Introduction

Breast cancer is the most common cancer in female individuals responsible for the high rate of malignancy [1,2,3]. Excluding basal cell and squamous cell skin cancers, breast cancer is the most commonly diagnosed cancer among women in the U.S. In 2022, there will be an estimated 287,850 new cases of invasive breast cancer diagnosed in women; 2710 cases diagnosed in men, and an additional 51,400 cases of ductal carcinoma in situ (DCIS) diagnosed in women. [4, 5]. Breast cancer is a heterogeneous solid tumor with > 20 distinct subgroups differing in appearance, clinical behavior, and genetics [6]. Breast cancer has been categorized into triple-negative breast cancer (TNBC), luminal A, luminal B, & HER2 + based on gene expression [7, 8]. Luminal A, the most common subtype, may be PR positive, ER positive or negative, HER2 negative, and EGFR and CK5/6 negative [9,10,11,12]. Lower expression of progesterone receptor (PR), estrogen receptor (ER), and high histologic grade are characteristics of luminal B tumors [13]. HER2 + subtype may be ER/PR negative, HER2 positive, and EGFR and CK5/6 negative [9, 10]. TNBC is the most aggressive type of breast cancer which accounts for nearly 20% of identified breast cancer cases & is characterized by the absence of progesterone (PR) receptor, estrogen receptor, and or under-expression of human epidermal growth factor receptor 2 (HER2) [13,14,15]. Among breast cancer isotypes, TNBC has very poor overall survival and enhanced metastatic potential [16,17,18,19,20,21].

To develop and survive, cancer cells frequently alter the human body's metabolism [22]. One of the hallmarks of tumor cells that are currently being realized is any abnormal change in fatty acid metabolism [11, 23,24,25,26,27]. The modification of fatty acid metabolism, necessary to achieve the high rate of fatty acid synthesis for cancer cell proliferation is one of the most intriguing modifications. Therefore, fatty acid metabolism needs to be reprogrammed for cancer cells to survive. Since it can alter cellular processes, fatty acid metabolic reprogramming is one of the characteristics of a cancer situation. Increased exogenous fatty acid intake, de novo fatty acid synthesis and fatty acid oxidation are all components of the reprogramming of fatty acid synthesis. For diagnosing and estimating the effectiveness of the therapy regimen for treating tumors, it is helpful to identify the biochemical targets in fatty acid metabolism. [28,29,30]. This dependency of cancerous cells on the altered fatty acid metabolism reveals that the proteins involved in this process may prove good therapeutic targets for the treatment of cancer [31,32,33].

Dysregulation of fatty acid metabolism is recognized as a component of malignant transformation in many different cancers, including breast; yet the potential for targeting this pathway for prevention and/or treatment of cancer remains unrealized. Evidence indicates that proteins involved in both the synthesis and oxidation of fatty acids play a pivotal role in the proliferation, migration, and invasion of breast cancer cells [25]. Thus targeting the fatty acid metabolism pathway and the genes related to it will prove an efficient treatment strategy for breast cancer patients. In this study, we evaluated the expression of fatty acid metabolism genes using various bioinformatic tools. We used the TCGA BRCA database obtainable from Gepia2 and UCSC XENA web servers to evaluate the expression of fatty acid metabolism genes in clinical samples of BC patients. Here we came up with the significant dysregulation of fatty acid metabolism gene expression in breast cancer patients. Furthermore, this dysregulated gene expression was also found to exert an influence on relapse-free survival (RFS) and overall survival (OS) in breast cancer patients. Our findings suggest that fatty acid metabolism gene expression is downregulated in different tumor types and normal activation of their expression may reduce abnormal cell proliferation and may give better treatment outcomes in certain cancers.

Results

Expression of fatty acid metabolism genes is dysregulated in breast cancer

The gene expression pattern of fatty acid metabolism was examined in BC using UCSC XENA. Fatty acid metabolism gene expression analysis shown by the TCGA BrCa database disclosed a notable gene dysregulation in BC (Fig. 1). Additional analysis of different fatty acid metabolism genes obtained from the Gepia2 dataset displayed a log2-fold change as shown in Table 1. FABP4, FABP5, PLIN1, PLIN2, PLIN4, PLIN5, LPIN1, MGLL, PNPLA2, PNPLA7, ACSL1, and ACOX2 were found to be downregulated (Fig. 2). The expression pattern of highly dysregulated fatty acid metabolism genes among different BC subclasses was analyzed using UALCAN, and it was found that the Luminal breast cancer patients show high expression of genes PLIN5, MGLL, PNPLA2, and ACOX2 as compared to their expression in TNBC patients. Further, luminal patients show a high expression of ACOX2 as compared to normal. Moreover, it is found that HER2 Positive patients show a very low expression of FABP5, PLIN1, PLIN2, PLIN5, ACSL1, MGLL, PNPLA2, and ACOX2 genes when compared to their normal levels. Adding to this, the expression of PLIN1, PLIN5, ACSL1, MGLL, PNPLA2, and ACOX2 is downregulated in TNBC patients when compared to normal levels (Fig. 3).

Fig. 1
figure 1

mRNA expression pattern of Fatty acid metabolism genes in breast cancer patients. Heat map showing the expression patterns of Fatty acid metabolism genes. Gene expression was drawn out using XENA. Contrasting the expression of fatty acid metabolism genes between GTEx samples (normal breast, n = 291), Solid Tissue Normal (tissue surrounding the tumor (n = 114), and primary tumor (cancer, n = 1097)

Full size image
Table 1 Log2-fold change of Fatty acid metabolism genes in breast cancer
Full size table
Fig. 2
figure 2

Fatty acid metabolism gene expression in Breast cancer. A. Bar graph of the expression pattern of fatty acid metabolism genes scrutinized using TCGA BRCA and GTEx databases by operating UCSC XENA. B. Box plots of the expression pattern of highly dysregulated fatty acid metabolism genes analysed in BC by making use of Gepia2 web source (p value > 0.05)

Full size image
Fig. 3
figure 3

Expression pattern of highly dysregulated Fatty acid metabolism genes in major BC subtypes. The expression analysis of the genes including A FABP5 B PLIN1 C PLIN2 D PLIN5 E ACSL1 F MGLL G PNPLA2 and H ACOX2 further explored using TCGA samples

Full size image

Dysregulation of fatty acid metabolism gene is associated with poor prognosis in BC patients

The Kaplan–Meier plotter, a bioinformatic tool for gene expression pattern information & survival probability of BC patients, was used to look over the survival time of highly dysregulated fatty acid metabolism genes. Based on median expression, BC patients were splitted into two groups viz: high expression group and low expression group. The relapse-free survival (RFS) examination was executed on 4929 patients with breast cancer for about 300 months, and it was reported that the patients with low expression of PLIN1, MGLL, ACOX2 have worse RFS than patients with increased levels of FABP5 mRNA, having log-rank p value 1.3e-06, Hr-value 0.78 for PLIN1, p value 0.1, HR-value 0.88 for MGLL, p value 1.6e-08, HR 0.75 for ACOX2, While as, patients with high expression of SLC27A6 and FABP5 show poor RFS with p value 0.17 and 1.8 e-13 respectively, Fig. 4A. The tie-up between gene expression of fatty acid metabolism genes and overall survival (OS) was scrutinized in 1879 patients, and it was disclosed that patients of breast cancer with increased expression of SLC27A6, and MGLL have a poor OS with p value 0.00058, 0.0024, and HR 1.78, 1.61, respectively, while as patients with low expression of FABP5, PLIN1, PLIN2, PLIN5 PNPLA2 and ACOX2 have worse OS with p value 0.074, 0.03, 0.15, 0.0012, 0.024, 0.029 and HR 0.75, 0.72, 0.79, 0.61,0.71, 0.81 respectively Fig. 4B.

Fig. 4
figure 4

Expression of dysregulated Fatty acid metabolism genes corresponds with survival outcomes. A Correspondence between expression of fatty acid metabolism genes with RFS in BC patients. B Relationship between expression of fatty acid metabolism genes with OS in BC patients

Full size image

Correlation of dysregulated fatty acid metabolism genes with clinical-physiological characteristics in BC

The bc-Gen EXminer web source was used to analyze the correlation between highly dysregulated fatty acid metabolism genes and ordinary pathophysiological characteristics in BC patients Fig. 5. FABP4 was shown to exhibit a higher expression in BC patients who expressed hormone receptors (Estrogen and progesterone receptors) (p value < 0.0001) and was observed to correlate with SBR grade 1(p value < 0.0001) and individuals with wild p53 had notably increased levels of FABP4 mRNA as compared to individuals with mutant p53 (p value < 0.0001) Fig. 5A. In comparison to FABP4, FABP5 expression levels were notably enhanced in BC patients lacking hormone receptors (Estrogen and progesterone receptors) (p value < 0.0001) however did not vary substantially from HER2 (p value of 0.0914). Enhanced FABP5 expression levels were found to be associated with SBR3 (p value < 0.0001). Furthermore, patients with mutant p53 had increased levels of FABP5 mRNA than individuals having normal-type of p53 Fig. 5B.

Fig. 5
figure 5figure 5figure 5

Clinicopathological parameters and fatty acid metabolism genes in BC. bC-Gen EXminer analysis of dysregulated fatty acid metabolism genes showed a tie-up with clinicopathological parameters of BC i.e., Hormonal status, Her2 enrichment, SBR grade, and p53 status. A FABP4 B FABP5 C PLIN1 D PLIN2 E PLIN4 F PLIN5 G MGLL H PNPLA2 I PNPLA7 J ACSL1 K ACOX2

Full size image

PLIN1 expression levels were high in BC patients who expressed estrogen and progesterone receptors exhibiting p value < 0.0001 and was observed to correlate with SBR1(p value < 0.0001). Additionally, individuals having mutant p53 (p value < 0.0001) had lower levels of PLIN1 mRNA than those with wild-type p53 Fig. 5C.

PLIN2 expression levels were higher in BC patients deficient in estrogen and progesterone receptors (p value < 0.0001). In contrast, PLIN2 expression was greater in HER2-augmented BCp-value 0.0003). PLIN2 expression was shown to be correlated with SBR3 (p value < 0.0001). Moreover, patients with wild p53 had very low levels of PLIN2 mRNA than those with mutant p53 who have notably higher levels of PLIN2 Fig. 5D.

PLIN4 and PLIN5 expression levels were high in BC patients who expressed estrogen receptor and progesterone receptor (p value < 0.0001). Further, it was reported that enhanced PLIN4 and PLIN5 expression was connected with SBR1 (p value < 0.0001) and individuals with wild p53 had significantly greater PLIN4 and PLIN5 mRNA levels than patients with mutant-type p53 (p value < 0.0001) Fig. 5E, F.

MGLL expression was high in ER + and HER2 positive patients (p value < 0.0001) while as the expression did not vary substantially from PR (p value = 0.8046) or SBR grade (p value < 0.0001). Further individuals with wild p53 showed increased levels of MGLL mRNA than those who had mutant p53 (p value = 0.013) Fig. 5 (G).

Expression levels of genes PNPLA2 and PNPLA7 were found to be high in BC patients who expressed hormone estrogen and progesterone receptors (p value < 0.0001). In respect of SBR grading, increased levels of PNPLA2 and PNPLA7 were found to be connected with SBR1 (p value < 0.0001). Moreover, individuals who had wild p53 showed greater levels of PNPLA2 and PNPLA7 in comparison to those with mutant p53 Fig. 5H, I.

ACSL1 gene expression appeared to be elevated in breast tumors deficient in hormone estrogen and progesterone receptors (p value < 0.0001). In contrast, HER2-augmented breast tumors (p value-0.0001) showed elevated levels of ACSL1. In respect of SBR grading, elevated ACSL1 expression was connected with SBR3 (p value < 0.0001). And individuals with mutant p53 had elevated levels of ACSL1 mRNA as opposed to those who had wild p53 (p value = 0.0517) Fig. 5J.

ACOX2 expression was high in patients with breast cancer who expressed progesterone and estrogen receptors (p value < 0.0001). ACOX2 expression was also found to be elevated in HER2-augmented breast tumorsp-value-0.0001). In respect of SBR grading, augmented ACOX2 expression was connected with SBR1 (p value < 0.0001), and individuals with wild p53 had remarkably higher levels of ACOX2 mRNA as opposed to patients with mutant p53 (p value < 0.0001) Fig. 5K.

Protein–protein interaction of Fatty Acid Metabolism Genes

A network of protein–protein interactions was constructed by connecting co-expressed 16 genes (nodes) with 62 protein–protein relationships (edges) in a string database. Also, the PPI network developed displayed an average node degree: 7.75, expected number of edges: 7, avg. local clustering coefficient: 0.81, and a PPI enrichment p value: < 1.0e-16 (Fig. 6A). The top five hub genes of the network depending on degree score were identified using Cytohubba as shown in Fig. 6B. The top genes of the network included ACSL1, LIPE, PNPLA2, FABP4, and PPARG.

Fig. 6
figure 6

PPI analysis of FAM genes A PPI constricted module of FAM genes using STRING. B Top 5 hub genes of PPI network identified using cytohubba

Full size image

Gene ontology enrichment and pathways analysis

By utilizing the Enricher database, we pointed out signalling cascades and gene ontology features linked to FABP4, FABP5, PLIN1, PLIN2, PLIN4, PLIN5, LPIN1, MGLL, PNPLA2, PNPLA7, ACSL1, and ACOX2. GO analysis in biological processes revealed that these genes were enriched in long-chain fatty acid transport, regulation of sequestering of triglyceride fatty acid transport, acylglycerol catabolic process, triglyceride catabolic process, triglyceride metabolic process, acylglycerol acyl-chain remodeling, positive regulation of sequestering of triglyceride, positive regulation of cold-induced thermogenesis and regulation of triglyceride catabolic process Fig. 7A. In molecular function (MF) terms, these genes showed enrichment in activities of lipase, carboxylic ester hydrolase, phospholipase, lysophospholipase, arachidonate-CoA ligase, acid -thiol ligase, acylglycerol lipase, acyl-CoA oxidase, oxidoreductase, triglyceride lipase, and long-chain fatty acid binding Fig. 7B. In respect of cellular location, the recommended GO was primarily enriched in lipid droplet, intracellular non-membrane abounded organelle, endoplasmic reticulum membrane, azurophil granule lumen, mitochondrial outer membrane, asymmetric synapse, postsynaptic density, organelle outer membrane, azurophil granule, vacuolar lumen, etc. Figure 7C.

Fig. 7
figure 7

Gene enrichment and pathway analysis. (A–C)G O analysis of the BP, CC, and MF and, (D) KEGG pathway enrichment analysis of FABP4, FABP5, PLIN1, PLIN2, PLIN4 PLIN5, LPIN1, MGLL, PNPLA2, PNPLA7, ACSL1 and ACOX2 genes in breast cancer

Full size image

Kyoto Encyclopaedia of Genes and Genomes (KEGG) web source was utilized to assess pathway studies. As per KEGG Human 2021, these genes are associated with the PPAR signalling pathway, regulation of lipolysis in adipocytes, thermogenesis, glycerolipid metabolism, peroxisome, primary bile acid biosynthesis, fatty acid biosynthesis, ferroptosis, fatty acid degradation, and adipocytokine signalling pathway Fig. 7D.

Discussion

Breast cancers are quite frequent among the female population and are responsible for increased tumor-related deaths in women [25], 34. While breakthroughs in early detection and therapy have improved the standard of life and overall survival in BC patients, the emergence of metastatic and resistant cancers demands immediate attention [26, 27]. Cancerous cells and their microenvironment including the stroma around them impact reciprocally via paracrine factors and juxtracrine interaction, promoting tumor development, metastasis, and therapy resistance [28]. The stromal cells have been discovered to generate epigenetic alterations in tumor cells and regulate gene expression, supporting tumor development and influencing prognosis [6, 29].

Several oncogenic signaling cascades regulate the lipid metabolism of transformed cells and any type of deviation in these pathways may lead to the development of tumors. Alterations in the metabolism of lipids in tumor cells are currently acknowledged as a trademark of transformed cells [30]. Reprogramming metabolic pathways by tumor cells is important for their proliferation and survival.

In the current study, we utilized various bioinformatic tools to puzzle out the diagnostic and prognostic significance, gene expression pattern, and functional role of the dysregulated fatty acid metabolism genes in BC patients. Our study reported that fatty acid metabolism genes are very much dysregulated in breast tumors with FABP4, FABP5, PLIN1, PLIN2, PLIN4, PLIN5, LPIN1, MGLL, PNPLA2, PNPLA7, ACSL1, and ACOX2 showing a fold change > ± 1.5.

Our study revealed that breast cancer patients with low levels of PLIN1, MGLL, and ACOX2 have poor Relapse Free Survival as compared to patients having increased levels of PLIN1, MGLL, and ACOX2 mRNA, while patients having high expression of SLC27A6 and FABP5 show poor RFS. Further, it was reported that BC patients having elevated expression of SLC27A6, and MGLL, have poor OS, while patients with low expression of FABP5, PLIN1, PLIN2, PLIN5 PNPLA2 and ACOX2 have worse OS.

Further investigation suggested that FABP4 expression was significantly higher in those BC patients who expressed hormone progesterone and estrogen receptors, depicting a negative relationship with hormone receptors, and was observed to correlate with SBR grade 1 and wild p53. On the other hand, FABP5 expression appeared remarkably high in those patients deficient in estrogen and progesterone receptors, however, did not vary substantially from HER2. In respect of SBR grading, elevated levels of FABP5 were found to be correlated with SBR3 and mutant p53.

PLIN1 expression was high in BC patients who had estrogen receptors and progesterone receptors, depicting a negative relationship with hormone receptors and was associated with SBR1 and wild p53. Experimentally PLIN1 was overexpressed exogenously in breast cancer cell lines which resulted in the inhibition of invasion, cell proliferation, migration, and in vivo tumor formation in mice models [31]. In contrast to this, expression levels of PLIN2 expression were enhanced in those patients not having estrogen and progesterone receptors and also enhanced in HER2-enriched breast cancers. In respect of SBR grading, elevated levels of PLIN2 appeared to be connected with SBR3 and mutant p53.

PLIN4 and PLIN5 expression was high in those patients having estrogen and progesterone receptors showing a negative relation with hormone receptors (p value < 0.0001). In respect of SBR grading, it was confirmed that elevated levels of PLIN4 and PLIN5 were connected with SBR1 and wild p53.

MGLL expression was high in ER + and HER2 positive patients while its expression did not vary very much from PR (p value = 0.8046) or SBR grade (p value < 0.0001) and individuals with wild-type p53 showed notably larger levels of MGLL mRNA as opposed to those with mutant p53.

PNPLA2 and PNPLA7 expression levels were elevated in BC patients having estrogen and progesterone receptors depicting a negative relation with hormone receptors. In respect of SBR grading, elevated levels of PNPLA2 & PNPLA7 appeared to be associated with SBR1 & wild p53.

Elevated expression of ACSL1 was reported in BC patients lacking progesterone and estrogen receptors, but its expression was augmented in HER2-enriched breast cancers. Concerning SBR grading, higher ACSL1 expression was found to be associated with SBR3 and mutant p53.

BC patients with estrogen and progesterone receptors exhibited elevated levels of ACOX2 depicting a positive relation. Its expression was also enhanced in HER2-enriched breast cancers. About SBR grading, elevated ACOX2 expression was connected with SBR1 & wild p53.

Also, the string analysis using the Cytoscape as export software revealed the top five hub genes associated with fatty acid metabolism in breast cancer. Further, Gene ontology analysis in biological processes revealed that these genes were enriched in long-chain fatty acid transport, acylglycerol catabolic process, triglyceride metabolic process, regulation of sequestering of triglyceride fatty acid transport, triglyceride catabolic process, acylglycerol acyl-chain remodeling, positive regulation of cold-induced thermogenesis, positive regulation of sequestering of triglyceride and regulation of triglyceride catabolic process. In molecular function (MF) terms, these genes showed enrichment in long-chain fatty acid binding and activities of carboxylic ester hydrolase, lysophospholipase, lipase, phospholipase, arachidonate -CoA ligase, acylglycerol lipase, acid-thiol ligase, CoA- ligase, triglyceride lipase, etc. About cellular location, the preferred GO was primarily enriched in lipid droplet, intracellular non-membrane abounded organelle, endoplasmic reticulum membrane, azurophil granule lumen, mitochondrial outer membrane, asymmetric synapse, postsynaptic density, organelle outer membrane, azurophil granule, vacuolar lumen, etc.

This dependency of transformed cells on the deviated metabolism of lipids demonstrates that proteins that function in lipid metabolism may be pinpointed as chemotherapeutic targets for the treatment of breast cancer [32, 33]

Materials and methods

Differential gene expression of fatty acid metabolism genes in breast cancers

The expression pattern of fatty acid metabolism genes was analyzed using the UCSC XENA web source and resulted in the production of a heatmap [35]. The Gepia2 online dataset (http://gepia2.cancer-pku.cn/) was utilized to show the fold change in differential expression levels of fatty acid metabolism genes in breast tumors [36]. This web source has 291 samples of GTEX & 1085 samples of BC. Additionally, our study also used UALCAN comprehensive web portal just to delineate the expression of highly dysregulated fatty acid metabolism genes in different subclasses of breast cancers [37].

Kaplan–Meier plotter

The Kaplan–Meier plotter, a bioinformatic tool for gene expression pattern information and survival probability of BC patients, was used to look over the survival time of highly dysregulated fatty acid metabolism genes [38]. This web server has OS data for 1879 patients and RFS data for 4929 patients in its mRNA gene chip BC dataset. Two groups of patients were created based on median gene expression. Among these two designated cohorts, consequences of dysregulated gene expression on OS and RFS were inspected from KM- survival curves. Moreover, the KM plotter was used to calculate log-rank p value and the hazard ratio intervals.

bc-Gen EXminer

The bc-Gen EXminer web source (http;//bcgenex.centregauducheau.fr/BC-GEM) was used to analyze the correlation between highly dysregulated fatty acid metabolism genes and ordinary pathophysiological characteristics in BC patients [39, 40]. This database was utilized to assess the correlation between expression levels of different fatty acid metabolism genes and different clinical parameters of breast tumor patients such as Her2 enrichment, p53 status, hormonal status, and SBR grade.

Protein–protein interaction of  Fatty acid metabolism genes

A protein–protein interaction (PPI) network of selected FAM genes was created using the Search Tool for the Retrieval of Interacting Genes (STRING database ver.11.0b), a biological database created to construct and study functional interactions between proteins, with a confidence score of 0.7 [41]. Using the Cytoscape program, the PPI network was further examined and visualized (version 3.8.2). To identify the noteworthy modules in the PPI network, the Cytoscape software's Molecular Complex Detection (MCODE) plug-in was added. The cytohubba plugin was used to extract the HUB nodes with the top 5° in the PPI network [42].

Functional enrichment of highly dysregulated fatty acid metabolism genes

The Mayaan laboratory database (https://maayanlab.cloud/Enrichr/)meant for gene ontology function and KEGG (Kyoto Encyclopaedia of Genes and Genomes) was utilized to reveal biological process, gene ontology features, and pathway enrichment analyses of FABP4, FABP5, PLIN1, PLIN2, PLIN4, PLIN5, LPIN1, MGLL, PNPLA2, PNPLA7, ACSL1 and ACOX2 [43]. A p value of < 0.05 was considered significant for pathways & functions in respect of KEGG & GO.

Conclusion

In this study, a correlation was assessed between expression levels of different fatty acid metabolism genes and prognosis among different molecular isotypes/subtypes of breast cancers. Our findings suggest that FAM genes viz, FABP4, FABP5, PLIN1, PLIN2, PLIN4, PLIN5, LPIN1, MGLL, PNPLA2, PNPLA7, ACSL1, and ACOX2 are downregulated in different subtypes of breast cancers, and may serve as ideal therapeutic targets for cancer treatment. The studies have revealed that PLIN1 overexpression done exogenously in breast cancer cell lines resulted in inhibition of proliferation, migration, invasion as well as in vivo tumor formation in mice models, hence normal activation of fatty acid metabolism pathways in tumors may reduce abnormal cell proliferation and may give better treatment outcomes. Therefore, targeting the fatty acid metabolism pathway and the genes related to it will prove an efficient treatment strategy for breast cancer patients.