect protein domains due to mutations caused by tumor and subsequent disruption in cancer-related pathways (Yang et al., 2016; Climente-González et al., 2017). According to the literature, apoptosis, ubiquitin, signaling, and spliceosomes were the most disrupted protein interactions (Kahraman et al., 2020). It has been reported that isoform switching leads to the loss of the DNA sequence that encodes for protein domains, promoting functional loss. The subsequent switches have functional consequences for cancer development and progression (Vitting-Seerup and Sandelin, 2017).

The present study has identified the differential usage of transcript isoforms among ERP and TNBC. It revealed isoforms that are significantly expressed and used by shortlisted downregulated genes (STMN1, MELTF, and CXCL8). CXCL8 encodes a protein that is involved in chemotaxis (Łukaszewicz-Zając et al., 2020). It transcribes five transcripts; among them, only one isoform transcript was significantly used. Differential gene expression plotting shows that CXCL8 is downregulated in ERP (Figure 6). On the other hand, there is in isoform usage an increased use of isoform ENST00000483500 in ERP. However, this isoform is non-coding due to retained introns and the unavailability of any domain, leading to functional loss of CXCL8 and immune suppression as a consequence of the non-recruitment of macrophages and neutrophils to the TME (Xiong et al., 2022). STMN1, a cytosolic phosphoprotein, is involved in microtubule destabilization by regulating the microtubule filament system and signal transduction (Bao et al., 2017). It transcribes four known and two novel isoforms (Figure 6). The isoform usage plot indicates that a single isoform (ENST00000485226) is significantly used in ERP, as compared to TNBC. However, it lacks a domain, which results in its non-coding behavior. Furthermore, STMN1 is repressed in ERP, as shown in differential gene expression plotting which promotes ERP progression due to the disruption of microtubules and their subsequent role in the growth of immune cells such as T-cells and natural killer cells (Zhang et al., 2022). MELTF, a cell surface glycoprotein, is involved in cellular iron uptake (Sawaki et al., 2019). MELTF could transcribe six novel and two known isoforms (Figure 6); however, increased use of the single isoform MSTRG.29921.1 has been identified in ERP. Moreover, this isoform contains nonsense codons that prematurely terminate translation—nonsense-mediated decay (NMD). Differential gene expression plotting also shows that MELTF is downregulated in ERP, leading to the decreased proliferation and maturation of immune cells such as lymphocytes due to decreased iron uptake (Roemhild et al., 2021). Gene Ontology analysis of shortlisted immune genes revealed that CXCR chemokine receptor binding, iron ion binding, and cytokine activity are the most dysregulated molecular functions (Figure 7). These functions mediate immune response by recruiting immune cells such as monocytes, T cells, lymphocytes, and natural killer cells, and assist their growth and proliferation. The downregulation of these functions promotes ERP BC due to suppressed immune response in TME (Bates et al., 2018).

This research therefore provides key insights into the genes that are differentially expressed in ERP. Moreover, DNALI1 is a novel gene that has not been previously reported and is involved in ERP BC development. Furthermore, the identification of three immune system-related genes (STMN1, MELTF, and CXCL8) reveals that the dysregulation of the immune system due to isoform switching is the major factor in ERP BC development and progression. Downregulation and isoform switching of key immune system genes suggest BC progression and possible metastasis due to the non-recruitment of cytokines in the TME.

ERP BC is characterized by the growth of tumor cells in response to estrogen hormone. The dysregulation of gene expression results in the development of significant biological changes that are key features of multiple human carcinomas such as prostate cancer, gastric cancer, hepatocellular carcinoma, and lung cancer. In this study, 9,771 DEGs were identified; among these, 86 genes were upregulated and 117 were downregulated. Six genes (FOXA1, RHOB, AGR2, ESR1, CBLC, and FOXC1) were found to be significantly associated with the development and progression of ERP BC. This study also identified a novel set of genes (DNALI1, TMSB15A, AR, TFF3, SYBU, CENPW, EN1, CDK6, MELTF, and CA9) not previously reported positive for estrogen receptors but that has been reported in other carcinomas. Moreover, alternative splicing and subsequent isoform expression in three downregulated immune system genes (STMN1, MELTF, and CXCL8) had been identified that were mainly responsible for ERP progression due to suppression of the immune system and the non-recruitment of cytokines against cancer cells. It was found that CXCR chemokine receptor binding, iron ion binding, and cytokine activity were the most dysregulated functions due to immune system suppression. This study reveals that dysregulation of the immune system due to isoform switching is the major factor in ERP BC development and progression. Therefore, these crucial immune system genes should be targeted as therapeutic biomarkers.