Cuproptosis-related genes predict prognosis and trastuzumab therapeutic response in HER2-positive breast cancer

Breast cancer is the most common diagnosed cancer, the HER2-positive subtype account for 15% of all breast cancer. HER2-targeted therapy is the mainstay treatment for HER2-positive breast cancer. Cuproptosis is a novel form of programmed cell death, and is caused by mitochondrial lipoylation and destabilization of iron-sulfur proteins triggered by copper, which was considered as a key player in various biological processes. However, the roles of cuproptosis-related genes in HER2-positive breast cancer remain largely unknown. In the present study, we constructed a prognostic prediction model of HER2-positive breast cancer patients using TCGA database. Dysregulated genes for cells resistant to HER2-targeted therapy were analyzed in the GEO dataset. KEGG pathway, GO enrichment and GSEA was performed respectively. The immune landscape of DLAT was analyzed by CIBERSORT algorithm and TIDE algorithm. HER2-positive breast cancer patients with high CRGs risk score showed shorter OS. DLAT was downregulated and correlated with better survival of HER2-positive breast cancer patients (HR = 3.30, p = 0.022). High expressed DLAT was associated with resistant to HER2-targeted therapy. Knocking down DLAT with siRNA increased sensitivity of breast cancer to trastuzumab. KEGG pathway and GO enrichment of DEGs indicated that DLAT participates in various pathways correlated with organelle fission, chromosome segregation, nuclear division, hormone-mediated signaling pathway, regulation of intracellular estrogen receptor signaling pathway, condensed chromosome and PPAR signaling pathway. There was a negative correlation between TIDE and DLAT expression (r = − 0.292, p < 0.001), which means high DLAT expression is an indicator of sensitivity to immunotherapy. In conclusion, our study constructed a four CRGs signature prognostic prediction model and identified DLAT as an independent prognostic factor and associated with resistant to HER2-targeted therapy for HER2-positive breast cancer patients.

The workflow of the study design was presented in Fig. 1.In this study, we identified 14 genes related to cuproptosis according to the literature 30 .The expression level of the 14 genes in HER2-positive BC were determined by the TCGA database.As shown in Fig. 2A, nine genes including DLAT, DLD, GLS, LIPT1, MTF1, PDHA1, PDHB, SLC25A3 and SLC31A2 were significantly downregulated, but the other 3 genes including ATOX1, CDKN2A and SLC31A1 were significantly upregulated in HER2-positive BC compared with normal tissues.For FDX1 and LIAS, however, there had no statistical difference between HER2-positive BC and normal breast tissues.Subsequently, the prognostic values of the 14 genes were evaluated in HER2-positive BC patients.The univariate Cox hazard model suggested a higher risk of death in the high expression of DLAT (HR = 3.30, p = 0.022) and SLC25A3 (HR = 2.90, p = 0.039) group (Fig. 2B).The cumulative OS curves provided a visualization of the prognosis with different DLAT and SLC25A3 expression (Fig. 2C).

The prediction model for HER2-positive BC
Given that CRGs had distinct expression between normal breast tissues and HER2-positive BC, and part of these genes had predictive value for the prognosis of HER2-positive BC patients.We attempted to construct the prognostic gene set for HER2-positive BC using the LASSO method.The prognostic model was established using the 14 cuproptosis related genes in HER2-positive BC patients (Fig. 3A, B).The risk score = (0.1843) * DLAT + (− 0.4056) * SLC31A2 + (0.7899) * SLC25A3 + (0.4987) * ATOX1.The risk score, survival status and expression of patients with HER2-positive BC were shown in Fig. 3C.The further OS curve analysis showed that patients in the low-risk group had a lower risk of death and longer survival time (HR = 0.31, p = 0.026, Fig. 3D).

DLAT is the potential gene resistant to HER2-targeted therapy
To identify whether the CRGs have the predictive value for resistance to HER2-targeted therapy, we first analyzed the GEO dataset, GSE121105 31 .Dysregulated genes for cells resistant to trastuzumab or trastuzumab + pertuzumab in combination were showed in Fig. 4A, B, respectively.Compared with the 14 cuproptosis related genes, only 4 genes showed differently expression in all 3 datasets (Fig. 4C).Further analysis revealed that DLAT was the only gene included both in CRG model genes and resistant genes (Fig. 4D).RT-qPCR found that DLAT expression was significantly down-regulated after transfected with siRNA targeting DLAT (Fig. 4E).MTT assay revealed that DLAT silencing sensitized the SKBR-3 cell to trastuzumab (Fig. 4F).

The relationship between DLAT expression and clinical characteristic of BC patients
We analyzed the association between prognosis and DLAT expression in BC patients, however, there had no difference of OS between high or low DLAT expression patients (HR = 1.35, p = 0.08, Fig. 6A).To visualize the attributes of individual patients and display any correlations with survival, we constructed the alluvial diagram (Fig. 6B).The expression level of DLAT in different BC subtype according the PM50 was showed in Fig. 6C, the DLAT had lower expression in Luminal A than Luminal B (p < 0.001), HER2-positive (p < 0.05) and Basal subtype (p < 0.001), while there had no significant difference among Luminal B, HER2-positive and Basal subtype.DLAT had higher expression in T4 than T1&T2&T3 subtype (p < 0.05, Fig. 6D) and higher expression in pre-menopause than post-menopause patients (p < 0.05, Fig. 6E).DLAT also had higher expression in ER negative (p < 0.001) and PR negative patients (p < 0.01), however, there had no difference between HER2-negative and HER2-positive patients (Fig. 6F).In the KM-plotter database, patients in the DLAT low expression group (HR = 1.36, p = 0.031) had significantly longer RFS than patients in the high expression group (Fig. 6G).In the TNM-plotter database, the expression of DLAT had significantly difference between HER2-positive BC and

GO, KEGG and GESA analysis for DLAT
To further investigate the potential role of DLAT for HER2-positive BC, we divided the HER2-positive BC patients into two groups according to the DLAT expression, namely, DLAT high group and DLAT low group.The upregulated and downregulated genes between the two groups were showed in Fig. 7A, under the condition of given threshold of FDR < 0.05 and fold change > 1. KEGG pathway and GO enrichment of DEGs indicated that DLAT participates in various pathways correlate with organelle fission (GO:0048285), chromosome segregation (GO:0007059), nuclear division (GO:0000280), hormone-mediated signaling pathway (GO:0009755), regulation of intracellular estrogen receptor signaling pathway (GO:0033146), condensed chromosome (GO:0000793) and PPAR signaling pathway (hsa03320) (Fig. 7B, C).By performing GSEA, we further verified the potential roles of DLAT associated molecules in the regulation of cell cycle checkpoints, class I MHC mediated antigen processing presentation, DNA repair, interferon signaling and organelle biogenesis and maintenance (Fig. 7D).

The immune landscape for DLAT
We used the CIBERSORT algorithm to assess the difference of immune infiltration in HER2-positive BC patients with high or low DLAT expression.There is obvious difference in the distribution of 7 out of 22 immunocytes.As compared to the DLAT low group, the infiltration of B cell naïve (p < 0.01) and M2 macrophages (p < 0.001) subtypes was higher, while the infiltrating of B cell memory (p < 0.01), T cell CD8 + (p < 0.05), T cell fh (p < 0.05), Tregs (p < 0.001) and Neutrophil (p < 0.01) subtypes was lower in DLAT high group (Fig. 8A).We analyzed the correlation between DLAT and immune checkpoint inhibitors related genes (Fig. 8B).In addition, the difference in sensitivity to immunotherapy between patients in the DLAT high and low groups was further investigated using the TIDE algorithm.The TIDE algorithm is a recently developed tool for determining the efficacy of tumor immune checkpoint therapy 32 .In the present study, we found there was a negative correlation between TIDE and DLAT expression (r = − 0.292, p < 0.001, Fig. 8C).

The predicted value of DLAT for HER2-low BC patients
We further established the prognostic nomogram to quantitatively predict the 1-, 2-and 3-year survival probability of HER2-low BC patients (Fig. 9A).The calibration curve of the nomogram demonstrated that it could predict the survival probability relatively well (Fig. 9B).Although there had no difference of OS between DLAT high and low groups for HER2-low BC patients (HR = 2.15, p = 0.24, Fig. 9C).However, the HR+, DLAT low patients had longer OS than HR−, DLAT high patients (HR = 8.61, p = 0.032, Fig. 9D).

Discussion
Cuproptosis is a newly defined RCD, which occurs via direct binding of copper to lipoylated components of the tricarboxylic acid (TCA) cycle and this results in lipoylated protein aggregation and subsequent iron-sulfur cluster protein loss leading to proteotoxic stress and ultimately cell death 19 .In this study, we identified a novel The prognostic model for HER2-positive BC was established according to the LASSO method.Four named ATOX1, DLAT, SLC31A2 and SLC25A3, were included in this prognostic model.DLAT gene was the E2 core component of the pyruvate dehydrogenase (PDH) mitochondrial enzyme complex, which encoding dihydrolipoamide acetyltransferase.Dihydrolipoamide acetyltransferase accepts acetyl groups and transfers them to coenzyme A 33,34 .DLAT plays an important role in glucose metabolism and TCA cycle 19,35 .Goh et al. found that DLAT had higher expression in eleven gastric cancer cells lines compared with non-cancer gastric epithelial cell line HFE145, and pyruvate level was increased when DLAT knockdown with siRNA 36 .Another research found that DLAT expression have positive relationship with PD-L1 in a clinical hepatocellular carcinoma (HCC) cohort, and DLAT was upregulated and correlated with poor prognosis 37 .Zhou et al. found that in the HCC cells, the proliferation and invasion ability was decreased once DLAT was knockdown, and the PI3K/Akt or Wnt/β-catenin signaling pathways were also significantly inhibited 38 .In our study, however, DLAT was downregulated in HER2positive BC patients compared with normal tissues.Patients in the low DLAT expression group had significantly longer OS than patients in the high expression group.The negative correlation between DLAT expression and  www.nature.com/scientificreports/neutrophils, macrophages and dendritic cells was found in the HCC cells 38 .The GSEA verified the potential roles of DLAT associated molecules in the regulation of cell cycle checkpoints, class I MHC mediated antigen processing presentation, DNA repair, interferon signaling and organelle biogenesis and maintenance.DLAT may play distinct roles between HCC and HER2-positive BC.Several potential mechanisms, such as reduced drug binding to HER2, constitutive activation of HER2 parallel or downstream signaling pathways, metabolic reprogramming or reduced immune system activation, have been identified as primary/secondary resistance to anti-HER2 agents 14 .Trastuzumab can bind with HER2 extracellular domain IV, which can further prevent the formation of HER2-HER2 homodimers and HER2-HER3 heterodimers 39 , this also leads to the inhibition of HER2 ectodomain shedding and prevent the expression of the p95-HER2 isoform 40 .The additional mechanisms including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).Trastuzumab was recognized by Fc receptors of the innate immune system cells, including natural killer cells, antigen presenting cells and effector immune cells, once it bound to HER2 on cancer cell membranes, and this reduced the bounding of trastuzumab with cancer cell 41,42 .In our study, we found that DLAT high expressed HER2-positive BC patients have shorter OS and associated with resistance to trastuzumab and trastuzumab + pertuzumab.We identified DLAT had positive correlation with DNA replication and tumor proliferation and DLAT also had higher expression in T4 stage than T1&T2&T3 stage.All the evidence suggests that HER2-positive BC patients with high DLAT expression have a higher degree of malignancy and get limited benefits from HER2-targeted therapy.In our vitro experiment, knocking down of DLAT with siRNA significantly improved the treatment effect of trastuzumab.In addition, we found that high expressed DLAT had lower TIDE score, which means it is sensitive to immunotherapy.Immunotherapy has changed the treatment of several cancer types, and it has already been approved as a standard of care for patients with TNBC 43,44 .Immunotherapy for HER2-positive BC have been an interesting field and it is currently under clinical investigation.Based on the high TMB and high levels of tumor-infiltrating lymphocytes (TILs) of HER2-positive BC, investigating of immunotherapy for HER2-positive BC is feasible 45,46 .In this study, we found that DLAT also had significant positive correlation with TMB.DLAT high expressed HER2-positive BC patients may benefit from HER2-targeted therapy combined with immunotherapy.
Disruption of mitochondrial activity leading to physiological imbalances and metabolic shifts in the cell, which contribute to cancer progression.One of the most important reasons is mutations in TCA cycle enzymes 47,48 .However, as part of the TCA cycle enzymes, the somatic mutation rate of DLAT was 0.2% according to this study.The typical PDH deficiency patients were caused by E1α mutations, however, two PDH deficiency patients with DLAT mutation reported by Head et al. were less severely and both have survived well into childhood 49 .Shatokhina et al. demonstrated that DLAT was down regulated after the control U87 glioma cells were exposed under glutamine deprivation 50 .In our study, the expression level of DLAT had no difference between HER2-negative and HER2-positive patients.Nevertheless, it was still ambiguous whether DLAT was aberrant regulated in HER2-positive BC patients under some specific conditions.TCA cycle genes including www.nature.com/scientificreports/IDH2, SUCLA2, SDHB, SDHD were downregulated after co-expression of HER2 and mucin 1 cytoplasmic domain (MUC1-CD).Interestingly, MUC1-CD induced reduction in TCA cycle genes was found to be significantly associated with poor survivals in patients with HER2-positive BC 51 .Chang et al. demonstrated that in trastuzumab resistant gastric cancer cell lines (NCI N87R and MKN45R), the activity of GATA binding protein 6 (GATA6) was enhanced prominently.In GATA6 knockout cell lines, the sensitivity to trastuzumab was improved and the mitochondrial function was inhibited 52 .As DLAT plays an important role in glucose metabolism and TCA cycle, it also acted as a CRGs.In our study, KEGG pathway and GO enrichment of DEGs indicated that DLAT participates in various pathways correlate with organelle fission, chromosome segregation, nuclear division, hormone-mediated signaling pathway, regulation of intracellular estrogen receptor signaling pathway, condensed chromosome and PPAR signaling pathway.Despite the fact that the expression of DLAT had no difference between HER2-negative and HER2-positive BC patients, the exact function of DLAT in HER2-positive BC need further investigation.In summary, we identified a novel prognostic model consisting of four CRGs for HER2-positive BC patients.In addition, DLAT was further confirmed to be downregulated and correlated with better survival of HER2positive BC patients.High expressed DLAT related to resistance to HER2-targeted therapy and sensitivity to immunotherapy.However, this study also had some limitations.Firstly, the 14 CRGs were selected from the literature and there may exist some other genes related with cuproptosis, those results we got just applicable at the present stage.Secondly, we did not explore the detailed function of the four CRGs, especially DLAT, in HER2positive BC.Furthermore, it is unclear how DLAT affects anti-tumor immunity in HER2-positive BC patients.

Cell culture
The human breast cancer cell lines SKBR-3 was obtained from the American Type Culture Collection (ATCC), and cultured in RPMI-1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Sigma-Aldrich, USA) at 37°C in a humidified incubator with 5% CO 2 .

Cell growth assay
Growth inhibition was assessed using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) Cell Proliferation and Cytotoxicity Assay Kit (Beyotime Institute of Biotechnology, Shanghai, China).Cells were diluted in 100 μl/well of maintenance cell culture media and plated in 96-well flat-bottom plates.The number of SKBR-3 cells per well used in the subsequent experiments were 5000.At 24 h after plating, cell culture media were replaced with 10% FBS-containing media with and without trastuzumab, followed by incubation for an additional 120 h.Trastuzumab concentrations ranged from 0.001 to 100 μg/ml.A total of 3 plate wells were set for each experimental point, and all experiments were carried out at least in triplicate.Data are expressed as percentage of growth relative to that of untreated control cells.

Figure 1 .
Figure 1.Workflow of the current study.All of the TCGA BRCA data used in this study are HER2-positive type, unless otherwise stated.

Figure 2 .
Figure 2. Expression difference and survival analysis of CRGs in HER2-positive breast cancer.(A) Expression difference of 14 CRGs in HER2-positive breast cancer and normal breast tissues.*p < 0.05; ***p < 0.001; ns, no significance.(B) The forest plot of univariate COX hazard model of the 14 CRGs.*p < 0.05.(C) The OS curves of DLAT and SLC25A3 in patients with HER2-positive breast cancer in the low and high expression group.

Figure 3 .
Figure 3. Construction of the cuproptosis-related prognostic signature.(A) LASSO regression analysis of 14 prognostic CRGs.(B) Cross validation method to select optimal genes.(C) Rank dot, scatter plots and heat map of the model gene expression.(D) Kaplan-Meier analyses of the OS between the high risk and low risk group.

Figure 5 .
Figure 5.The genetic characteristic of DLAT.(A) The somatic mutation of DLAT.(B) The relationship between DLAT and MMR related genes.(C) The relationship between DLAT and TMB, TSI, stemness score.(D) The relationship between DLAT and DNA replication, tumor proliferation, angiogenesis.

Figure 6 .
Figure 6.The clinical characteristic of DLAT.(A) The OS curves of breast cancer patients with DLAT low and high expression group.(B) The alluvial diagram of DLAT with clinical characteristic.(C) DLAT expression in different PM50 subtype.*p < 0.05; ***p < 0.001; ns, no significance.(D) DLAT expression in different T stage.*p < 0.05.(E) DLAT expression in different menopause status.*p < 0.05.(F) DLAT expression in different ER status, PR status and HER2 status.**p < 0.01; ***p < 0.001; ns No significance.(G) The RFS curves of HER2positive BC patients with DLAT low and high expression group.(H) DLAT expression between normal breast tissues and HER2-positive BC tissues.(I) DLAT expression among normal breast tissues, HER2-positive BC tissues and metastatic tissues.

Figure 7 .
Figure 7. Functional analysis for DLAT.(A) The volcano map for DEGs in DLAT high or low group.(B) The GO enrichment and KEGG pathway for DEGs in DLAT high or low group.(C) The netplot of KEGG pathway of the DEGs.(D) The significantly enrichment pathway in DLAT high or low group according to the GSEA.

Figure 8 .
Figure 8.The immune landscape for DLAT.(A) The infiltrating levels of immune cells in high and low DLAT expression groups in HER2-positive breast cancer patients.*p < 0.05; **p < 0.01; ***p < 0.001.(B) The correlation between DLAT and immune checkpoint inhibitors related genes.(C) TIDE predicted response to immunotherapy.

Figure 9 .
Figure 9.The predicted value of DLAT for HER2-low breast cancer patients.(A) The nomogram for predicting HER2-low breast cancer patients' 1-, 2-, and 3-year OS probability.(B) The 1-, 2-, and 3-year calibration curves of the nomogram.(C) The OS curves of HER2-low breast cancer patients between DLAT low and high groups.(D) The OS curves of HER2-low breast cancer patients between HR+, DLAT low and HR−, DLAT high groups.
rate with the actual one of 1-, 3-, and 5-year survival rates.The nomogram, calibration curve and survival curve were drawn by R package 'survival' and 'rms' .